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UNIVERSITV OF HAWAI'I LIBRARY Investigating the Regulation of Fatty Acid Degradation in Pseudomonas aeruginosa A THESIS SUBMITTED TO THE GRADUATE DMSION OF THE UNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN MICROBIOLOGY AUGUST 2006 By David Tran Nguyen Thesis Committee: Tung T. Hoang, Chairperson Dulal Borthakur Paul Patek
180

Investigating the Regulation of Fatty Acid Degradation in ...€¦ · regulation of fatty acid degradation in P. aeruginosa. Using transcriptional fusions, the regulation of fatty

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Page 1: Investigating the Regulation of Fatty Acid Degradation in ...€¦ · regulation of fatty acid degradation in P. aeruginosa. Using transcriptional fusions, the regulation of fatty

UNIVERSITV OF HAWAI'I LIBRARY

Investigating the Regulation of Fatty Acid Degradation in Pseudomonas aeruginosa

A THESIS SUBMITTED TO THE GRADUATE DMSION OF THE UNIVERSITY OF HAW AI'I IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

IN

MICROBIOLOGY

AUGUST 2006

By

David Tran Nguyen

Thesis Committee: Tung T. Hoang, Chairperson

Dulal Borthakur Paul Patek

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We certify that we have read this thesis and that, in our opinion, it is satisfactory in scope

and quality as a thesis for the degree of Master of Science in Microbiology.

THESIS COMMITTEE

Chairperson

~~

ii

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Abstract

Although well established in Escherichia coli, the regulatory mechanisms

involved in controlling fatty acid metabolism in Pseudomonas aeruginosa are poorly

understood. Fatty acid metabolism consists of anabolic and catabolic pathways that play

several critical roles. Fatty acid degradative processes in P. aeruginosa have been shown

to play an important clinical role, contributing to the degradation of lung surfactant

component phosphatidylchoJine, whereas fatty acid biosynthetic processes contribute to

the synthesis of acyl homoserine lactones (AHLs) quorum sensing molecules involved in

virulence response and expression.

Experiments were conducted to investigate regulatory mer.hanisms and the

regulation of fatty acid degradation in P. aeruginosa. Using transcriptional fusions, the

regulation of fatty acid biosynthesis and degradation were studied and approaches were

utilized to identify the probable transcriptional regulators. The regulatory potential of

these probable transcriptional regulators were then assessed through gene-fusion and

DNA-binding studies.

During the course of this work, a genetic tool was developed, which may

contribute to future work on the regulatory processes in P. aeruginosa. The development

of this tool and its potential applications are included in this thesis.

iii

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Acknowledgements

I would like to thank Dr. Hoang for providing me with the opportunity to study in

Hawai'i and introducing me to the world of science and research. Thank you for pushing

me to my limits and challenging me, mentally, physically and emotionally. I leave not

only having gained knowledge in the field of science but also having learned a lot about

life and myself in general. You have prepared me for what lies ahead and have provided

me with the drive to take on any challenges I may encounter in the future, for that I am

thankful.

Thank you to Drs. Patek and Borthakur for taking the time to serve on my

committee, for their support and insights during my studies. I would also like to thank Dr.

Michael Schurr for providing me with the microarray data and thus the foundation for

studying the regulation of fatty acid metabolism. Thank you to Dr. Sung-Eun Lee for his

help with liquid-cbromatography-mass-spectroscopy and for taking time to help me with

my studies.

I would like to thank my labmates both former and current for their infinite

patience and understanding. I would like to thank Xiaojun (Steven) Lu, Yun Kang, and

Alton Wong for their support, encouragement and help in the lab. Thank for making me

laugh and smile when I needed it the most. I would especially like to thank Mike Son

who has been more than just a labmate and friend but also a brother. Thank you, not just

for your help with the microarray analyses or the growth curves but also for being a role

model and someone I can really look up to. I will always be grateful for all those times

you looked out for me and helped me when I was down. Things would have definitely

been a lot tougher had it not been for your companionship, thank you Mike.

iv

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I would also like to thank my God-sister, chi Khanh for welcoming me into her

home and into her family when mine was thousands of miles away. Thank you for

making me feel "at home" away from home, which is the greatest gift I can ever ask for.

You have truly been my "oxygen tank." I am eternally grateful for what you have done

for me and will never forget your kindness and your generosity.

Thank you to my parents, Lan Tran and Dinh Nguyen, for your unconditional

love and support. I am grateful and will always be, for the all the opportunities that you

have given me in life. You have always been there for me, given me strength when I am

weak, cure me when I am ill and pick me up when I fall, I cannot thank you enough for

all that you have done. I love you mom and dad.

Last but certainly not least, I would like to thank my brother and sisters,

and all my friends back home for their encouragement and support. I would like to

particularly thank my sister, chi Van, for giving me the strength to carry on all those

times when things felt hopeless and for patiently listening to all my problems. Thank you

for looking out for me even though you are an ocean away and for the being the best

sister a little brother can ask for.

v

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Dedication

This work

is dedicated to my

parents

Lan Tran and Dinh Nguyen

and to my

brother and sisters

anh Viet, chi Van

and

chi Anna

vi

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Table of Contents

Approval Page ..................................................................................... ii

Abstract ............................................................................................. iii

Acknowledgements ............................................................................... .iv

Dedication .......................................................................................... vi

Table of Contents .................................................................................. vii

L· fF' .. 1st 0 19ures ...................................................................................... XII

List of Tables ....................................................................................... xvi

List of Abbreviations .............................................................................. xvii

Chapter One: Introduction......... ...... ...... ....... ..... ...... ... ........ ........•....•.•.• 1

1.1 Microbiology of Pseudomonas aeruginosa .. ............................................. 1

1.2 P. aeruginosa Infections and Virulence Factors .......................................... 1

1.3 P. aeruginosa and Cystic Fibrosis (CF) ................................................... 6

1.4 Fatty Acid Metabolism and Regulation ................................................... 9

1.5 Transcriptional Regulators......... .. .... .................. ......... ........................ 17

1.5.1 TetR-type Regulators ............................................................ 18

1.5.2 LysR-type Regulators ........................................................... 19

1.5.3 IclR-type Regulators ............................................................ 20

1.5.4 GntR-type Regulators ........................................................... 21

1.6 Specific Aims of Research .................................................................. 23

vii

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Chapter Two: Materials and Methods ..............•.•...................................•.. 24

2.1 Media ........................................................................................... 24

2.2 Reagents ........................................................................................ 25

2.3 Bacterial Strains........................... ......... .......................................... 25

2.4 Bacterial Plasmids ............................................................................ 28

2.5 DNA Techniques.......................................................................... ... 30

2.5.1 Plasmid DNA Isolation ......................................................... 30

2.5.2 Isolation of P. aeruginosa Chromosomal DNA ............................. 30

2.5.3 Restriction Digests .............................................................. 31

2.5.4 Extraction of DNA from Agarose Gels ....................................... 31

2.5.5 Ligations .......................................................................... 31

2.5.6 Preparation of Competent Cells and Transformation ...................... 32

2.6 Polymerase Chain Reaction (PCR) Techniques ........................................... 32

2.6.1 PCR amplification ............................................................... 32

2.6.2 Oligonucleotides ................................................................ 33

2.7 Genetic Techniques ........................................................................... 34

2.7.1 Biparental Matings ............................................................... 34

2.7.2 Construction of Transcriptional Fusions in P. aeruginosa

and E. coli ........................ ................................................ 35

2.7.2.1 P. aeruginosa ............................................................ 35

2.7.2.2 E.coli.. ................................................................... 36

2.7.3 Gene Replacement ............................................................... 36

2.8 Transposon Mutagenesis ..................................................................... 38

viii

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2.9 Construction of Expression Vectors ........................................................ 39

2.1 0 Electrophoresis Mobility Shift Assays (EMSA) ........................................ .40

2.10.1 Crude Extract Preparation ...................................................... 40

2.10.2 Expression and Purification ofORF PA3508 for

EMSA Studies ................................................................... 40

2.1 0.3 Streptavidin Magnetic Particle Purification ................................. .42

2.10.4 Ammonium Sulphate Precipitation ........................................... .43

2.10.5 Biotin-labelling ...•.............................................................. 43

2.1 0.6 Gel Shifts ......................................................................... 44

2.11 ~-Galactosidase Assays ..................................................................... 45

2.11.1 Inverse Regulation Studies... .................. .................. ............. 45

2.11.2 Monitoring the Regulation ofjadBA5 in iacZTranscriptionai

Fusion Strains .................................................................... 45

Chapter Three: Inverse Regulation of Fatty Acid Degradation and

Biosynthesis .................................................................... 47

3.1 Introduction ................................................................................... .47

3.2 Results .......................................................................................... 49

3.2.1 Construction ofP/adBs-lacZ, P/adE"iacZ and P/abA-lacZ

Transcriptional Fusions in P. aeruginosa .................................... 49

3.2.2 Analysis ofjad andjab Regulation Using Transcriptional

Fusion .............................................................................. 52

ix

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Chapter Four: Bioinform.atic Approach ..............•...................................... 56

4.1 Introduction .................................................................................... 56

4.2 Results .......................................................................................... 57

4.2.1 Construction ofPAOl-attB:: PfadB5-1acZl4fadR::Gm

(PAl 627, PA4769, and PA5356) by Gene Replacement .................. 57

4.2.2 Characterizing PAOI-attB:: PfadB5-1acZlt.fadR::Gm

(pAI627, PA4769, and PA5356) by f3-Galactosidase Assays ............. 58

4.2.3 Construction OfPfadB.5"lacZTranscriptional Fusion in E. coli

(HPS I-A.aItB: :pCD13PSK -P fadB.5"lacZ) ..••.•......•.••••..••.•.•....•.••.•...• 63

4.2.4 Assessing the Regulatory Potential ofPA1627, PA4769,

and PAS356 by Expressing the Respective Gene Products

in the E. coli PfadBs-lacZ Transcriptional Fusion Strain ..................... 64

4.2.5 DNA Binding Studies ........................................................... 71

Chapter Five: Protein purification and EMSA .......................................... _. 78

5.1 Introduction .................................................................................... 78

5.2 Results .......................................................................................... 80

5.2.1 Analysis of Promoter Regions (PfadB5. PfadE. and PfabA) .••..••.•••.•••...... 80

5.2.2 Protein Purification Using Streptavidin Magnetic Particles ............... 80

5.2.3 DNA-binding Studies Using Consensus Sequence-Purified

Extract ............................................................................. 82

5.2.4 Identification of DNA-binding Protein by LC-MS .......................... 86

x

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5.2.5 DNA-binding Studies Using E. coli Lysate .................................. 86

5.2.6 Assessing the Regulatory Potential ofPA1627, PA4769,

and PA5356 by Expressing the Respective Gene Products

in the E. coli PfadBS'iacZTranscriptional Fusion Strain ..................... 90

5.2.7 Ammonium Sulfate Precipitation ............................................. 93

Chapter Six: TransposoD Mutagenesis ...................................................... 96

6.1 Introduction .................................................................................... 96

6.2 Results .......................................................................................... 98

6.2.1 Transposon Mutagenesis and Insertion Sites ................................. 98

6.2.2 Characterizing PAOl-attB:: PfadB5-lacZl4fadR::Gm

(pA2601, PA3006, and PA3508) by ~actosidase Assays ............. 100

6.2.3 Assessing the Regulatory Potential of PA260 1 , PA3006,

and PA3508 by Expressing the Respective Gene Products

in the E. coli P fadB5-1acZ Transcriptional Fusion Strain ..................... 105

6.2.4 DNA-Binding Studies using E. coli Lysate (pUC18 and

PA2601, PA3006, and PA3508 derivatives) ................................. 108

6.2.5 A Closer Look at PA3508 ...................................................... 108

Chapter Seven: Dis<:DSsion .......•..••••••.•.•..•....•..••...•.....••••.•••........••.•.••••••. 121

Appendix I .....................................................................•................... 139

Referenees ....................................................................................... _.143

xi

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List of Figures

Figure ~

1. PC structure and action of phospholipase and lipase ........................ 8

2. Fatty acid biosynthesis and degradative pathways .......................... 10

3. Model for FadR regulation ...................................................... 15

4. Schematic representation of mutant construction by

gene replacement... ........... .......... ......... .................. ... ......... 37

5. Mini-CTX2-mediated integration ofP/adB5"iacZ, P/adE"iacZ,

and p/abA-iacZ at the attB site of P. aeruginosa •.. .....•...••••.............. 51

6. PCR amplification ofP/adB5-iacZ, P/adE"iacZ, and P/abA-iacZ

in respective P. aeruginosa transcriptional fusion strains .................. 53

7. Inverse regulation studies in P. aeruginosa transcriptional

fusion strains ..................................................................... 54

8. PCR amplification from chromosomal DNA of

PA01-attB::P jwJJJ5"iacZ and mutant derivatives

(~AI627::GmR, ~A4769::GmR, and ~A5356::GmR) ................. 59

9. Schematic representation of gene replacement mutagenesis

strategy... . .. .. .. .. . .. . . . . .. . ..... . . . .. . .. . . .. . . . . ... . . .. .. .. ... .. .... . .. . . . . .. ... 60

10. Growth curves of P. aeruginosa transcriptional fusion strains

PAOl-attB::P/adBs-iacZ and mutant derivatives

(~AI627::GmR, ~A4769::GmR, and ~A5356::GmR) ................. 61

II. Transcriptional regulation of P/adB5-iacZ fusion in the

P. aeruginosa transcriptional fusion strain

xii

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PAO l-attB:: PjadBj-lacZ and mutant derivatives

(M'AI627::GmR, M'A4769::GmR

, and M'A5356::GmR) ................ 62

12. PCR amplification ofPjadB~lacZ in respective E. coli

transcriptional fusion strains ................................................... 65

13. Growth curves of E. coli transcriptional fusion strains

expressing PA1627 or PA5356 strain ......................................... 67

14. Growth curves of E. coli transcriptional fusion strains

expressing PA4769 .............................................................. 68

15. Transcriptional regulation ofPjadBj-lacZ fusion in E. coli

transcriptional fusion expressing PA1627 or PA5356 ...................... 69

16. Transcriptional regulation ofPjadB~lacZ fusion in E. coli

transcriptional fusion strain expressing PA4769 ............................ 70

17. EMSA studies with pUC18-PA1627 lysate .................................. 72

18. EMSA studies with pUC18-PA4769 lysate .................................. 73

19. EMSA studies withpUCI8-PA5356 lysate .................................. 75

20. EMSA studies with pUCI8-PA5356lysate

(using PjadB5, PjabA and glpD as probes) ....................................... 77

21. Alignment of putative consensus sequences ................................. 79

22. Promoter region offadBA5,fadE, andfahAB ................................ 81

23. Schematic representation of streptavidin magnetic particle

protein purification strategy .................................................... 83

24. Streptavidin magnetic particle purified extract .............................. 84

25. EMSA studies with streptavidin magnetic particle-purified

xiii

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extract ............................................................................. 85

26. EMSA studies with pUCI8-PA5525 lysate .................................. 88

27. EMSA studies with pUC 18-PA5525 lysate

(using PjadB5, PpM and glpD as probes) ....................................... 89

28. Growth curves of E. coli transcriptional fusion strains

expressing PA5525 .............................................................. 91

29. Transcriptional regulation of P jadBs-IacZ fusion in E. coli

transcriptional fusion strain expressing PA5525 ........................... 92

30. EMSA studies using ammonium sulfate precipitated extracts ............ 95

31. Schematic representation oftransposon mutagenesis

strategy. .. .. . .. . . ...... . . ... ... . .. .. . . . .. .. .... . . . .. .. . . . . .. . .... .. . .. .... .. . ..... 97

32. Transposon insertion sites grouped according to functional

class ................................................................................ 101

33. Growth curves of P. aeruginosa transcriptional fusion strains

PAOl-attB::PjadBs-1acZ and mutant derivatives

(M'A2601::Tn, M'AJ006::Tn, and M'AJ508::Tn) ........................ 103

34. Transcriptional regulation ofPjadBs-1acZ fusion in the

P. aeruginosa transcriptional fusion strain

PA01-attB:: PjadBS-1acZ and mutant derivatives

(M'A2601 ::Tn, M'AJ006::Tn, and M'AJ508::Tn) ........................ 104

35. Growth curves of E. coli transcriptional fusion strains

expressing PA2601, PAJ006 or PA3508 ..................................... 106

36. Transcriptional regulation of PjadBS-1acZ fusion in E. coli

xiv

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transcriptional fusion expressing PA2601, PAJ006

or PAJ50S ........................................................................ 107

37. EMSA studies with pUC1S-PAJ006 and

pUC1S-PAJ50S lysates .......................................................... 109

3S. EMSA studies with pUC1S-PA2601Iysate .................................. 110

39. Organization of genes surrounding PA350S locus .......................... 112

40. EMSA studies with pUC lS-P AJ50S lysate (gradient)............... ...... 113

41. EMSA studies with pUC1S-PAJ50S lysate

(gradient using PjabA) ••••••••••• ••••••••••••••••••••••••••••••••••••••••••••••••• 115

42. Overexpression ofHiS(;-tagged PAJ50S in E. coli ........................... 116

43. Purification ofHis6-tagged PAJ50S .......................................... 117

44. EMSA studies with HiS(;-tagged PAJ50S with PjadBSand PjabA ............ 119

45. EMSA studies with HiS(;-tagged P AJ50S

(using PjabA and glpD as probes) .............................................. 120

46. Dyad symmetry element in PjadBs ••• •••••••••••••••••••••••••••••••••••••••••• 132

47. Schematic representation ofFRT-lacZ integration .......................... 140

xv

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List of Tables

Table ~

1. Prokaryotic Regulator Families ................................................ 18

2. List of Bacterial Strains ......................................................... 26

3. List ofBacteria1 Plasmids ...................................................... 28

4. Oligonucleotides ................................................................. 33

5. Microarray Data:fab genes (LB vs. C16:01 C 18:1<\9) ••••••••••••••••••••••••••• 48

6. Microarray Data:fad genes (C16:01 C 18:1<\9 vs. LB) ............................ 48

7. Transposon Insertion Sites ..................................................... 99

xvi

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ACP

ADP

AMP

Ap

ATP

bp

Cb

CbR

CF

CFTR

em

DDW

dNTP

DNA

DIT

EDTA

EMSA

FAS

g

Gm

GmR

h

List of Abbreviations

acyl carrier protein

adenosine diphosphate

adenosine monophosphate

ampicillin

adenosine triphosphate

base pair

carbenicillin

carbenicillin resistant/resistance

cystic fibrosis

cystic fibrosis transmembrane regulator

centimeter

deionized distilled water

deoxynucleoside triphosphate

deoxyribonucleic acid

dithiothreitol

ethylenediaminetetraacetic acid

electrophoretic mobility shift assay(s)

fatty acid synthase

gram

gentamycin

gentamycin resistant/resistance

hour

xvii

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HTII

IPTG

kb

kDa

Km

I

LPS

LTTR

LB

M

MDRPA

mg

min

m1

mM

ng

OD

ORF

PC

PCR

pmol

PMSF

PIA

helix-turn-helix

isopropyl-~-D-thioga1actospyranoside

kilobase(s)

kiloda1ton

kanamycin

liter

lipopolysaccharide

LysR-type transcriptional regulator

Luria-Bertani

molar

multiple drug resistant Pseudomonas aeruginosa

milligram

minute

milliliter

millimolar

nanogram

optical density

open reading frame

phosphatidylcholine

polymerase chain reaction

picomole

phenyJmethylsulfonyl fluoride

Pseudomonas isolation agar

xviii

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PMN polymorphonuclear leukocyte

RBS ribosomal binding site

RNAP RNA-polymerase

rpm revolution per minute

RT room temperature

s second

SDS sodium dodecyl sulfate

Sp streptomycin

Sue sucrose

SucR sucrose resistant/resistance

TAE tris-acetate-EDTA

TBE tris-borate-EDTA

Tc tetracycline

TdT tenninal deoxynucleotidyl transferase

Tn transposon

Tris-Hel tris-hydrochloride

)1 micro

)1g microgram

III microliter

U unit

UV ultraviolet

v volume

w weight

xix

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X-Gal

°C

%

5-bromo-4-chloro-3-indoyl-f3-D-galactoside

degrees Celsius

percentage

xx

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Chapter One: Introduction

1.1 Microbiology of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a versatile Gram-negative bacterium that inhabits

many diverse environments such as soils, lakes, and mineral water and in association

with plants, but is also known to be an opportunistic pathogen of humans (36). Being an

opportunistic pathogen, P. aeruginosa is different from other human pathogens, given its

ability to colonize and cause disease in warm- and cold-blooded vertebrates, insects,

aquatic animals and plants (1,26, 102). P. aeruginosa is rod-shaped and ranges from 1 to

3 IJ.Ill in length and from 0.5 to 1 IJ.Ill in width (26, 9). The bacterium can be motile,

possessing one to three polar flagella (7, 19). The ability of P. aeruginosa to live in

diverse environments is partly due to its ability to inhabit in a range of temperatures,

capable of growing at temperatures from 5 to 42'C (19). A prominent characteristic of the

bacterium is its dimorphic lifestyle, existing as planktonic (individual free-swimming)

cells or as microcolonies (aggregation of cells) enclosed in an exopolysaccharide­

protected biofilm (15, 16).

1.2 P. aeruginosa Infections and Virulence Factors

P. aeruginosa is a ubiquitous pathogen capable of infecting virtually all tissues in

the human body (75). The bacterium causes a wide range of infections from minor skin

infections to more serious and sometimes life-threatening complications (127). Among

those particularly at risk of P. aeruginosa infections are patients with severe burns, are

immunocompromised such as AIDS sufferers, are neutropenic due to chemotherapy, or

have cystic fibrosis (CF) (123, 132). Recently, P. aeruginosa has been determined to be

1

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the leading cause of nosocomial infections and in many hospitals has become the most

common Gram-negative bacterial species associated with serious hospital-acquired

infections, particularly within intensive care units (85). The ability for P. aeruginosa to

cause such variety of human infections is related to three factors (127): i) it harbors an

arsenal of virulence factors, ii) it is resistant to a wide spectrum of antibiotics (56) and iii)

its nutritional versatility, making the bacterium extremely adaptive, which contributes to

its ability to colonize a variety of human tissues.

A variety of virulence factors contribute to the success of Pseudomonas infections.

The most prominent factors include biofilm formation, pili, flagella, lipopolysaccharide

(LPS), proteases, quorum sensing, exotoxin A and other enzymes secreted by the type III

secretion system (75). Pili and flagella are necessary for adhesion and colonization and

their importance as virulence factors is exemplified in burn wounds. Experiments

comparing infection of burn wounds by pilus- and flagella-deficient P. aeruginosa strains

clearly demonstrate that bacteria deficient in either of these structures have reduced

virulence, both in their ability to persist and disseminate (110). Their role as adhesins is

also evident in corneal infections. illcerative keratitis, a rapidly progressing inflammatory

response to bacterial corneal infection has been called the most destructive bacterial

disease of the human cornea (70). It has been demonstrated that both pili and flagella are

required for infection by binding to the host cell glycosphingolipid asialo-GMI (42). This

binding event is essential for epithelial cell invasion and cytoxicity that lead to corneal

infections (14).

Dissemination of P. aeruginosa is aided by the production of proteases (92).

Proteases generally serve to destroy physical barriers and to compromise host immune

2

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effectors (75). Elastase, for instance, has been shown to degrade col1agen and non­

col1agen host-protein, thus disrupting the integrity of the host basement membrane (2).

Studies have also demonstrated that elastase inhibits monocytes chemotaxis, which

adversely affect the early clearance of P. aeruginosa by phagocytosis, as wel1 as the

subsequent presentation of bacterial antigens to the host immune system (61).

Studies from Frank and col1eagues (129) have reported the presence of a type III

secretion system in P. aeruginosa that appears to playa significant role in virulence (111).

The type 111 secretion system delivers many important virulence factors such as ExoS,

ExoT and ExoU into mammalian cel1s. Pseudomonas also produces several other

exotoxin proteins that are thought to be major determinants of virulence (75). Exotoxin A,

for instance, has been extensively studied and determined to block protein synthesis

within targeted cel1s via ADP-ribosylation of protein elongation factor-2 (38, 83).

Administration of the purified toxin has been shown to result in rapid destruction of

corneal epithelial cel1s fol1owed by chemotaxis of polymorphonuclear leukocytes (PMNs)

to the site of infection and eventual corneal ulceration (57). Endotoxins are also

important virulence determinants. The production of LPS by P. aeruginosa plays a key

role in pathogenesis. LPS consists of a hydrophilic moiety consisting of the O-specific

chain (O-antigen) and the core oligosaccharide, covalently linked to a lipophilic moiety

(Lipid A) that anchors LPS to the outer membrane. Lipid A constitutes the endotoxic

portion of the LPS molecule (115), while the other components contribute to other

aspects of pathogenesis. The O-antigen of LPS-smooth strains are capable of resisting

complement-mediated phagocytosis, while LPS-rough strains evade host defenses owing

to the loss of a ligand that is important for host clearance (35).

3

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P. aeruginosa controls the production of many of its extracellular virulence

factors by a mechanism that monitors bacterial cell density and allows communication

between bacteria by cell-to-cell signaling. Bacteria are capable of sensing their

environment, process information, and react appropriately through the phenomenon

called quorum-sensing. Central to this cell-to-cell signaling system is the lasR gene that

encodes a protein critical for the initiation of the quorum-sensing response, which is

responsible for virulence factor production and biofilm formation. Recently, a study on a

P. aeruginosa strain, with a disruption in the lasR regulatory gene was shown to be

incapable of disseminating to distal host sites from a colonized burn wound (108). The

large number of virulence factors and its ability to produce them in a coordinated, cell­

density--dependent manner surely contribute to the success of P. aeruginosa as a

pathogen.

Due to the high intrinsic resistance of P. aeruginosa to antimicrobial agents,

effective antibiotic therapy has been problematic (107). This intrinsic resistance is

attributable to biofilm formation, low permeability of the outer membrane, the presence

of ~-lactamases (45) and multi drug efflux pumps (65). Biofilm formation significantly

increases resistance to antibiotics compared to what is normally observed in planktonic

cells. Studies have shown that when cells exist in a biofilm, they can become 1 0-1000

times more resistant to the effects of antimicrobial agents (101). Several mechanisms

contribute to this resistance including physical and chemical diffusion barriers, slow

growth of the biofilm due to nutrient limitation and induction of a general stress response

(77). The low permeability of the outer membrane is in part due to the highly charged

bacterial surface of P. aeruginosa, which is stabilized by divalent cations and is thought

4

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to reduce the passage of hydrophobic antibiotics (95). Small hydrophilic antimicrobial

agents, such as ~-lactams may enter the bacteria via porin proteins but at low frequency,

compared to E. coli for instance, P. aeruginosa has only 1 to 5% the permeability for ~­

lactams (43). Resistance to ~-lactams is additionally heightened by the presence of

periplasmic ~-lactamases (44). The presence of various efflux systems including mexAB­

oprM (74), mexCD-oprJ (97) and mexEF-oprN (96), which are capable of exporting

structurally unrelated antibiotics, allow P. aeruginosa to be resistant to multiple drugs.

The emergence of multi-drug resistant P. aeruginosa (MDRPA) is currently becoming a

major concern in the hospital setting, as effective antimicrobial options are severely

limited. MDRP A are resistant to at least three drugs in the following classes: ~ -lactams,

carbapenems, aminoglycosides, and fluoroquinolones and the number of MDRPA

isolates have been increasing. The emergence of MDRPA isolates during therapy was

reported in 27-72% of patients with initially susceptible P. aeruginosa isolates (87).

The genus Pseudomonas is notable for the large number and variety of

compounds that can serve as carbon and energy sources and P. aeruginosa is no

exception. P. aeruginosa has the ability to metabolize various compounds for carbon and

energy sources including mannose, fructose, glucose, ribose, xylose, glycerol, acetate and

many C4 to Cs fatty acids utilizing the Entner-Douddoroff pathway, the pentose

phosphate pathway and the tricarboxylic acid cycle (13). In addition, P. aeruginosa can

grow at temperature ranging from 5-42"C (19). Considered by many to be an aerobic

organism, P. aeruginosa is also capable of growing anaerobically if certain substrates are

available, for example, nitrates or arginine. Genetic exchange, including transformation,

5

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transduction, and conjugation, help P. aeruginosa adapt to changing conditions by

acquiring new genetic information (124).

1.3 P. aeruginosa and Cystic Fibrosis (CF)

P. aeruginosa is most prominently associated with causing significant morbidity

and mortality in cystic fibrosis (CF) patients. CF is an autosomal recessive disorder

resulting from mutation of the cyclic AMP (cAMP)-regulated chloride ion channel

protein known as the cystic fibrosis transmembrane conductance regulator (CFTR) (107).

The electrolyte imbalance that results causes dehydration within the lungs and the

production of a viscous mucous which significantly impairs mucociliary clearance

mechanisms, allowing persistant bacterial colonization (64). Although CF patients can

become infected with other microorganisms such as Burkholderia cepacia complex,

Staphylococcus aureus, Haemophilus injluenzae, and atypical mycobacteria, P.

aeruginosa predominates and more than 80% of CF patients over the age of 26 years are

colonized by P. aeruginosa (33). Recent evidence suggests that the CFTR protein, in

addition to its role in salt transport, may influence P. aeruginosa lung infection directly

through its role as an epithelial cell receptor (94). Mutations in CFTR are known to cause

under-sialylation of epithelial cell surface receptors, which increases P. aeruginosa

adherence to host tissue (27, 67). Cbronic infection with P. aeruginosa accounts most of

the pulmonary deterioration and mortality in CF patients, and P. aeruginosa is usually the

only pathogen recovered postmortem from the sputum and lung tissue (32).

The genome of P. aeruginosa contains 5570 genes (118), and analysis of the

annotated sequence reveals the presence of a substantial number of genes encoding

6

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putative factors that may facilitate the colonization of different habitats (17, 118). Its rich

gene pool contributes to it ubiquitous nature and enables it to colonize multiple niches

and utilize a variety of compounds as energy sources (117). Although the factors that

enable P. aeruginosa to colonize different tissues are generally understood, the sources of

energy that are available to sustain P. aeruginosa in relatively nutrient-poor environments

such as the lung, in particular the lung of CF patients has not been addressed. How P.

aeruginosa is capable of reaching high cell density required for the expression of

virulence factors remains to be answered.

The working hypothesis is that pulmonary lung surfactant may provide this

necessary source of energy. Pulmonary lung surfactant is synthesized by alveolar type II

epithelial cells and is secreted into the alveolar lumen and into other parts of the lung.

Lung surfactant is essential for the proper functioning of the lung in several respects by i)

maintaining alveolar patency thus preventing small airway collapse (29), ii) promoting

mucociliary clearance (60, 82), iii) acting as part of the innate immune response and host

defense to prevent pulmonary infections (62, 79, 98), iv) possessing anti-inflammatory

properties (132) and v) scavenging oxygen-radicals and enhancing antioxidant activities

inside the cell (78). It consists of surfactant proteins but is composed primarily of lipids

of which phosphatidylcholine (PC) is the predominant component, making up -80% of

the complex mixture (4, 51). The action ofphospholipases and lipases on PC, which are

both a part of P. aeruginosa virulence arsenal (3, 126), liberate lipid components that can

potentially serve as sources of carbon in the lung. It has been demonstrated that P.

aeruginosa phospholipase C cleaves the amphipathic PC molecule (72), and although the

action of lipases on PC has not been investigated, a similar action can be inferred (Figure

7

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Upases

1

Phospholipase C

Figure 1. Phosphatidylcholine (PC) structure showing cleavage sites of lipases and phospholipase C. Cleavage by lipases release glycerophosporylcholine and two fatty acids. Cleavage by phospholipase C liberates diacylglycerol and phosphorylcholine. R and R' represent the acyl fatty acid tails of PC.

8

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1). The metabolism of PC offers a very rich nutrient source for P. aeruginosa allowing it

to sustain itself and multiply in the lung. It has been suggested that the action of these

enzymes on PC contribute significantly to the pathology associated with P. aeruginosa­

related lung infections (72), thus the degradation of PC not only affords high cell-density

growth by liberating fatty acids as carbon and energy sources but also directly contributes

to disease.

1.4 Fatty Acid Metabolism and Regulation

Fatty acid metabolism is a fundamental component of the cellular metabolic

network and fatty acids are the essential building blocks for membrane phospholipid

formation (133). Fatty acids are synthesized for incorporation into phospholipid

membranes while exogenously supplied fatty acids can serve as carbon and energy

sources when coupled to the citric acid cycle via ~-oxidation (59). Most bacteria

synthesize fatty acids using a series of discrete proteins, each catalyzing one reaction in

the pathway (133). The bacterial system, also known as dissociated, type II fatty acid

synthase (F ASII), is a collection of individual enzymes encoded by separate genes.

Figure 2 outlines the major steps in the P. aeruginosa FASII (52). Much like in E. coli,

P. aeruginosa FASII is can be divided into two separate stages, composed ofan initiation

step followed by cycles of elongation. Acetyl-CoA carboxylase (AccABCD) catalyzes

the first committed reaction of fatty acid biosynthesis, forming malonyl-CoA from acetyl­

CoA. The malonyl moiety from the malonyl-CoA product is then transferred to ACP

(acyl carrier protein) by malonyl-CoA:ACP transacylase (FabD). Fatty acid synthesis is

initiated by the Claisen condensation of malonyl-ACP with acetyl-CoA catalyzed by ~-

9

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R - _0. R -

0C0A a~

Figure 2. Fatty acid biosynthesis and degradative pathways. (A) The fatty acid biosynthesis (Fab) pathway of P. aeruginosa (55) showing the biosyntheses of the two virulence controlling acylated-homoserine lactones (N-(butyryl)-L-homoserine lactone and N-(3-oxododecanoyl)-L-homoserine lactone). Although the complete fatty acid degradation (Fad) pathway(s) of P. aeruginosa have not yet been established, (B) shows the well known long-chain (~C12) Fad pathway of E. coli (12) and references therein; figure adapted from reference (12). Regulatory mechanisms of Fab and Fad by FadR from bacteria other than E. coli, including Pseudomonas, have not been characterized. In E. coli, FadR inversely regulates both Fab and Fad by up-regulating fab-genes and repressing fad-genes in rich media without long-chain fatty acid. However, in the presence of fatty acid (~C12),fab-genes are down-regulated and fad-genes are induced (23).

10

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ketoacyl-ACP synthase III (FabH) to fonn acetoacetyl-ACP. Four enzymes then catalyze

each round of elongation. The ~keto group is reduced by the NADPH-dependent ~­

ketoacyl-ACP reductase (FabG), and the resulting ~-hydroxyacyl-ACP is dehydrated by

the ~-hydroxyacyl-ACP dehydratase (FabA or FabZ) to enoyl-ACP. The final step in the

cycle is catalyzed by the NAD(p)H-dependent enoyl-ACP reductase (FabI or FabJ),

which produces an acyl-ACP. Additional cycles of elongation are initiated by the ~­

ketoacyl-ACP syntase (FabB or FabF), which elongates the acyl-ACP by two carbons to

from a f3-ketoacyl-ACP (52). Elongation ends when the fatty acyl chain reaches the

appropriate length that can be used for membrane phospholipid or lipopolysaccharide

synthesis (133). In addition to contributing to membrane phospholipid components, P.

aeruginosa like many Gram-negative pathogenic bacteria utilizes the fatty acid

biosynthesis pathway to synthesize acylated homo serine lactones (AHLs), which are used

to monitor cell density and to regulate many virulence factors by quorum sensing and

response (39, 71, 90, 93). The AHLs derive their invariant lactone rings from S­

adenosylmethionine (SAM) and their variable acyl chains from the cellular acyl-ACP

pool.

While the fatty acid degradative pathway (Fad) in P. aeruginosa has not been

characterized, the pathway is well established in E. coli and can serve as a basis for

understanding the ~-oxidative systems of other bacteria. The pathway by which E. coli

degrades fatty acids is substantially similar to the ~-oxidative pathways present in the

mitochondria of mammals and other eukaryotic organisms (12) and is shown in Figure 2.

The synthesis of at least five proteins involved in fatty acid degradation is coordinately

induced when long-chain fatty acids are present in the growth medium (12). The fad-

11

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regulon is primarily responsible for the transport, activation, and ~-oxidation of both

medium-chain (C7 to CII) and long-chain (C12 to CIS) fatty acids, and their expression is

specificaIIy controlled by the fadR gene product. The first step of fatty acid degradation is

the activation of free fatty acid to an acyl-CoA thioester by acyl-CoA synthetase (FadD)

as shown Figure 2. In E. coli, purifications studies have confirmed the existence of a

single acyl-CoA synthetase with broad specificity for medium- and long-chain fatty acids

(63, 89). In contrast, present mutational analysis studies in our laboratory suggest the

existence of at least four probable acyl-CoA synthetases in P. aeruginosa. Two operator

sites for the FadR repressor in the regulatory region of fadD have been identified and

confirmed by DNase footprinting experiments in E. coli (6). Promoter mapping studies of

the potentiaifadDs in P. aeruginosa are currently underway in our laboratory to identify

putative operator sites.

Knowledge of the next enzyme involved in the ~-oxidation pathway, acyl-CoA

dehydrogenase (fadE) has up until recently been very limited. FadE is responsible for the

first step of the ~-oxidation cycle of fatty acid degradation in E. coli, oxidizing acyl -CoA

thioester to 2-enoyl-CoA by transferring two electrons from the substrate to a flavin

adenine dinucleotide (FAD) cofactor (Figure 2). Much of the available genetic and

biochemical information specific to the E. coli acyl-CoA dehydrogenase reaction has

come from the doctoral dissertation ofK. Klein (K. Klein, Ph.D. Dissertation, Universitat

zu KoIn, KoIn, Germany, 1973). Although probable mutants (fadE) lacking

dehydrogenase activity have been isolated and mapped (63) the identity offadE in E. coli

remained a mystery. Recently, however, the identity of the fadE in E. coli was

determined through transcriptional array analysis of the FadR regulon (11).

12

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FadBA is the only known operon of the fad-regulon in E. coli, and encodes the

other p-oxidation enzymes that are a part of a multi-enzyme complex having broad

substrate specificity (12). The complex has been shown to have an (J.2P2 structure and is

associated with five enzyme activities (Figure 2), 3-ketoacyl-CoA thiolase, enoyl-CoA

hydratase, L-3-hydroxyacyl-CoA dehydrogenase, cis-tl3-trans- ~-enoyl-CoA isomerase,

and 3-hydroxyacyl-CoA epimerase (5, 86, 91, 99). Saturated fatty acid oxidation requires

enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA

thiolase activites whereas unsaturated fatty acids require two additional activities cis-tl3-

trans- ~-enoyl-CoA isomerase and 3-hydroxyacyl-CoA epimerase encoded by the

complex. With each cycle of the p-oxidation pathway, the activated fatty acyl-CoA loses

a two-carbon fragment as acetyl-CoA and reduces one molecule of FAD and one

molecule of NAD. The acetyl-CoA generated by CoA-dependent thiolytic cleavage is

metabolized by the TCA cycle whereas the shortened fatty acyl-CoA molecule re-enters

the degradation cycle.

PC metabolism as mentioned above liberates lipid components that can

potentially serve as a carbon and energy sources for P. aeruginosa, thus the p-oxidation

pathway may contribute to the degradation of the PC molecule allowing the bacterium to

survive in the lung of infected individuals. Recent studies in our laboratory support this

hypothesis showing that p-oxidation plays an important role in PC metabolism in vivo.

This potential for lung surfactant PC to serve as a carbon and energy source in the lung,

in addition to the involvement of fatty acid metabolism in quorum-sensing and virulence

expression makes fatty acid metabolism regulation in P. aeruginosa a topic of interest

(Figure 2). In E. coli, expression of the fatty acid p-oxidative or degradative (fat!) genes

13

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of the fatty acid degradation regulon is regulated at two levels. Firstly, most other carbon

sources are preferred over fatty acids, thus fad gene expression is under general

regulation by the global cAMP-dependent catabolite repression system. Secondly, fad

gene expression is under the control of a specific regulatory mechanism exerted by the

transcriptional factor, FadR, which plays a dual role in fatty acid metabolism regulation,

influencing both fatty acid degradation as well as biosynthetic processes (' coordinated

inverse' regulation) (Figure 2). FadR acts as a transcriptional repressor ofthefadregulon,

repressing genes essential for fatty acid transport, activation and ~-oxidation, including

fadL, fadD, fadE, fadBA, fadH, fadJ and fad! (11). Concurrently, FadR acts a

transcriptional activator of key enzymes required for unsaturated fatty acid biosynthesis,

fabA andfabB (10, 49). FadR has also been shown to be required for the activation of the

iclR gene, which encodes a specific repressor of the glyoxylate (ace) operon (40). Thus,

FadR regulates the conversion of fatty acids to acetyl-CoA and the utilization of this fina1

product in the citric acid cycle (59).

FadR is a helix-tum-helix DNA binding protein of GntR family (46) with a

molecular weight of 26.6 kDa The regulatory protein exists as a homodimer in solution

and binds DNA as a dimer (104). The following model describes the mechanism ofFadR

regulation of the fatty acid metabolism. In the absence of an exogenous source of long­

chain fatty acids, FadR is bound to all of its cognate operators acting both to repress

transcription of the fad genes and to activate the transcription ofthefab genes (Figure 3).

Upon addition of an exogenous supply of long-chain fatty acid, the fatty acid enters the

cell and is converted to its acyl-CoA thioester. The acyl-CoA binds to FadR resulting in a

conformational change that causes FadR to dissociate from all its operators. Dissociation

14

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+ Fatty

()--==:-0 Acid

• ladBA

Oc FadR () lad84

0 --==:-ladE .cRNA ladE

polymerase

• cAcyl-CoA () ~ •

fabAB - Fatty fabAB Acid

Figure 3. Model of fatty acid metabolism regulation in E. coli. The cell on the left depicts basal level expression of the FadR-reguiated processes. The fadR gene (not shown) produces FadR, which binds its cognate operators, repressing transcription of fadBA andfadE, while activating transcription from thefabAB promoter (sawtooth lines). The cell on the right depicts a cell induced by addition of fatty acid. The fatty acid is converted to acyl-CoA upon transport into the cell, which binds to FadR and releases it from its cognate operator DNAs. This release results in the de-repression of fadBA and fadE and decreased transcription of fahAR This figure is adapted from Figure I of reference (17).

15

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results in the induction of the fad genes because RNA polymerase can now fully use the

fad promoters, and the fab genes are repressed because there would be no DNA-bound

FadR to aid in RNA polymerase function (18). The recently described crystal structure of

FadR in the presence of its target DNA and its cognate inducer supports this model and

reveals the molecular basis of acyl-CoA-responsive regulation. The crystal structure of

FadR reveals a two domain dimeric molecule where the N-terminal domains bind DNA,

and the C-terminal domains bind acyl-CoA. The DNA binding domain has a winged­

helix motif, and the C-terminal domain resembles the sensor domain of the Tet repressor

(127). Analysis of the FadR-DNA complex reveals a novel winged helix-tum-helix

protein-DNA interaction, involving sequence-specific contacts from the wing to the

minor groove of the target DNA (122). Three residues, His-65, Arg-35 and Arg-45,

which are invariant in the known sequences ofFadR (Haemophilus injluenzae and Vibrio

cholerae) appear to interact directly with the target DNA, while other conserved residues

appear to be involved in indirectly stabilizing protein-DNA interaction through salt

bridge and electrostatic interactions (127). The binding of acyl-CoA results in dramatic

conformational changes throughout the protein, the net effect of which is the

rearrangement of the DNA binding domains in the dimer resulting in a separation of the

DNA recognition helices and loss of DNA binding (122). Thus, binding of the effector

molecule controls the separation between the recognition helices, and thus the ability to

interact with the DNA helix. Mutagenesis experiments have identified Gly-216, Glu-218,

Ser-219, Trp-223, and Lys-228 as components of the acyl-CoA binding site (127). The

transcription factor most similar to FadR from a structural and mechanistic point of view

is the tetracycline repressor, TetR (122). However, unlike TetR and many other DNA-

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binding proteins, FadR does not autoregulate its synthesis (49). FadR regulation seems

likely in bacteria closely related to E. coli but whether it exists in more divergent bacteria

remains to be addressed. A FadR-like protein has been discovered H injluenzae that

shares 47% identity to the E. coli FadR (103, 104) and incomplete open reading frames

(ORFs), which strongly match the E. coli FadR sequence, has been reported in V.

cholerae and V. alginolyticus. Thus it would appear that FadR regulation might be

broadly distributed in Gram-negative bacteria

1.5 Transcriptional Regulators

Prokaryotic transcriptional regulators are classified into families on the basis of

sequence similarity and structural and functional criteria. Most microbial regulators

involved in transcriptional control are two-domain proteins with a signal-receiving

domain and a DNA-binding domain, which transduces the signal. Structural analyses

have revealed that the helix-tum-helix (HTH) signature is the most recurrent DNA­

binding motif in prokaryotic transcriptional factors (105). Almost 95% of all

transcriptional factors described in prokaryotes use the HTH motif to bind to their target

DNA sequences (105). Table 1 lists some of the important families of microbial

transcriptional regulators, whether the members are preferentially repressors or activators,

some of the functions regulated by each family and the active conformations. Further

discussion of transcriptional regulators will focus on GntR, IcIR, TetR, and LysR-type

regulators because of their direct relevance to this study.

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Table 1. Prokaryotic regulator families

Family Action Some Regulated Function Active Conformation

*LysR activator/repressor carbon and nitrogen metabolism tetramer

*TetR repressor biosynthesis of antibiotics, efflux dimer pumps, osmotic stress, etc.

*IclR repressor/activator carbon metabolism, efflux pumps tetramer

*GntR repressor/activator general metabolism dimer (padR)

AsnC activator/repressor amino acid biosynthesis octamer

MarR activator/repressor multiple antibiotic resistance dimer

Crp activator/repressor global responses, catabolite repression dimer and anaerobiosis

>I< Correspond to families relevant to this study. This table has been adapted from reference (105).

1.5.1 TetR-type regulators

TetR family members are particularly abundant in microbes exposed to

environmental changes, such as soil microorganisms, plant and animal pathogens,

extremophiles and methanogenic bacteria (105). In P. aeruginosa PAOl, there are 38

transcriptional regulators that are members of the TetR family. Generally, members of the

TetR family are involved in the adaptation to complex and changing environments. All

members of the family whose functions are known are repressors and probably function

in a similar way. The binding of an inducer molecule to the non-conserved domain causes

18

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confonnational changes in the conserved DNA-binding domain that result in the release

of the repressor from the cognate promoter (105).

1.5.2 LysR-type regulators

LysR-type transcriptional regulators (LTIRs) comprise the largest family of

prokaryotic regulatory proteins identified thus far (47, 113). The family includes over 100

members and has been identified in diverse bacterial genera (121). Although a large

group of L TIRs regulate a single target operon, such as CatR, which specifically controls

the calBCA expression for catechol metabolism in Pseudomonas putida, some members

are capable of controlling more than one. The NahR protein for instance, is a master

regulator for the regulon of naphthalene degradation and controls both the nah and sal

operons required for metabolism of naphthalene to salicylate and pyruvate, and salicylate

conversion respectively (112,131).

Generally, the gene for L TIRs lies upstream of its target-regulated gene cluster

and is transcribed in the opposite direction, although exceptions do exist (121). L TIRs

usually act as transcriptional activators for their target metabolic operons in the presence

of a chemical inducer and are capable of repressing their own expression. Both

autorepression and activation of the catabolic operon promoter are exerted on the same

binding site (121). Since most studies have focused on the mechanisms of target gene

activation, relatively little is known about repression mechanisms (121).

L TIRs appear to be genera11y composed of 394 to 403 amino acid residues with a

molecular mass of between 32 and 37 kDa and evidence thus far supports LTIRs

functioning as tetramers (121). Members of the LysR family have a conserved domain

19

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organization. The DNA binding region with a predicted HTII motif is located in N­

terminus of the protein, while inducer recognition and multimerization domains are

located in the C-terminus (121). Because of its tetrameric form, LTIRs interact with

several sites on the DNA of the promoter region and the activator binding sites appear to

be consistently upstream of the transcription start site. ClcR for example, which is

involved in regulating the clcABD operon for cblorocatechol metabolism, protects a 27-

bp region from -79 to -53 and a 10-bp region from -37 to -28 relative to the start site

(121).

1.5.3 IelR-type regulators

IciR-type regulators have a similar structure as the LTIRs (113) but rather

dissimilar amino acid sequences (121). Generally, Ic1R-type regulators are transcriptional

repressors (50), however, those that are involved in regulating catabolic pathways have

all been described as activators. PcaU of P. pulido is an activator for the protocatechuate

pathway but in the absence of its inducer is capable of acting as a repressor (120). Similar

to LTIRs, the gene for Ic1R-type regulators generally lies upstream of its target gene

cluster and is transcribed in the opposite direction. Like L TIRs, IclR -type regulators are

capable of repressing their own expression (22).

The size ofIclR-type regulators is around 238 to 280 amino acids with molecular

masses between 25 to 30 kDa. IclR family members have an HTII DNA binding motif in

the N-terminal domain (21) and a C-terminal domain that is involved in subunit

multimerization and effector binding (66). The IclR-type regulators seem to exist as a

dimer of a dimer or in other words as tetramers in its active DNA binding form (121).

20

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Because the two subunits within one IclR dimer only interacts at the interface of their

DNA binding domains, the distance between the HTH motifs within one dimer is

relatively short and results in a structure favorable for binding relatively short (12- to 14-

bp) palindromic DNA sequences (121).

There is no clear consensus on the binding site for IclR regulators but it appears

that it usually lies upstream of the transcription start site. For example, MhpR and PeaR,

which are involved in controlling protocatechuate and 3-(3-Hydroxyphenyl)-propionic

acid metabolism respectively, both bind upstream of their target operon start sites. IclR­

type regulators bind their promoter DNA in the absence of effector, and adding effector

does not appear to enhance protein-DNA interactions in some representative IclR family

members. The addition of chemical effectors does however enhance formation of

regulator-DNA-RNA polymerase (RNAP) complexes compared to the situation without

(41) suggesting that the role of the regulatory protein may be to recruit RNAP to the

promoter.

1.5.4 GntR-type regulators

Members of the GntR family are generally transcriptional repressors in the

absence of pathway substrates (121). As is seen in family members that control the

degradation of aromatic compounds, the presence of pathway substrates relieves

repression through the interaction of the regulator with its effector molecule (31).

Although most members of the GntR family are negative regulators, BpbR2, which is a

part of the biphenyl catabolic regulon seems to act as an activator. Not much is known

about the mechanism through which BpbR2 activates transcription, however, gel shift

21

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assays have demonstrated that purified BpbR2 binds to its own promoter region more

strongly in the presence of inducers than in their absence (125).

GntR-like regulators are between 239 and 254 amino acids long with a molecular

mass of around 27 kDa (121). Haydon and Guest first described bacterial regulators of

the GntR family as having conserved N-terminal DNA binding domains but variable C­

terminal effector binding and oligomerization domains (46).

Simple hindrance for RNAP binding or open-complex formation seems to be the

general repression mechanism of GntR-like regulators (121). GntR proteins bind either

the promoter region or between the transcription and translation start sites. One member

of the family, BphS of Pseudomonas spp., which regulates (chIoro )-biphenyl metabolism,

binds to four region of its cognate promoter with varying affinities (88).

22

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1.6 Specific Aims of Research

Current knowledge of fatty acid metabolism in P. aeruginosa is limited to the

fatty acid biosynthetic pathway, not much is known about fatty acid degradation or how

fatty acid metabolism is regulated. The objective of this study is to investigate the

regulation of fatty metabolism in P. aeruginosa and use various approaches to attempt to

identify a regulator of the fatty acid degradation pathway. The specific aims of this study

are:

1. Construct transcriptional fusion strains that would aid in the study of fatty acid

metabolism in P. aeruginosa.

2. Use the transcriptional fusion strain to investigate how fatty acid biosynthesis and

degradation are regulated.

3. Employ different strategies to identify a regulator of fatty acid degradation and

assess their potential to regulate the fadBA5 operon. The strategies are as follows:

a. Use bioinformatics to identify potential regulators based on homology to E.

coli FadR.

b. Use different protein purification schemes to isolate and identify a protein

that interacts with thefadBA5 operon.

c. Use transposon mutagenesis to screen and identify potential transcriptional

regulators ofthefadBA5 operon.

23

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Chapter Two: Materials and Methods

2.1 Media

The following media were used during the course of the experiments described in

this thesis. Luria-Bertani (LB) broth consisted of 1.0 % tryptone, 0.5 % yeast extract, and

0.05 % NaCI and was purchased pre-formulated from Teknova (Half Moon Bay, CA).

The same formulation containing 1.5 % agar was used for preparation of LB agar plates.

Pseudomonas isolation agar (PIA) was purchased from Difco Laboratories. M9 media

was used as described in Sambrook et al., 2001 (109). M9 media ingredients per liter

include 6 g Na2HP04, 3 g KH2P04, 0.5 g NaCI. and 1 g NILC!. The medium was

autoclaved and MgS04 and eaCh were added to 1 mM and 0.1 mM final concentration

after cooling.

When necessary, respective media were supplemented with antibiotics after being

allowed to cool following autoclaving. All antibiotics were purchased from Teknova.

Ampicillin (Ap) was dissolved in deionized distilled water (DDW) at a concentration of

100 mg/ml, filter sterilized through a 0.22 J.lffi filter and stored frozen in aliquots at -

20·C. Carbenicillin (Cb) was dissolved in DDW at a concentration of 500 mglml and

stored in aliquots at - 20·C. Gentamycin (Om) was dissolved in sterile DDW at a

concentration of 40 mg/ml, filter sterilized and stored at 4·C. Streptomycin (Sp) was

dissolved in DDW at a concentration of 25 mg/ml, filter sterilized and stored at 4 ·C.

Kanamycin (Km) was dissolved in sterile DDW at a concentration of 35 mg/ml, filter

sterilized and stored at 4·C. Tetracycline (Tc) was dissolved in 70% ethanol at a

concentration of 10 mg/ml and stored at -20·C. Ap was used in E. coli at a final

concentration of 100 Ilg/ml, while Om, Sp, and Tc were used at a final concentration of

24

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15 flglml,25 flglml, and 10 flgIml respectively. Km was used at a final concentration of

35 flglml. In P. aeruginosa Cb was used at 500 flgIml final concentration, while Gm and

Tc were used at 150 flglml and 200 flglml respectively.

2.2 Reagents

Reagents used for agarose gel electrophoresis were high and low-melting point

agarose from Sigma. The buffers used were IX TAE (0.04 M Tris-acetate, 0.001 M

EDTA, pH 8.0) and O.5X TBE (0.045 M Tris-bomte, 0.001 M EDTA, pH 8.0). TAE was

routinely used for agarose gel electrophoresis and TBE was used for acrylamide gel

electrophoresis followed by electroblotting and gel shift assays. The 6X DNA loading

buffer and 2X protein loading buffer was prepared as described in Sambrook et al. 200 I

(l09).

X-Gal (5-bromo-4-chloro-3-indoyl-~-D-galactoside) (Sigma, St. Louis, MO) was

dissolved in dimethylformamide at a concentration of20 mglml and stored as aliquots at-

20°C. The X-Gal indicator was incorpomted into PIA media by adding 80 flgIml final

concentration to cooled media after autoclaving.

DNA standards were purchased from New England Biolabs (NEB), and dissolved

at a concentration of 0.1 flgIfll in TE buffer (10 mM Tris-HCI, pH 8.0, 1 mM EDTA)

containing IX DNA loading buffer then stored at 4°C. For use as standards, 5 fll of the

DNA solution was loaded per lane.

2.3 Bacterial Strains

25

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The bacterial strains utilized in the various experiments described in this thesis are

listed in Table 2.

Table 2. List of Bacterial Strains

Strain Genotype Reference/Source

E.coli

DHSa (f.lSOdlacZLJM15, a(lacZY A-argF)U169 Bethesda Research deoRrecAl endAl hsdR17 (rK-mK+) Laboratories, supE44).-thi-l gyrA96 relAl Gaithersburg, MD

ER2S66-mob F- 'f...-jhuA2 [Ion] ompT lacZ::17 gene 1 gal T.Hoang sulAll L1(mcrC-mrr) 114::1810 R(mcr-73::miniTnlO)2 R(zgb-120::TnlO)l (Tets) endAl [dcm]recA::RP4-2Tc::Mu Km

ER2S66 F- 'f...-jhuA2 [Ion] ompT lacZ::17 genel gal New England sulA11 L1(mcrC-mrr) 114::1810 R(mcr- Biolabs, Beverly, 73::miniTn10)2 R(zgb-120::TnlO)l (Tets) MA endAl [dcm]

SMI0-Apir thi thr leu tonA lacY supE recA::RP4-2- (SI) Tc::Mu Km ').pir

HPSI e14- (mcrA) recAl endAl gyrA96 thi-1 T.Hoang hsdR17 supE44 relAl a(lac-proAB) rif zxx::miniTn5Lac4 (lacP'" lacZaM1S)

HPSl::pir116 el4- (mcrA) recAl endAl gyrA96 thi-l T.Hoang hsdR17 supE44 relAl a(lac-proAB) rif zxx::miniTn5Lac4 (lacP'" lacZaMlS) pir116

HPSl- HPSI with'f...attB::pCD13PSK- this study

26

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Strain AattB::pCD13PSK­P/adBs-lacZ

P. aeruginosa

PAOl

PAOl-attB::P/adBAS­lacZ

Genotype

prototroph

PAOt withattB::P/ad.w-lacZ

PAOl-attB::P/attE-1acZ PAOl with attB::PjattE-1acZ

PAOl-attB::P/abAB" lacZ

PAOl-attB::P/adBS­lacZlM'A1627::GmR

PAOl-attB::PjadBS­lacZlM'A4769::GmR

PAOl-attB::P/adBS­lacZl M' A5356::GmR

PAOl-attB::PjadBS­lacZlM'A2601::Tn

PAO l-attB::P/adBs­lacZl M' A3006::Tn

PAOl-attB::PjadBS­lacZlM'A3508::Tn

PAOl with attB::P/abAB"lacZ

PAOt with attB::PjadBs-lacZ M'A1627::GmR

PAOt with attB::P/adBs-1acZ M'A4769::GmR

PAOl with attB::P/adBs-1acZ M'A5356::GmR

PAOt with attB::P/adBs-lacZM' A2601 ::Tn

PAOI with attB::PjadBs-1acZM'A3006::Tn

PAOl with attB::PjadBs-1acZM' A3508::Tn

27

Reference/Source

M. Vasil

this study

this study

this study

this study

this study

this study

this study

this study

this study

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2.4 Bacterial Plasmids

The bacterial plasmids used and constructed in this study are listed in Table 3.

Table 3. List of Bacterial PIasmids

Plasmids Properties Reference/Source

pPS856 ApR, GmR; GmR-FRT cassette vector (53)

pBTIO GmR; Himar-l mariner mini transposon (69) vector

pPICK KmR; temperature sensitive (100)

pTZ120 ApR; lacZ operon fusion vector (114)

pTZ120-PjadBs-1acZ ApR; l20-bp HindIII and blunt fragment this study ofPAOl chromosomal DNA cloned into HindIII + SmaI cut pTZ120

pTZ120-PjadE""lacZ ApR; 270-bp HindIIl and blunt fragment this study ofPAOl chromosomal DNA cloned into HindIII + SmaI cut pTZ120

pTZ120-P jabAe-1acZ ApR; 320-bp HindIII and blunt fragment this study ofPAOl chromosomal DNA cloned into HindIII + SmaI cut pTZ120

pEX18T ApR; oriT + sacB +, gene replacement (53) vector

pEX18T-PA1627 ApR; 955-bp EcoRI- HindIII fragment of this study PAOI chromosomal DNA cloned into EcoRI+ HindIII cut pEX18T

pEX18T-PA4769 ApR; 1.4-kb EcoRI - HindIII fragment of this study PAOl chromosomal DNA cloned into EcoRI+ HindIII cut pEX18T

EEX18T -P A5356 ApR; l.3-kb EcoRI- HindIII fragment of this study

28

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Plasmids Pro~erties Reference/Source PAD 1 chromosomal DNA cloned into EcoRl + Hindfll cut pEX18T

pEX18T-PA1627::Gm ApR, GmR ; l.l-kb PstI fragment of this study pPS856 cloned into PstI cut pEX18T::PA1627

pEX18T-PA4769::Gm ApR, GmR; l.l-kb SmaI fragment of this study pPS856 cloned into SgfI (blunt) cut pEX18T::PA4769

pEX18T-PA5356::Gm ApR, GmR ; l.l-kb SmaI fragment of this study pPS856 cloned into MluI (blunt) cut pEX18T::PA5356

mini-CTX2 Tef; integration vector (54)

mini-CTX2-PjadB5-1acZ TetR; 3.5-kb Hindfll andAjlll (blunt) this study fragment ofpTZ120::PjadB5cloned into Hindfll + SmaI cut mini-CTX2

mini-CTX2-PjadE"lacZ Tef; 3.6-kb Hindfll and Ajlll (blunt) this study fragment ofpTZ120::PjadE cloned into Hindfll + SmaI cut mini-CTX2

mini-CTX2-PjabAB-lacZ Tef; 3.7-kb Hindfll andAjlll (blunt) this study fragment ofpTZ120::PjabA cloned into Hindfll + SmaI cut mini-CTX2

pCD13PSK SpR; integration vector (100)

pCD13PSK-PjadB,-lacZ SpR; 4.6-kb SapI (blunt) fragment of (100) mini-CTX2::PjadB5-lacZ cloned into NspI cut (blunt) pCD13PSK

pUC18/19 ApR; cloning vectors (130)

pUC18-PA2601 ApR; 1.4-kb EcoRl- Hindfll fragment of this study PAD 1 chromosomal DNA cloned into EcoRl+ Hindfll cut pUCl8

pUC18-PA3006 ApR; 1.3-kb EcoRl- Hindfll fragment of this study PAOl chromosomal DNA cloned into EcoRl+ Hindfll cut pUC18

29

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Plasmids

pUC I 8-PA3508

pUC18-PA5525

pUC I 8-PA1627

pUC I 9-PA4769

pUCI8-PA5356

pET28a

pET28a-P A3508

2.5 DNA Techniques

Properties Reference/Source

ApR; 1.4-kb EcoRI- HindIll fragment of this study PAD I chromosomal DNA cloned into EcoRI+ HindIll cut pUCl8

ApR; 1.3-kb EcoRI- HindIll fragment of this study PADI chromosomal DNA cloned into EcoRI+ HindIll cut pUCl8

ApR; 955-bp EcoRI- HindIll fragment of this study PAOl chromosomal DNA cloned into EcoRI+ Hint/ill cut pUCl8

ApR; l.4-kb EcoRI- HindIll fragment of this study PAD I chromosomal DNA cloned into EcoRI+ HindIll cut pUCl9

ApR; 1.3-kb EcoRI- HindIll fragment of this study PAOl chromosomal DNA cloned into EcoRI+ HindIll cut pUCl8

KmR; T7 expression vector Novagen, Madison, WI

ApR; 1.4-kb EcoRI-NdeI fragment of this study PADI chromosomal DNA cloned into EcoRI+NdeI cut pET28a

2.5.1 Plasmid DNA Isolation

For plasmid DNA isolations, standard plasmid purification or 'mini-prep' (Zymo

Research Corp.) was performed as directed by the manufacturer.

The protocol above was routinely used for isolation of high-copy number

plasmids. For the isolation of low-copy number plasmids, essentially the same protocol

was utilized except nuclease (QIAGEN) was added prior to the washing step to minimize

30

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DNA degradation. The isolation procedure was then continued essentially as described

above.

2.5.2 Isolation of P. aeruginosa Chromosomal DNA

P. aeruginosa chromosomal DNA was isolated using the lsoQuick Nucleic Acid

Extraction kit (ORCA Research, Bortell, W A) utilizing buffers and protocol described by

the manufacturer.

2.5.3 Restriction Digests

Restriction digests were performed using enzymes purchased from New England

Biolabs (NEB). Buffers from the enzyme supplier were used and reactions were

incubated at 37'C or as specified by the supplier for a minimum of 1 hr. Blunt-ending

was carried out by adding 1 ¢ of2 mM dNTPs and 3 U ofT4 DNA polymerase directly

to the digest, followed by an additional incubation at 37·C for 30 min.

2.5.4 Extraction of DNA from Agarose Gels

DNA samples were usually ran on 1 % agarose gels and viewed under UV

following ethidium bromide staining. Bands of desired sizes were excised from the

agarose gel and DNA extracted utilizing the Zymo Gel Extraction protocol then stored at

- 20·C. These extractions were performed on any DNA fragments separated on agarose

for the purpose of ligation, biotin labeling or sequencing of PCR products.

2.5.5 Ligations

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Ligations were performed by using a 4:1 (insert:vector) ratio. T4 DNA ligase was

purchased from Invitrogen or NEB. Ligation reactions were incubated overnight at RT in

IX ligase buffer, using 1 U of T4 DNA ligase. Samples were usually used for

transformation immediately or stored at - 20'C for later use.

2.5.6. Preparation of Competent Cells and Transformation

Cultures of the desired strains were grown overnight at 37'C in 5 ml LB broth.

The entire overnight culture was used to inoculate 500 ml of fresh LB media and grown

with shaking at 37'C to log phase (OD6oo - 0.5). Cells were then immediately harvested

by centrifugation (20 min, 7500 rpm, 4 'C) in pre-chilled sterilized centrifuge tube using a

Beckman floor model centrifuge. The supernatant was decanted and the cell pellet

resuspended in 40 ml ice-cold sterile 0.1 M MgCh and then chilled on ice for 5 min. The

cells were then sedimented as before, the supernatant decanted and the cell pellet

resuspended in 20 ml ice-cold sterile TG-salts (75 mM CaCh, 6 mM MgCI2. 15%

glycerol). After incubation on ice for 20 min, the suspension was sedimented as described

above and the pellet resuspended in 10 ml ice-cold TG-salts. Competent cells were

chilled on ice from 4 h to overnight, and then aliquots of 200 JJl were transferred into pre­

chilled microfuge tubes and stored at - SOT. For transformation, 10 JJl of ligation mixture

or 5 JJl of plasmid DNA preparation was added to 0.2 ml of competent cells transferred

into a thin-walled glass tube sitting on ice. After incubation for 30 min on ice, the

suspension was heat-shocked for 2 min at 42 'C, then 1 ml of LB broth was added and the

tubes were shaken at 37'C for I h before plating 150 JJl aliquots onto selective media

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2.6 Polymerase Chain Reaction (PCR) Techniques

2.6.1 PCR amplification

Standard reaction mixtures contained IX Pfo buffer, 200 !JM dNTPs, 30 pmol of

each oligonucleotide, PAOl chromosomal DNA/plasmid template, 5 U of TaqlPfu.

General reaction conditions were as follows: 94SC for I min, followed by 34 cycles of

94SC for I min, 50-70"C for 30 s, and 70-72"C for I minlkb. PCR were carried out

using the EppendorfMastercycler gradient.

2.6.2 Oligonucleotides

The oligonucleotides used in this study are listed in Table 4.

Table 4. Oligonucleotides

Primer Name

lacZ-fusion construction #274-fabAB(p1 )-Hindill #277:fabAB-PEI #384-fadE-HindIIl #385:fadE-SmaI #287:fadBA5(p4)-Hindill #289:fadBA5-P5 #45I-M13-FP2

Bioinformatics #534-HindIIl-PAI627 #535-PAI627-EcoRI #536-HindIIl-PA4769 #537-PA4769-EcoRI #538-EcoRI-P A5356 #539-P A5356-HindIII #582-5'-1627 #583-1627-3'

Regulator purification #499-PfadB5-concatamer

Sequence·

5' -CTGCAAGCTTATCGGGAGAACTGCCTGCAG-3' 5' -AATCCCCGGGCTGCAGGGTTGTGGCGGTTC-3' 5' -CGTGAAGCTTGGCCTTCAGGGTCTGCGGATC-3' 5' -TTGGCCCGGGTTTGTCACGTT ATAGGCG-3' 5' -TGAATAAGCTTGCGCAGAGGGCCTGGAGGAG-3' 5' -GCAAACGCTCGA TTCATACGCC-3' 5'-GATTAAGTTGGGT AACGCCAG-3'

5'-TAAGTTAAAGCTTTCCGGGCTTGCAGCTGC-3' 5'-TATAAGAATTCGCGCACCTGGTTGAGCAGG-3' 5'-ATGGTAAGCTTCCTCTCI I I IICGCGCTGG-3' 5'-TAAGAA TTCACCTCTATCCCTCAAGCGCGAC-3' 5'-ACTAGAATTCACCTTCAGCCGATGGCAGAG -3' 5'-ATTTAAGCTTGCGGCGAGGGTAGCACGTTC -3' 5' -ATTCATCCGATGATTGGATG-3' 5' -ATTCAAGGCGTGAGTCATG-3'

5' -CTGCCGGAATGTGTGCGCACCCAA)3-3'

33

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Primer Name #504-PfadB5-reverse #561-KpnI-PA5525 #562-P A5525-HindIII

Transposon Mutagenesis #550-EcoRI-PA2601 #551-PA2601-HindIII #546-EcoRI-P A3006 #547-PA3006-HindIII #544-EcoRI-PA3508 #545-PA3508-HindIII #559-NdeI-P A3508

Sequencing #139-Taq-up #524-HIBI7 #463-GmR-RT #525-3' -Om-reverse #526-marinerTn-reverse

EMSA Studies

Sequence-5' -biotin-TTGGGTGCGCACACA TTCCG-3' 5'-ACCGGTACCCGCGGCAGCAAC-3' 5'-AAAGCTTCTGTTCGCTGCATTGC-3'

5'-AAATGAATTCGACCACCGAGAAGCGCTGCC-3' 5'-AAGCTTTTCCGTCTTTTCGTTTCCCAAGGC-3' 5'-AGATGAATTCGGCGACTTGAAGCCGAGTTC-3' 5'-T AAATAAGCTTGCCGTCACGGGCCTCAGAC-3' 5'-ATTAGAATTCGGTATCGACGGCATGGCCAG-3' 5'-TTAATAAGCTTCGGCGAAGGCGCGCACCTG-3' 5'-ACCGCCATATGGATAAGTCAG-3'

5' -GGGCATATGCTGCCCCTCTTTGAGCC-3' 5' -CGGATTTCCGGATNGA YKSNGGNTC-3' 5' -GAGCAGCCGCGTAGTGAG-3' 5' -AAGTACCGCCACCTAAC-3' 5 '-CTGTATGGAACGGGATG-3 ,

#563-glpD-SG-For 5' -ACGCCCA TTTCAAGCAGCAA-3' #564-glpD-SG-Rev 5' -TGCTTGCACAGGTAGTCCACTT-3' - Restriction sites are underlined

2.7 Genetic Techniques

2.7.1 Biparental Matings

Plasmids containing the oriT were transferred from E. coli strains SMIO-lpir or

ER2566-mob to P. aeruginosa strains by biparental mating (116). This involved growing

both donor and recipient strains to early log-phase with shaking at 37·C. Then, 0.5 ml of

each culture was mixed in the same 1.5 ml microfuge tube and centrifuged at 8,000 rpm

for 1 min at RT to pellet the cells. The supernatant was decanted, leaving -50 )Jl of

residual liquid in the tube which was used to resuspend the cell pellet. The suspension

was then spotted onto a pre-warmed 1 em diameter cellulose acetate membrane filter

(Sartorius, Germany) placed onto an LB plate. Following ON incubation at 3TC, the

34

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celIs were resuspended from the filter by vortexing in 1 ml of LB and 150 jJl of the celI

suspension plated onto PIA + Om medium. The plates were incubated at 37'C until

colonies appeared.

2.7.2 Construction of Transcriptional Fusions in P. aeruginosa and E. coB

2.7.2.1 P. aeruginosa

The PfahA-lacZ transcriptional fusion in P. aeruginosa was constructed in several

steps. The fabAB regulatory region, encompassing nucleotides 23-339 upstream of the

translational initiation codon, was amplified using primers 274 and 277 and PAOl

chromosomal DNA isolated with the IsoQuick Nucleic Acid Extraction Kit (ORCA

Research Inc., BothelI, WA). Cycle conditions for this and subsequent amplifications are

94.5'C for 1 min folIowed by 30 cycles of 94.5'C for 1 min, 58'C for 30 s and 70'C for

30 slkb using Pfu polymerase (Stratagene, La JolIa, CA). The 320-bp fragment was

digested with HindlII and cloned into pTZ120 (114) digested with HindIlI and SmaI to

yield pTZI20-PfahA. This created a transcriptional fusion of the PfahA with the lacZ-gene

on pTZ120. Similarly, the PfllllE"iacZ and PftulBS""lacZ transcriptional fusions were

constructed by amplifying the fadE and the fadB5 promoters; encompassing nucleotides

78-347 and 148-266 upstream of the respective start codon, and PCR were performed

using primers 384 and 385 and primers 287 and 289 from PAOl chromosomal DNA. The

resulting 268-bp and 120-bp fragments were cloned into pTZ120 as described above to

yield pTZ120-PfadE and pTZI20-PfadBS, respectively. For integration into the P.

aeruginosa genome, the PfabA-lacZ, PfadE"/acZ and PftulBS""/acZ fusions were digested with

AjlIII, blunt-ended, and digested with HindlII; these fragments were then sub-cloned

35

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between the HindIJl and SmaJ sites of mini-CTX2 (54) to yield mini-CTX2::PfabA-laC4

mini-CTX2::PfadE"iacZ, and miniCTX2::PfadBs-iacZ, respectively. Chromosomal

integration of these mini-CTX2 lacZ-fusion vectors in P. aeruginosa and excision of

unwanted plasmid sequences were performed essentially as described in (54). Insertions

at the chromosomal attB-locus was verified by PCR using primer 451 along with primer

274,287, or 384 forfabA,fadB5, andfadE respectively.

2.7.2.2 E. coli

Construction of the transcriptional fusion in E. coli utilized the miniCTX2::PfadBS­

lacZ vector created for the construction of the P. aeruginosa transcriptional fusion. The

PfadBS-1acZ fusion was excised from miniCTX2::PfadBs-lacZ using SapI, then blunt-ended

using T4 DNA polymerase. The resulting fragment was then cloned into pCD13PSK,

which was digested with NspI and blunt-ended with T4 DNA polymerase. The resulting

construct, pCD13PSK-PfadBs-iacZ was transformed into strain HPSlIpPICK via heat

shock and blue-white selection performed on LB + Sp + X-Gal at 30° C. After ON

incubation, plates were transferred to 37°C to cure the cells of the pPICK plasmid and

blue colonies selected. Successful integration of the PfadBs-lacZ fusion at the lattB site

was verified by PCR.

2.7.3 Gene Replacement

For gene-replacement, the previously described strategy utilizing the gene

replacement vector pEX18T was employed (53). PotentiaifadR loci were amplified using

oligonucleotides that contain EcoRI and HindIJl restriction sites, which allowed for

36

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bIa sacB • •

PAI627.:6m pEX18T::PA1627::Gm

PA162T FRT FRT 'PA1627

PAOI. :"PfadBAli-IacZ chromosome PAI627

1 st homologous recombination event selected on Gm-containing media

FRT FRT on7 sacB b/a 0';

----~=x() .()C~--==~·~==~·~===·~~====~~ PAI627' 6m B 'PA1627 PAI627

I

2nd homogous recombination event selected FRT FRT on media containing 6% sucrose + Gm

--c::::a:O • ()C::J--PA1627' 6mB 'PA1627

Figure 4. Schematic representation of mutant construction by gene replacement. The gene replacement vector, pEX18T, harboring the inactivated gene of interest (pA1627) is introduced into the PAOl transcriptional fusion strain. The first recombination event leads to the formation of a merodiploid that is selected for on PIA + Om media. The vector backbone is removed via sacB-mediated recombination on PIA + Om + 5% sucrose media leaving the inactivated gene of interest in the chromosome.

37

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subsequent digestion of the PCR product. The resulting EcoRl-HindIII was cloned into

the EcoRl-HindIlI-cleaved gene replacement vector pEX18T. Central regions of the

potentiaifadR loci were then replaced with a GmR-cassette obtained from pPS856. The

resulting constructs were transformed into the mobilizer strain ER2566-mob and

conjugally transferred by biparental mating into PAOl-attB::PJadBS'lacZ. Successful gene

replacement at the PAOI fadR loci was obtained in several steps (Figure 4). First, a

fadR::GmRfadR+ merodiploid strain due to integration of the non-replicative pEX18T

plasmid into the chromosome was obtained after selection on PIA + Gm media. Second,

colonies obtained on PIA + Gm were streaked on PIA + Gm + 5% Suc to force a second

recombination event leading to the removal of the plasmid backbone. Carbenicillin

sensitivity was tested by patching onto PIA + Om and PIA + Cb. Lastly, GmR SucR CbS

patches were then restreaked onto PIA + Gm + 5% Suc media and after the appearance of

isolated colonies, were verified for the insertion of GmR -cassette at the fadR loci by PCR

The following primers were used for verification: primers 582 and 583; primers 536 and

537; and primers 538 and 539 were used for PA1627, PA4769, and PA5356 respectively.

2.8 Transposon Mutagenesis

The lacZ transcriptional fusion strain PAOl-attB::PJadBS'lacZ was subjected to

transposon mutagenesis using the mini transposon vector, pBT20. The transposon in

pBT20 is catalyzed by the Himar-l mariner transposase. The pBT20 vector was

conjugally transferred by biparental mating into PAOl-attB::PJadB;-lacZ essentially

following the protocol described in Kulasekara et aI., 2005 (69). The donor strain (SMIO­

').pir) harboring the pBT20 vector and recipient PAOl-attB::PJadBj-IacZ were scraped

38

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from overnight plates, LB + Ap and PIA respectively, and resuspended in 2 mI of LB.

The concentration of the cell suspension was adjusted to OD600 of 40 for the donor and

OD6OO of 20 for the recipient. Next, 25 j.ll of donor and recipient were mixed and spotted

onto a dry LB agar plate and incubated at 37°C for 4 h. Mating mixtures were scraped

and resuspended in 3 mI of LB. Then, 300 j.LI from this suspension was plated onto PIA +

X-Gal and incubated at 37°C.

Transposon insertion sites were determined by sequencing the flanking region of

the transposon using a semi-random PCR method utilizing nested primers (primer 524 or

139 along with the nested primers i) 463, ii) 525, then iii) 526). Touchdown PCR method

consisted of two phases. Phase I consisted of an initial step of 95°C, followed by 25

cycles of denaturation at 95°C for 45 s, annealing at variable temperatures for 45 s, and

extension at 72°C for 2 min. The annealing temperature was set at 60°C in the first cycle

and, at each of the 24 subsequent cycles; it was decreased by 0.5°C increments per cycle.

Phase 2 consisted of 25 cycles of 95°C for 45 s, 50°C for 45 s, and 72°C for 2 min (73).

Alternatively; the touchup PCR method was used, which consisted of 3 phases. Phase I

consisted 10 cycles of 95°C for 30 s, 42°C for 25 s, and 72°C for 2 min. Phase 2 consisted

of 30 cycles of 95°C for 30 s, 52°C for 25 s, and 72°C for 2 min. Phase 3 consisted of 34

cycles of 95°C for 30 s, 54°C for 30 s, and 72°C for 1.5 min. PCR products were extracted

and sequenced using primer 526 at local facilities.

2.9 Construction of Expression Vectors

Potential fadR loci were amplified from P AOl chromosomal DNA using

oligonucleotides that contain EcoRI and HindIII restriction sites, which allowed for

39

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subsequent digestion of the PCR product. The EcoRl-HindIII digested PCR product was

cloned into the EcoRl-HindIII-cleaved cloning vector pUCI8/19. Alternatively, thefadR

loci were obtained from the various previously constructed gene replacement vector

derivatives, pEXI8T:;fadR. The resulting constructs were then transformed into HPSI­

AattB::pC013PSK-PjadBs-IacZ by heat shock and selected on LB + Ap. Transformants

were verified by digestion using restriction sites unique to the insert and vector.

2.10 Electrophoresis Mobility Shift Assays (EMSA)

2.10.1 Crode Extract Preparation

Cultures of the desired strains were grown overnight at 37°C in 5 mI of LB. A

portion of the culture was used for a 1: I 00 inoculation into fresh LB media and grown

with shaking at 37°C to log phase (00600- I). The cells were harvested by centrifugation

(10 min, 8,000 rpm, 4°C) in a Beckman floor model centrifuge. The supernatant was

decanted and the pellet resuspended either in MCAC-O buffer (20 mM Tris-HCI pH 7.9,

0.5 M NaCl) containing 10 J1lImI lysozyme, 1 mM PMSF, 1 mM EOTA, 0.1 % Triton X-

100 or 20 mM Tris-HCI pH 8, 0.5 mM EOTA, 1 mM OTT, 5% (v/v) glycerol containing

SO mM NaCl, 1 mM PMSF and 10 J1lImI lysozyme for (NRt)2S04 precipitation. The cell

suspension was then subjected to cycles of freeze-thawing at -80°C until the bacterial

cells were lysed. Clarified lysate was obtained by ultracentrifugation (2 h, 47,000 rpm,

4°C) in a Beckman floor model ultracentrifuge. The clarified lysate was then dialyzed

against 1 L of 10 mM Tris-HCI, pH 7.5 three times prior to subsequent studies.

2.10.2 Expression and Purification ORF PA3508 for EMSA Studies

40

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The ORF PA350S was expressed and purified using the T7 RNA polymerase

expression system. Plasmid pET2Sa-PA350S was constructed by amplifying the PA350S

region from pUCIS-PA350S using primers 544 and 559 listed in Table 4. This fragment

was then digested with NdeI and HindIII and subcloned in-frame into the pET2Sa

expression vector digested with the same enzymes, which produced a His6-tag at the N­

terminus. The vector was then transformed into the E. coli expression strain ER2566 by

heat shock described above in Section 2.5.6. The host strain ER2566 (Table 2) contains

the gene for T7 RNA polymerase and is under the control of the IPTG-inducible lac

promoter (119). The transformed ER2566 strain containing pET2Sa-PA350S was grown

in 5 rnl LB + KID overnight at 37°C. The entire overnight culture (5 rnl) was used to

inoculate 500 rnl of pre-warmed (37°C) LB + KID and grown with shaking at RT for S h

(OD600 - 0.1). The culture was then moved and grown with shaking at 37°C until OD6OQ-

0.7. Cells were then transferred back to RT and grown for 1 h prior to inducing with 1

mM IPTG. After growing at RT for an additional S h, cells were harvested by

centrifugation WC, S,OOO rpm, 10 min) and resuspended in MCAC-O buffer containing

10 fJ.gfmllysozyme, 7.5 units of DNAse I, 1 mM PMSF, 1 mM EDTA and 0.1% Triton x-

100. The cell suspension was then subjected to cycles of freeze-thawing at -SO°C until the

bacterial cells were lysed. Clarified lysate was obtained by ultracentrifugation (2 h,

4S,000 rpm, 4°C) in a Beckman floor model ultracentrifuge. HiS6-tagged PA350S was

purified on Ni+-NTA column. Column was washed with 300 rnl of MCAC-40 + 1 mM

PMSF and eluted with 5 rnl of MCAC-200 buffer. Purified protein sample was then

dialyzed against 1 L of 10 mM Tris-HC1, pH 7.5 three times prior to EMSA studies.

41

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Protein concentration was detennined by Bradford assay (8) using BSA as a

standard.

2.10.3 Streptavidin Magnetic Particles Purification

P. aeruginosa PAOl clarified lysate was prepared as described in Section 2.10.1.

In addition, the clarified lysate was concentrated down to 2 ml with an Amicon Ultra

10,000 MW spin column (Millipore Corporation, Bedford, MA). Concatamers of the

fadB5 consensus sequence, amplified with primers 499 and 504 (60 pmole each), was

used to purify the regulator from P. aeruginosa clarified lysate. PCR conditions to

generate concatamers of the fadB5 consensus sequence were 94.5"C for 2 minutes

followed by 34 cycles of 94.5'C for 45 s, 55'C for 40 s and 68'for 45 s + 5 s/cycle using

Pfu (Stratagene, La Jolla, CA). Concatamers of various sizes in the PCR reaction were

purified away from primers using the DNA CleaniConcentrator™-5 kit (Zymo Research

Corporation). The biotinylated PCR product was used to purify the regulator from P.

aeruginosa clarified lysate with Roche streptavidin magnetic particles and a magnetic

particle separator (Roche, Indianapolis, IN) as directed by the manufacturer; Briefly,

biotinylated PCR product (-240 pmole) was incubated with 1 mg of streptavidin

magnetic particles in 250 j.tl of TENIOO binding buffer (10 mM Tris-HCI, 1 mM EDTA,

100 mM NaCl, pH 7.5) for 30 min, then washed twice with equal volumes of TENIOOO

washing buffer (10 mM Tris-HCI, 1 mM EDTA, 1 M NaCl, pH 7.5). DNA-bound

streptavidin magnetic particles were then incubated with 175 j.tl clarified lysate and 50 j.tl

protein-binding buffer composed of 20 mM Hepes, pH 7.6, 1 mM EDT A, 10 mM

(NI4):zS04. 1 mM OTT, 1% Tween 20 (w/v), 150 mM KCl containing 12.5 !Jog of

42

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poly(dI-C) and 0.125 Ilg of poly-L-lysine for 60 minutes at 4°C. Following binding,

particles were washed three times with equal volumes of protein-binding buffer and

eluted in a total volume of 45 J.1l with 2.0 M KCl in protein-binding buffer (20 roM

Hepes, pH 7.6,1 roM EDTA, 10 roM CNH4)2S04.1 roM DTT, 1% Tween 20 (w/v), 150

roM KCI). The partially purified lysate was then dialyzed against 30 roM Tris-HCl, pH

8.0, 0.2 roM EDT A, 200 roM NaCI. Recovered lysate was then used in EMSA studies.

2.10.4 Ammonium Sulfate Precipitation

Crude extract was prepared as described in Section 2.10.1 and subjected to

ammonium sulfate precipitation. After ultracentrifugation, the crude extract was treated

with a saturated solution of CNH4)2S04 to precipitate proteins at 30-60% in 5%

increments and at a final precipitation step at 80%. Precipitate was allowed to form at 4°C

for 1 h under gentle stirring with a magnetic stir bar. Protein precipitated at each step was

recovered by centrifugation (10 min, 12,500 rpm, 4°C) and resuspended in 20 roM Tris­

HCl pH 7.5. Resuspended protein was then dialyzed three times against 1 L of 10 roM

Tris-HCl, pH 7.5 prior to EMSA studies.

2.10.5 Biotin-labelling

DNA fragments used for EMSA were labeled with biotin at the 3' end using the

Biotin 3' End DNA Labelling Kit from Pierce (Rockford, IL). Briefly, the promoter

regions of fadB5 and fabA, and other DNA fragments of interest were amplified from

PAOl chromosomal DNA (PfadB5 and PfabA were amplified as above for lacZ gene fusions

with primers 287 + 289 and 274 + 277 respectively; primers 563 + 564 were used to

43

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amplify glpD), and ran on 2% agarose gel. DNA bands were extracted as described in

Section 2.5.4 and the concentration adjusted to 1 ).1M using the Beckman

Spectrophotometer. On ice, the following components were added: 25,.u ofDDW, 10,.u

of SX TdT (terminal deoxynucleotidyl transferase) Reaction buffer, 5 ,.u unlabeled DNA

(1 ).lM), 5 111 biotin-N4-CTP (5 ).lM), and 5 111 TdT (2 Villi). Reactions were then

incubated at 37"C for 30 min. Following incubation; reactions were terminated by adding

2.5 ,.u 0.2 M EDTA. TdT was extracted from the reaction by adding 50 ,.u

chloroform:isoamyl alcohol (24:1). The mixture was then vortexed briefly and

centrifuged (2 min, 13,200 rpm, Rn in a microcentrifuge to separate the phases. The

aqueous phase was removed and stored at -20°C until use.

2.10.6 Gel Shifts

Concentrated protein preparations were incubated with the PfadB5 and the PfabA

regulatory fragments or glpD (amplified as above for lacZ gene fusions with primers 287

+ 289 and 274 + 277; primers 563 + 564 for g/pD), and gel mobility shift was performed

to demonstrate DNA binding. The amplified fragments were biotin labeled at the 3'-end

using the Pierce biotin 3'-end labeling kit (Rockford, IL) described in Seetion 2.10.5.

The labeled DNA was purified through a Zymo DNA CleaniConcentrator™-5 column.

Binding conditions for DNA and purified proteins were 20 mM Hepes, pH 7.6, I mM

EDTA, 10 mM (N~)2S04. 1 mM DIT, 1% Tween 20 (w/v), 150 mM KCl, 1 )1g of

poly(dl-C) and O.ll1g ofpoly-L-lysine, the reaction was incubated at RT for 30 minutes.

Each binding reactions were loaded on a 8% native polyacrylamide gel and ran for 120

minutes at 100 V. Gels were blotted onto Immobilon™-Ny+ membranes (Millipore,

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Bedford, MA) with a Biorad Mini Trans-Blot Cell (Hercules, CA). Detection of

biotinylated DNA was performed with the New England BioLabs Phototope-Star®

Chemiluminescent Detection Kit. Chemiluminescent detection of membranes was

performed on the Biorad ChemiDoc EQ System.

2.11 p-Galactosidase Assays

2.11.1 Inverse Regulation Studies

IJ-Galactosidase activities were measured for the three integrated gene fusions

(P/abA-1acZ. p/tUJE-1acZ and PfadBs-lacZ) in three different growth conditions. Cells

harboring the fusions were grown ON at 37°C in PIA medium. ON cultures were washed

once with one volume of IX M9 buffer and resuspended in equal volume of the same

buffer. Resuspended cultures were then diluted 200-fold into fresh LB or IX M9 buffer

with 0.4% (w/v) CI6 (palmitate) or 0.4% (w/v) CIS (oleate). Growth-curves were

performed for each media, and growth-phases were determined from these growth-curves

to control for differences in cell-densities. One ml cell cultures were frozen at various

time, and p-galactosidase assays were performed at log-phase from the same cultures

used for growth-curves. P-Galactosidase assays were performed in triplicate and

displayed as Miller Units (80).

2.11.2 Monitoring the Regu1ation ofjadB5 in lacZTranscriptional Fnsion Strains

P-Galactosidase activities were measured for the P. aeruginosa or E. coli

transcriptional fusion strains and mutant derivative strains grown in LB or LB + AP,

respectively. Cells harboring the fusions were grown ON at 37°C in LB medium. ON

45

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cultures were used to inoculate (1 :200 dilution) into fresh LB medium (50 ml) Growth­

curves were performed and growth-phases were determined from these growth-curves to

control for differences in cell-densities. One ml cell cultures were frozen at various times,

and ~-ga1actosidase assays were performed at various growth phases (early-log, mid-log,

late-log and early-stationary phases for P. aeruginosa strains; early-log, mid-log, and

early stationary phases for E. coli strains) from the same cultures used for growth-curves.

~-Galactosidase assays were performed in triplicate and displayed as Miller Units (80).

46

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Chapter Three: Inverse Regulation of Fatty Acid Metabolism in P. aeruginosa?

3.1 Introduction

It bas been shown that fatty acid biosynthesis and degradation are regulated in a

coordinated manner in E. coli by a transcriptional regulator, FadR. In the absence of

exogenous long-chain fatty acids, FadR is bound to all of its cognate operators repressing

the transcription of/ad genes while concurrently activating the transcription of/ab genes

(Figure 3). When an exogenous source of long-chain fatty acid is available, the fatty acid

enters the cell and is converted to its acyl-CoA thioester. The acyl-CoA then binds to

FadR resulting in a conformational change that causes FadR to dissociate from all of its

operators resulting in the induction of/ad genes and the repression of/ab genes (18). This

observation implies that fatty acid biosynthesis processes are downregulated upon the

addition of long-chain fatty acids while fatty acid degradative processes are upregulated.

Similarly, in the absence oflong-chain fatty acids, fatty acid biosynthetic genes should be

upregulated while fatty acid degradative genes are downregulated. Demonstration of such

a regulatory mechanism in P. aeruginosa would suggest that a FadR homologue might

exist in P. aeruginosa. Analysis of previously collected microarray data (Tables 1 and 2)

reveal that the fatty acid degradative (feu!) genes in P. aeruginosa are upregulated and the

fatty acid biosynthetic (fab) genes are downregulated when growth on oleic acid (CIS) as

the sole carbon source is compared to growth in Luria-Bertani (LB). Conversely, the

inverse relationship is observed when growth on LB is compared to growth on oleic acid.

Verification of this relationship using transcriptional gene fusions in P. aeruginosa will

further support the hypothesis that fatty acid metabolism is coordinately regulated and

validate the search for a FadR-like protein in P. aeruginosa

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TABLE 5. P. aeruginosafab-genes expressed two-fold or greater when grown to mid-log phase in LB versus palmitic acid (CI6:O) as identified using microarrays

Accession Number PA0730 PA1609* PA1610'" PA2552 PA2553 PA2964 PA2965* PA2967* PA2968*

PA2969

PA3643 PA3644 PA3645*

PA3639'"

Gene Fold 1. •

N Ch 1 P-Value Descnption

ame ange phaG 5.2 (7.5) 3.85E-05 hydroxyacyl-ACP:CoA transacylase PhaG fabB 2.1(2.6) 3.68E-02 p-ketoacyl-AGP synthase I fahA (2.9) NO P-hydroxydecanoyl ACP dehydrase fadB3 2.2(2.7) 1.66E-02 probable acyl-CoA dehydrogenase fadA3 2.8 3.63E-02 probable acyl-CoA thiolase pabC 3.5 1.8E-02 <4-amino-4-deoxychorismate lyase fabFl 2.5 6.6E-03 P-ketoacyl-[acyl-carrier-protein] synthase II fabG 7.3 (4.9) 1.35E-3 3-oxoacyl-[acyl-carrier-protein reductase fabD 5.7(3.7) 1.06E-2 malonyl-CoA-[acyl-carrier-protein]

transacylase plsX 6.7(2.1) 2.98E-2 fatty acid/phospholipid biosynthesis protein

PlsX lpxB 3.1 lpxA 2.7 fabZ 2.5

8.2E-4 5.1E-4 1.3E-2

lipid A-disaccharide synthase UDP-N-acetylglucosamine acyltransferase (3R)-hydroxymyristoyl-[ acyl-carrier-protein] dehydrase

accA 2.6 (2.0) 9.09E-3 acetyl-coenzyme A carboxylase carboxyl transferase (alpha subunit)

PA4847'" accB 5.4(4.3) 1.96E-3 biotin carboxyl carrier protein (BCCP) PA4848'" accC 5.6(4.1) 5.00E-05 biotin carboxylase 1 Fold change values were averaged over a minimum of three different GeneChips" for each condition (C,,~ and LB); in parentheses are average fold change for two pair-wise comparisons of oleate (CIS:I",) versus LB, and hence P­values were not determined (NO) 2 P-values (~ O.OS) were generated from a minimum of six out of nine possible pair-wise comparisons for LB versus elM

• Genes involve in fatty acid biosynthesis (Fab)

TABLE 6. P. aeruginosafad-genes expressed two-fold or greater when grown to mid-log phase in palmitic acid (CI6:0) versus LB as identified using microarrays

Accession Gene Fold P-Value1 Description

Number Name Change1

PA0506oI< fadE 7.3(4.6) 2.81E-05 probable acyl-CoA dehydrogenase PA0508oI< fadE 17.2(15.2) 4.8E-04 probable acyl-CoA dehydroger!ase PA1736'" fadAl 3.8 (3.3) 1.09E-03 probable acyl-CoA thiolase

PA1737* fadBl (2.1) NO probable 3-hydroxyacyl-CoA dehydrogenase

PA1748* fadB (2.5) NO probable enoyl-CoA hydratase/isomerase

PA2550* fadE (5.4) NO probable acyl-CoA dehydrogerla8e

48

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Accession Number PA28IS*

PA2893*

PA30 13 *

PA3014*

PA3333**

PA3334*'" PA34S4*

PA3924*

PA392S* PA443S*

PA5188'"

Gene Name fadE

{adD3

{adE5

{adA5

{abH2

{adA

{adD4

{adA fadE

(adE

Fold Change! 3.2 (2.2)

(2.1)

S.7

6.4

11.S

31.4 4.0 (S.4)

(2.1)

P-Valuez Description

l.lE-03

ND

probable acyl-CoA dehydrogenase Probable very-long-chain acyl-CoA synthase

8.14E-OS fatty-acid oxidation complex beta­subunit

3.13E-OS fatty-acid oxidation complex alpha-subunit

1.8IE-02 3-oxoacyl-acyl-carrier-protein condensing enzyme

1.7E-02 1.17E-02

ND

probable acyl carrier protein probable acyl-CoA thiolase probable medium-chain acyl-CoA ligase

2.8(3.1) I.SE-04 probable acyl-CoA thiolase 10.7 (12.8) S.98E-03 probable acyl-CoA dehydrogenase

9.0 9.59E-03 probable 3-hydroxyacyl-CoA dehydrogenase

1 Fold change values were averaged over a minimum of three differeut GeneChips® for each condition(C16~ and LB); in parentheses are average fold change for two pair-wise comparisons of oleate (ClS:l",) versus LB, and hence P­values were not determined (ND) 2 P-values (S 0.05) were generated from a minimum of six out of nine possible pair-wise comparisons for C 16,o versus LB • Genes involved in fatty acid degradation (Fed) " Genes involved in fatty acid biosynthesis (Fab)

3.2 Results

3.2.1 Construction of P fadBS-1acZ, PfadE"lacZ and P fabA-facZ Transcriptional

Fusions in P. aeruginosa

Three fatty acid metabolism gene/operons were used to investigate the inverse

regulation of fatty acid degradative and biosynthetic processes in P. aeruginosa. The

ORF PA281S encodes a probable acyl-CoA dehydrogenase and was included in this

study because of its significant homology to the E. coli fadE (67% similarity and SI %

identity). ThefadEA5 operon (pA3013/14) encodes the large (l subunit and the small P

subunit respectively, of the fatty acid oxidation multi-enzyme complex that is responsible

49

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for catalyzing five key f3-0xidation reactions (5, 86, 91, 99). It has been previously shown

that a mutation in the fadBA5 operon results in a significant growth defect when grown

on minima! media plus fatty acid (oleic acid and palmitic acids) compared to wild-type.

Therefore, its relationship to fatty acid metabolism has been demonstrated. The fabAB

operon (PAl 6 1 0/09) is part of the fatty acid biosynthetic pathway and encodes ~­

hydroxydecanoyl-ACP dehydrase and ~-ketoacyl-ACP synthase I, respectively. The

importance of the fabAB operon to fatty acid biosynthesis has been established in P.

aeruginosa and therefore was selected to be in this study. From previous promoter

mapping data acquired from our laboratory using primer extension, the transcript start

sites of the above gene/operons are known and the region encompassing the promoter

region was fused to lacZ reporter gene to create the transcriptional fusions used in this

study. The transcript start site for fadB5 was substantiated using a promoter prediction

program (http://mendel.cs.rhul.ac.uklmendel.php), which predicted the same transcript

start site as determined by primer extension with a reasonable score value (37). Briefly,

the various promoter regions were amplified by PCR and cloned into pTZ120, which

harbors the lacZ gene, to create the lacZ transcriptional fusion. The lacZ fusions were

then subcloned into the mini-CTX2 plasmid, which was then used to integrate the fusion

as a single copy in the chromosome of P. aeruginosa PAOI at the defined neutral CTX

phage attB site (Figure 5). To verify the successful integration of the lacZ transcriptional

fusions at the allB region, chromosomal DNA of the various integrants following Flp­

mediated excision of plasmid sequences, were PCR-amplified using primers up- and

down-stream of the attB site. Although, these primers amplify a -270-bp fragment from

wild-type PAO 1, the size of the amplified PCR product from the integrants was - 4.5-kbp,

SO

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_n c::

tRNA Ser

• altO -u .n~

FRT PfadBAfi-laaZ;;P /iiT

onT

int uti tet • •

PAOl Chromosome

mini-ClX2::PfadBA5-lacZ

! step 1 . int-mediated recombination

altO''P /8t . int 'T n r- n attP"B -00 ---+- on ---+- on 0 ~ • 0-

FRT FRT PradBAfi-laaZ I I

! step 2. Rp recombinase-mediated excision of unwanted plasmid sequences

altO''P attP"B -oon c:: • n 0-

FRT PfadBAfi-laaZ

Figure 5. Mini-CTX2-mediated integration of PjadBAS-1acZ at the attB of P. aeruginosa. The steps leading to the isolation of unmarked integrants are illustrated. Step 1 depicts the integrase-mediated integration of the mini-CTX2::PjadIW-1acZ plasmid at the attB site following transfer into a P. aeruginosa recipient strain. Step 2 shows the Flp-mediated excision of plasmid sequences leading to the removal of genes and associated promoter sequences that might interfere with expression of gene fusion. This figure was adapted from Figure 2 of reference (54).

51

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confirming successful integration of the lacZ fusion. Additionally, integration of the

various fusions were also confinned using another set of primers, one primer that anneals

to the lacZ, region, primer 451, together with a primer that anneals to the promoter region

of the fadB5, fadE and fabA, primers 274, 287, and 384 respectively (Figure 6). The

results from these PCR -amplifications confinn the successful integration of the lacZ­

fusions (PAOl-attB::P/adBs-1acZ, PAOl-attB::P/adE"'lacZ, and PAOl-attB::P/abA-IacZ) and

the various constructs were then used for subsequent studies.

3.2.2 Analysis offad andfab Regulation Using Transcriptional Fusions

To address the question of whether fatty acid degradation and biosynthesis are

inversely regulated in P. aeruginosa, the PAOl integrated transcriptional fusion strains

were grown in 3 conditions (LB, 0.4% (w/v) CI6 and (palmitate), and 0.4% (w/v) CIS

(oleate)) and p-galactosidase activity measured as described in Section 2.11.1. Since LB

media is relatively poor in fatty acid content compared to minimal media supplemented

with C16 0rC18. p-galactosidase activity ofthefahA promoter is expected to be high inLB

whereas fadE and fadB5 should be relatively low. On the other hand, when grown in the

presence of CI6 or CIS, the f3-galactosidase activity of the fadE and fadB5 promoters

should be high relative to thefahA promoter activity. The results from the experiment are

shown in Figure 7. In media that contains fatty acids, both fadB5 and fadE were

upregulated relative to the fabA operon. This relationship was observed regardless of

whether CI6 0rCIsfatty acids were used. In contrast, thefahAB operon is upregulated in

the absence of fatty acids while the fadBA5 operon and fadE gene are both

downregulated. The relationship between these fatty acid biosynthetic and degradative

52

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4

3

2

1.5

1

0.5 -

1 2 3 4 5 6 7 8 9 10 11 12 13

1000

800

500

400

300

Figure 6. Verifying Pjad85-laeZ, PjabAB-laeZ, and PjadE-laeZ fusion integration in the P. aeruginosa PAO] chromosome by PCR. The laeZ fusions were integrated using the mini-CTX2 system and plasmid sequences removed by Flp-mediated site-specific recombination. Primers annealing to the respective promoter regions (primers 287, 274, and 384 for PladB5, PjabAB, and PjadE respectively) and the laeZ gene (primer 45 1) were used in PCR to verify the presence of the fusion in the chromosome. Three independent isolates were confirmed. Lanes I and 13, 100 bp ladders; lanes 2-4, Pladr1aeZ PCR product (- 360 bp); lanes 5 and 9, I kb DNA ladders; lanes 6-8, PjabAn-lacZ PCR product (- 425 bp); lanes 10-1 2, PjadB5-laeZ PCR product (- 220 bp).

53

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(A) PJabA-lacZ (B) 600 PJadB5-lacZ

...... ~ ~ ~

.-1:1 500 ~ .. .. .. = ::!

6 .-6 300

f £ .j!; 200 .. t < < - - 100 " " C C

ai 0 cO.. 0 LB C 16:0 C 18:1&9 LB C16:0 C18:1&9

(e) PfadE-lacZ

~ .-1:1 ~ .. .. = .-6 f .. < -" C ai

LB C 16:0 C 18:1&9

Figure 7. Inverse regulation ofJabA,JadB5,JadE promoters in LB, palmitic acid (C16:0),

and oleic acid (C18:1&9). J3-Galactosidase activities (n=3) of P. aeruginosa PAOl carrying (A) PfabA-lacZ, (B) PfadBS-1acZ, and (C) PfadE"lacZ were measured at log phase.

S4

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operon/genes is consistent with the microarray data previously collected in our laboratory

(Tables 5 and 6).

55

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Chapter Four: Bioinformatic Approach

4.1 Introduction

FadR regulation seems likely for bacteria closely related to E. coli, certainly FadR

homologues have been discovered in Salmonella, Shigella and Erwinia (18) among

others but similarities in more divergent bacteria have also been found. Haemophilus

injluenzae encodes a FadR-like protein that is 47% identical to the E. coli FadR and

amino acids known to play important roles in the E. coli protein (103, 104) are conserved

in the H. injluenzae protein. Additionally, incomplete open reading frames (DRFs) have

been reported for Vibrio choierae and v: alginoiyticus, which closely match the E. coli

FadR sequence. Therefore, FadR regulation may be broadly distributed in Gram-negative

bacteria. A BLAST search for FadR homologues in the P. aeruginosa PADI genome

revealed several proteins with some similarities (pAI627, PA4769 and PA5356) albeit

with slightly low sequence identity (-25 %) to the E. coli FadR. Sequence analysis of

these potentialfadRs reveals they contain an N-termina1lITH-containing region of GntR­

like bacterial transcription factors. GntR-like proteins can be divided into six different

sub-families, one of which is the FadR subfamily (106). Although there are only modest

similarities between these genes and the E. coli FadR, it may prove worthwhile to

investigate the regulatory roles of these proteins in P. aeruginosa.

Since fadB5 has been previously established to be important in fatty acid

degradation and the respective Pseudomonas transcriptional fusion has been

demonstrated to be functional (See Section 3.2.2), the PjodBs-lacZ fusion was specifically

selected to be used in this and subsequent studies. The objective of this study was to

construct isogenic mutations at the various loci (pAI627, PA4769, and PA5356) in a

56

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P fadB5-1acZ transcriptional fusion background and characterize the mutants by ~­

galactosidase assays. In addition, to investigating these potential FadRs in a P.

aeruginosa background, any interaction between the gene product of these loci and the

promoter region of fadE5 will be isolated by performing experiments in an E. coli

background. Similar as with P. aeruginosa, E. coli PJadBS-1acZ transcriptional fusion will

be constructed and the effect of the expression of the various potential FadRs will be

investigated via ~-ga1actosidase assays. Lastly, the lysate of the various E. coli

transcriptional fusions will be extracted and tested for its ability to interact directly with

thefadB5 promoter region by electrophoresis mobility shift assays (EMSA). Collectively,

the results from these experiments are expected to yield insights into the relationship

between these various loci identified by bioinformatics and the regulation offadB5.

4.2 Results

4.2.1 Construction ofPA01-attB::PJadBs-lIlcZl4fadR::Gm (pAI627, PA4769 and

P A53S6) by Gene Replacement

To assess the regulatory potential of the gene products ofPA1627, PA4769, and

PA5356 mutations were introduced at these loci in the PAOI-attB::PfadB5-1acZ fusion

strain using the gene replacement strategy illustrated in Figure 4. Briefly, the 'fadR'

regions amplified from PAOl chromosomal DNA and were cloned into the gene

replacement vectorpEXl8T. A region of the gene was then replaced with a GmR-cassette

derived from the vector pPS856. The plasmid-borne fadR::Gm deletions were then

transferred into the PAOI-attB::PJadBs-1acZ fusion strain by biparental mating as

described in Section 2.7.3. Insertion of the GmR -cassette into the respective 'fadR' genes

57

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in the PAC 1 chromosome was verified by peR using primers up- and down-stream of the

respective genes (Figure 8). Amplification ofPA1627, PA4769 and PA5356 from wild­

type PAOl gave peR products of sizes -708 bp, - 1433 bp, and - 1271 bp respectively,

while the corresponding mutants gave peR products of sizes - 1955 bp, - 2433 bp, and -

2271 bp. The shift in the peR product that observed after gene replacement is due to the

insertion of the -1 kb GmR -cassette.

4.2.2 Characterizing PA01-attB::PfadBs-iacZl4{adR::Gm (pAI627, PA4769

and P A5356) by Jl-galactosidase assays

To investigate the effect of mutations in PA1627, PA4769, and PA5356, the

mutated fusion strains were grown in media in which the PfadB5 promoter has been shown

to be repressed (see Section 3.2.2). The rationale is that if one of these genes encodes a

repressor of the fadBA5 operon, an insertion mutation would prevent the gene product

from being expressed leading to an apparent de-repression (Figure 9). This de-repression

can be directly observed through 13-galactosidase assays. Briefly, the Pseudomonas

transcriptional fusion strain PAOl-attB::PfadB5-1acZ along with the mutated derivatives

(APAI627::Gm, APA4769::Gm, and APA5356::Gm) were grown in LB, media in which

the PfadB5 promoter was shown to be repressed and 13-galactosidase activities measured at

various growth phases and compared. The results from these experiments are shown in

Figures 10 and 11. Figure 10 shows the growth curve for the various Pseudomonas

transcriptional fusion strains together with the mutated derivatives and the time points at

which culture was collected for 13-galactosidase assays. 13-Galactosidase activities were

58

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kb

to 8

6 5 4

1

'2

1', -

1 -

n .~ -

1 2 3 4 5 6 7

Figure 8. Verifying insertion of GmR-cassette fo llowing gene replacement at PA1627, PA4769, and PA5356 in P. aeruginosa transcriptional fus ion strain PAOl-attB ::PjadB5-laeZ. Primers up- and down-stream of the repective ORFs were used to verify the insertion of the GmR-cassette. Lane I, I kb DNA ladder, lane 2, PA1627 amplified from PAOI -atrB: :PjadB5-lacZ; lane 3, PAI627 amplified from mutant; lane 4, PA4769 amplified from PAOI -aIlB::PjadB5-lacZ; lane 5, PA4769 ampli fied from mutant; lane 6, PA5356 amplified from PA01-atrB::PjadB5-lacZ; lane 7, PA5356 amplified from mutant.

59

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Under non-inducing conditions - absence 01' exogenous !'ally acids (ac;+coA)

WTcel/

RNApolymerllSe r 0 = FadR

~ CD: !'adR PlHdBA5-lacZ

Gene Rqplacement mutant

x I'adR.:6m PlHdBA5-1acZ

Figure 9. Principle of the gene replacement mutagenesis screen strategy used to find the FadR-like protein in P. aeruginosa. Upper panel depicts wild-type PAOl-attB::PjadBs­lacZ cell in the absence exogenous fatty acid. FadR is expressed from the JadR loci and binds to the promoter region of the JadBA5-lacZ fusion repressing transcription of lacZ. The absence of p-galactosidase results in low p-galactosidase activity. Bottom panel shows the disruption of the JadR loci by insertion of GmR-cassette under the same conditions. Inactivation of JadR relieves repression of JadBA5-lacZ, allowing for the expression of p-galactosidase, which leads to high (3-galactosidase activity.

60

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6,-----------------------------------,

s

4

--c-- PA01-attB::PfadB5-IacZ

=0 3 <> 1627::Gm

~ §

····0··· 4769::Gm

>1 5356::Gm 2

O~-----,r_----_r------r-----~------, o w w ~ ~ ~

Time (h)

Figure 10. Growth curves of P. aeruginosa transcriptional fusion strains PAOl-attB:: PfadB5-1acZ and mutant derivatives grown in LB media. Mutants were constructed by insertion of a GmR-cassette by gene replacement at PA1627, PA4769 and PA5356. Indicated are the time points at which ~-galactosidase activities were measured, EL, early-log; ML 1, mid-log 1; ML2, mid-log 2; LL, late-log, and ES, early-stationary.

61

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I

• • • • • •

I~ E E ':I E E E J E E E ':I E E E i E E E III C!! .!! C!! C!! C!! C!! C!! C!!

~ III III III III III III

~§ ~ I l ~ .. iii t.:i .. iii ;.:. ~ 3i t\i ii i N ... I ... N ... ~ Ie I;: ... I;: iii ... I;: iii ... ... I;: iii ...

~ ~

~ ~ ~ ...

~

~ ~ i ~ ~ ~ ~ ~ ~

~ c c c ~ : : :

Early log Mid log1 Mid log 2 Late log Early Stationary

Phase/Strain

Figure 11. Transcriptional regulation of p/adB5-lacZ fusion in the P. aeruginosa transcriptional fusion strain PAOI-attB::P/adBs-1acZ and insertion mutant derivatives (APAI627::Gm, APA4769::Gm and APA5356::Gm) grown in LB. ~-galactosidase activities were measured at five time points, EL, early-log; MLl, mid-log I; ML2, mid­log 2; LL, late-log, and ES, early-stationary. The values represent the mean :!: the standard deviation (n = 3).

62

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measured at early-log, two points during mid-log, late-log and early-stationary. It is

apparent from the growth curve that mutations at the various loci do not affect growth in

LB media Growth of all the mutants is quite comparable to that of the wild-type fusion

strain, both in terms of growth rate and fina1 cell density. The results also show that there

are no significant differences in the level of f3-galactosidase activities across all growth

phases (Figure 11). The activity the fadB5 promoter of the wild-type strain was low as

would be expected in media lacking fatty acids, and remained low through out growth.

Inactivation of the various fadR candidates did not relieve repression of the fadB5

promoter and similar to wild-type, f3-galactosidase activity also remained low over the

various growth phases. The results from these experiments revealed that mutations at

these loci did not significantly affect f3-galactosidase activities compared to the wild-type

fusion strain.

4.2.3 Construction of p/adBS-IacZ transcriptional fusion in E. coli

(HPSl-lattB::pCD13PSK-P/adBs-lacZ)

The objective of this study is to isolate any potential interaction between the

various FadR candidates and the fadB5 promoter by performing experiments in a

different host system. By conducting studies in E. coli, not only does this allow the

isolation of Pseudomonas DNA-binding protein and its cognate target but provides

potential insights into whether other transcription factors are required for DNA binding.

Briefly, PfadBs-lacZ fusion was obtained from the previously constructed vector

miniCTX2::PfadB5-lacZ and cloned into the E. coli integration vector pCD13PSK to create

pCD13PSK-PfadBs-lacZ as described in Section 2.7.2.2. The integration vector was then

63

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introduced into the E. coli strain lIPSI by triparental mating, pCDl3PSK-P/adBS"

lacZJER2S66-mob, pPICKlER2S66-mob and the recipient lIPS 1 strain (100). Following

curing of the integration helper plasmid, successful integration of the P/adBS"lacZ fusion

was confirmed by PCR-amplification. Primers annealing to the fadB5 promoter region

(primer 287) and lacZ gene (primer 451) were used to amplify a -220 bp product from E.

coli chromosome to confirm the presence of the reporter fusion (Figure 12). Since the

expression of p-galactosidase should be constitutive since the fadB5 is not repressed.

Successful integration was also selected by the production of blue pigment on media

containing the chromogenic substrate for p-galactosidase, X-Gal, 5-bromo-4-chloro-3-

indoyl-P-D-galactoside. Together, these results verified the successful integration of the

fusion in the E. coli chromosome and the E. coli transcriptional fusion strain were then

used for subsequent studies.

4.2.4 Assessing the regulatory potential ofPA1627, PA4769, and PAS3S6 by

expressing the respeetive gene products in the E. coO PfadBS"lacZ

transcriptional fusion

The purpose of this study is to determine whether the gene product of PAl 627,

PA4769 or PA5356 is capable of interacting with thefadB5 promoter by expressing these

genes individually in the E. coli transcriptional fusion strain lIPSI-AattB::pCDl3PSK­

PfadBS"lacZ. The rationale is that if the gene product ofPA1627, PA4769 or PA5356 binds

to the fadB5 promoter then the normally constitutive expression of J3-galactosidase in the

E. coli system should be repressed. Briefly, PA1627, PA4769 and PA5356 were

amplified from PAOl chromosomal DNA using primers with incorporated EcoRI and

64

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900 -ROO -700 --600

500 -400 -300 -200 -

100 -

1 2 3

Figure 12. Verifying PjadB5-lacZ fusion integration in the E. coli HPS I chromosome by PCR. The lacZ fusions were integrated at the )..atlB using the integration vector pCD 13PSK-PjadB5-lacZ. Primers annealing to the promoter region of PjadB5 and the lacZ gene were used in PCR to veri fY the presence of the fusion in the chromosome. Two independent isolates were confinned. Lanes I, PjadB5-lacZ PCR amplified from HPS 1-wuB: :pCD 13PSK-PjadB5-lacZ (isolate I) ; lane 2, I kb DNA ladder; lane 3, PjadB5-1acZ PCR amplified from HPSI-)..attB::pCDI3PSK-PjadB5-lacZ (isolate 2).

65

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HindIII restriction sites. The amplified products were then digested with EcoRI and

HindIII and directionally cloned into the cloning vector pUC 18/19 digested with the same

enzymes. Each of the amplification products included upstream regions of the respective

genes, thereby including promoter elements that drive the expression of the genes. In

addition, the inducible lac promoter supplied by the cloning vector also contributes to the

expression. The pUC18/l9 vectors and their derivatives (pUC18-PA1627, pUC19-

PA4769, and pUC18-PA5356) were than introduced into the E. coli transcriptional fusion

strain HPSl-1attB::pCD13PSK-PjadBs-lacZ by biparental mating (Section 2.7.1) The

reporter strains were then grown in LB + Ap to maintain the plasmid and induced with 1

mM IPTG after 1 h of growth. The growth curve of the E. coli fusion strains are shown in

Figure 13 and 14. All of the strains grew similar both in terms of growth rate, as well as

final cell density, regardless of the construct introduced. P-Galactosidase activities were

measured at three different time points during growth, at mid-log, early-stationary, and

late-stationary phases shown in Figure 13 for PA1627 and PA5356 and Figure 14 for

PA4769. During mid-log phase, the p-galactosidase activity of the control strain

containing the pUC18/l9 plasmid is fairly high as expected and is comparable to the

derivatives containing PA1627, PA5356, and PA4769 (Figures 15 for PA1627 and

PA5356 and Figure 16 for PA4769). At later stages of growth, during early and late

stationary phases, the p..galactosidase activities of all the strains increase but more slowly

in the derivatives strains expressing the various FadR candidates. Both PA1627 and

P A5356 cause the most significant decrease in p-galactosidase activities, with activities

approximately 70-80% of the control strain, the greatest difference being observed during

late stationary phase. PA4769 on the other hand did not significantly reduce the

66

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3

ML

2 ~

0i-----~r------r------,_----~ o 10 20 30 40

Time (h)

---0--

····0····

pUC18

pUC18-PA1827

pUC18-PA5356

Figure 13. Growth curves of E. coli transcriptional fusion strains HPS 1-AattB::pCD13PSK-P.fadlIS"lacZ harboring pUCI8 or derivatives encoding PA1627 or PA5356, grown in LB + Ap media Cultures were induced with 1 mM IPTG at time = 1 h. Activities were measured at three time points during the course of growth, ML, mid-log; ES, early-stationary, and LS, late-stationary.

67

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3

2 ---0-- pUC19

~ pUC19-PA4769

O~=--r--~----~--~---r--~ o 5 10 15 20 25 30

Time(h)

Figure 14. Growth curves of E. coli transcriptional fusion strains HPSl­AattB::pCD13PSK-P/adBs-IacZ harboring pUCl9 or derivative encoding PA4769, grown in LB + Ap media. Cultures were induced with 1 mM IPTG at time = 1 h. Activities were measured at three time points during the course of growth, ML, mid-log; ES, early­stationary, and LS, late-stationary.

68

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3000

T

2500 r:ID i T :::::::: " w ~ .....•.• .. 2000 - :::::::: T II """" ..

:i:i:i:i

:::::::

~ 11 :i:i:i:i

.•..... T

m :~:;:;: $

f 1500 - :::::::: ~I~~

I!!!!!!! it: ~~~~~~~~ :.:.:.:.

W: % :;:~:~:; .....•.• :::::::: ........ . :.:.:.: . ....... I~~~~ :!:!:!:!

@jlll Col

I~~~~ .......• ::::::: mr1 :~:~:~:~

........ -< :.:.:.:. :1:j:~:I - 1000

tm~ ~~I~ :::::::: :~:l:~:~

!!I!I!I " :.:.:.:. ;~mm :::::::: ........

~~mm ~~I~l :;:;:;:;

~

!IIIIII: l~~l~l~l

:.:.:.:.

:i:!:!:! a:!I.

!I!lil!

.:.:.:.:

:!:!:!:! 500 - ~~~~llll ~l~l~l~l ;~mm ~:~:m

:!:!:!:! :::::::: :~:~:l:~ ........

iiiii!i ~~I~~l~~ ........ :::::::

~~I~~ :;:;:;:; :::::::: :::::::: ::::::::

~~~l~~~ 1t :::::::: ••..•.•. :::::::: ::::;::: :m:l:l :.:.:.:.

0 :::;:;:: ::::;::: . • • . . • . .. ... a .. ... ... .. Iii a .. III .. III ID .. t.l t.l :: t.l :;) .. :;) .. :;) .. ...

~ ~ ... ~ f ... ~ ! .. cI. .. .. .. .. .. .. t.l t.l t.l t.l t.l t.l :;) i :;) :;) i i ... ... Go

Mid-log Early Late Stationary Stationary

Phase/Strain

Figure 15. Transcriptional regulation of PjadB5"lacZ fusion in the E. coli transcriptional fusion strain HPSI-l..attB::pCD13PSK-PjadBs-IacZ in the presence of pUCl8 encoding either PA1627, PA5356 or the empty vector, grown in LB + Ap media. Cultures were induced with I mM IPTG I h following inoculation. Activities were measured at three time points during the course of growth, mid-log, early-stationary, and late-stationary. The values represent the mean ± the standard deviation (n = 3).

69

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'";j' ... . - 1500-

~ .. ~ 1000-..., ~ :~ ... .. -< 500--COl ~ A

... ... ... ... ... III ... CD ... CD ... U ~ U ~ CJ ~ ::l ::l ::l ... f ... f ... f • • • ... ... ... ... ... ...

CJ U U ::l ::l ::l ... ... ...

Mid-log 8uIy Late SIaIIonmy S\alionmy

PbastlStrain

Figure 16. Transcriptional regulation of PjadBs-[acZ fusion in the E. coli transcriptional fusion strain HPSI-AattB::pCD13PSK-PjadBs-[acZ in the presence of pUCl9 encoding PA4769 or the empty vector, grown in LB + Ap media. Cultures were induced with 1 mM lPTG 1 h following inoculation. Activities were measured at three time points during the course of growth, mid-log, early-stationary, and late-stationary. The values represent the mean ± the standard deviation (n = 3).

70

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constitutive expression of the JadB5 promoter, remaining fairly similar to the control

strain throughout all phases of growth.

4.2.5 DNA Binding Studies

To establish more definitive evidence of interaction between the various FadR

candidates and the promoter region of JadB5, electrophoresis mobility shift assays

(EMSA) were performed. Crude cell extract from the E. coli transcriptional fusion strains

(HPSI-J..attB::pCD13PSK-P/adBS"lacZ) expressing the various FadR candidates from the

cloning vector pUCl8/19 (Section 4.2.4) were used in the gel shift experiments. Briefly,

the E. coli transcriptional fusion strains were grown in LB + Ap and induced with 1 mM

IPTG after reaching log phase (OD 600 - 1). The culture was then allowed to grow for

another 4 h to reach stationary phase at which point the cells were harvested for lysate

extraction. Prior to use in gel shift experiments, the lysate was dialyzed against 10 mM

Tris-HCI buffer and concentrated 100 fold. A 120 bp fragment encompassing the

promoter region ofJadB5 was PCR-amplified then labeled with biotin at the 3' end and

used as a probe in the gel shift experiments. The dialyzed and concentrated clarified

lysate were incubated with labeled P fadB5 and subjected to electrophoresis in a native 8%

acrylamide gel. The results from the gel shift experiments using lysate from pUCI8-

PA1627, pUC19-PA4769, and pUC18-5356 harboring cells are shown in Figures 17, 18

and 19 respectively. In Figure 17, lysate from pUC18 and pUC18-PA1627 harboring

cells were incubated with the labeled promoter region of JadB5. The banding pattern

observed when pUC18-PA1627 lysate was incubated with P/adB5 is comparable to when

the control pUC18 vector lysate is used and the P /adB5 fragment does not appear to shift.

71

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DNA + -Extract - +

1 2

+ +

3

-+

4

+ +

5

Figure 17. Gel shift assay experiment performed with 120 bp PladB5 labeled with biotin at the 3' -end (see Section 2.10.5). Crude extract was obiained from E. coli transcriptional fusion (HPS I-AauB::pCDI3PSK-PladB5-lacZ) culnll'es harboring pUCI8 or pUCI8-PAI627 during stationary phase and incubated with labeled probes. Lane I, biotin­labeled P/adB5 alone; lane 2, pUC 18 extract alone; lane 3, biotin-labeled P/odB5 incubated with pUCl8 extract; lane 4, pUC18-PA1627 extract alone; lane 5, biotin-labeled P/adB5 incubated with pUC18-PA1627 extract.

72

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DNA + - + - +

Extract - + + + +

1 2 3 4 5

Figure 18. Gel shift assay experiment performed with 120 bp Pjad8s Iabeled with biotin at the 3' -end (see Section 2.10.5). Crude extracts were obtained from E. coli transcriptional fusion (HPSI-},attB::pCDI3PSK-PjadBS-lacZ) cultures harboring pUCl9 or pUCI9-PA4769 during stationary phase and incubated with labeled probes. Lane I, biotin­labeled PjadBS alone; lane 2, pUCI9 extract alone; lane 3, biotin-labeled PjadB5 incubated with pUCl9 extract; lane 4, pUC19-PA4769 extract alone; lane 5, biotin-labeled PjadBS incubated with pUCI9-PA4769.

73

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Figure 18 shows the results for the pUC19-PA4769 experiments. When lysate extracted

from pUC19-PA4769 harboring cells is incubated with promoter region ofJadB5, the

band corresponding to JadB5 is absent and appears higher up in the gel. Although this

would suggest that there is some interaction between the gene product of PA4769 and P

jadB5, the control experiment shows a similar banding pattern. Both pUCI9 and pUCI9-

PA4769 lysates were able to shift P jadB5 and the shifted band appeared at the same

position on the gel for both lysates. When lysate from cells harboring the pUC18-PA5356

vector was used a similar disappearance in the PjadB5 band was observed and the PjadB5

band does appear to shift Figure 19. Although a similar disappearance in the PjadBS band

was observed with the pUCI8 vector control lysate, the shifted band using the pUCI8-

PA5356 lysate was more discrete suggesting specific interactions.

To further investigate the potential interaction between the gene product of

PA5356 and PjadB5further, gel shift experiments were repeated using both the promoter

region of a fatty acid biosynthesis operon PjabA and an internal region of the glpD gene.

FadR in E. coli has been demonstrated to be able to bind not only to the promoter regions

of fatty acid degradative genes but also to the JabAB operon (10). Therefore to test

whether the gene product of PA5356 has a similar ability, PjadA was included in this

experiment. As a control, a random internal DNA sequence within the glycerol

metabolism gene, glpD was selected. The interaction between a transcriptional regulator

and its binding site is fairly specific and a change in a single base pair can lead to

disruption of this interaction. Therefore although there are regions within glpD that are

homologous to that of PjadB5, the difference should be great enough to prevent any non­

specific interaction while maintaining a high degree of stringency. The results from the

74

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DNA + - + - + Extract - + + + +

1 2 3 4 5

Figure 19. Gel shift assay experiment performed with 120 bp PjadB5 1abeled with biotin at the 3' -end (see Section 2.10.5). Crude extract was obtained from E. coli transcriptional fusion (HPS I-AGUE: :pCD 13PSK-PjadB5-IacZ) cultures harboring pUC18 or pUC 18-PA5356 during stationary phase and incubated with labeled probes. Lane I, biotin­labeled Pjad85 alone; lane 2, pUC 18 extract alone; lane 3, biotin-labeled Pjad85 incubated with pUC 18 extract; lane 4, pUC 18-PA5356 extract alone; lane 5, biotin-labeled Pjad85 incubated with pUC18-PA5356 extract.

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experiment with PfadB5 along with PlabAB and glpD are shown in Figure 20. It is evident

from the results that the gene product ofPA5356 is capable of binding to the promoter

region of PfadB5 and PfabA since incubation of the lysate with the respective DNA

fragments results in the different migration pattern and complete disappearance of the

DNA band. Althougb the DNA band does disappear, a discrete shifted band was not

observed. A similar result was observed when glpD was incubated with the lysate.

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DNA - + + + + + + Extract + - + - + - +

1 2 3 4 5 6 7

Figure 20. Gel shift assay experiment performed with 120 bp PladD5, 320 bp PlabAD, or 400 bp glpD fragment labeled with biotin at the 3' -end (see Section 2.10.5). Crude extract was obtained from E. coli transcriptional fusion (HPS I-ADlIB::pCD 13PSK-P/adD5-lacZ) cultures harboring pUC 18-PA5356 during stationary phase and incubated with labeled probes. Lane I, pUC 18-PA5356 extract alone; lane 2, biotin-labeled P/ad85 alone; lane 3, biotin-labeled Plad85 incubated with pUC18-PA5356 extract; lane 4, biotin-labeled PlabAB alone; lane 5, biotin-labeled PlabAB incubated with pUC 18-PA5356 extract; lane 6, biotin­labeled glpD alone; lane 7, biotin-labeled glpD fragment incubated with pUC18-PA5356 extract.

77

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Chapter Five: Protein purification and EMSA

5.1 Introduction

Transcriptional regulators control the expression of their cognate genes through

the interactions with the operator sites of these genes. FadR binds to specific sequences

found upstream of fabA and the fad genes and this has been readily demonstrated in E.

coli by gel shifts and protection assays (23, 24, 48, 49). Binding of a transcriptional

regulator to the operator site is generally facilitated by the recognition of a consensus

sequence in the operator region, which ensures that a regulatory protein is interacting

with its intended targets. The interaction between a regulatory protein and its consensus

sequence is generally strong and using consequence sequences in the purification scheme

has been successfully adapted to isolate a number of transcriptional regulators (34).

Analysis of a number of fatty acid degradative and biosynthetic genes in P. aeruginosa

has revealed the presence of a strong consensus-like sequence (fabAB,fadE andfadBA5)

(Figure 21). Alignment of these putative consensus sequences shows a significant degree

of conservation comparable to the consensus sequences used by FadR to regulate fatty

acid metabolism in E. coli. These observations in addition to the fact that DNA-binding

approaches have been successful in the isolation of transcriptional regulators, suggest that

it may be possible to exploit the strong interactions between the transcriptional regulator

and its operator site to isolate the FadR homologue(s) in P. aeruginosa. Purification of

proteins from raw P. aeruginosa lysate will support the involvement of binding­

consensus sequences in fatty acid metabolism regulation. It is presumed that the isolated

protein will bind mutually to fatty acid degradative and biosynthetic genes since this is

the case in E. coli, making such an observation in P. aeruginosa would

78

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(A) P. aeruginosa

+1 Gee G G A T""T""""G=T A GIGlT A G G A C T~'21 ~GCCGGAATGTGT~CGCACCCAM .GCCGGAATGATCTACGACAAIGl~ ftGCGGGAATGAAC~ATTACCT~~

GCCGGAATGNNNGNNNACCNG

(8) E. coli

~AG""T""",,G~G~T=-:C=AGACCTCCT

ATCTGGTACGACCAGAT AACTG~TCGGACTTGTT AGCTGGTATGATGAGTT GGCTGGTCCGCTGTTTC ~GCTGGTCCGACCTATA CACTGGTCTGATTTCTA ~ACTCATCGGATCAGT ANCTGATCNGACNNNTT

PfadE

PfadBA

PfabA

P1-fadD

P2-fadD

P1-fadL

P2-fadL

P- IclR

Consensus

P-fadE

P-fadBA 5

P1-fabAS (constitutive)

P2-fabAS

Consensus

Figure 21. Putative P. aeruginosa PAOl Fab-Fad consensus regulator binding-site (A) relative to the E. coli consensus for FadR binding-site (B). The E. coli sequences were compiled previously (10) where there are 12 conserved nucleotides out of 17. The P. aeruginosa sequence has 14 conserved nucleotides out of 21. The numbers in (A) correspond to position relative to the mapped transcriptional start sites. The presence of putative consensus sequences in both fab- and fad-genes reinforce the hypothesis that common regulator is involved in the regulation of fatty acid biosynthetic and degradative processes.

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5.2 Results

5.2.1 Analysis of Promoter Regions ofPladBS. PlabAB and PladE

The fact that binding sites for repressors typically overlap or are downstream of

the transcriptional start site along with the fact that binding sites for activators are

generally upstream of the start site served as starting points for promoter analyses. Based

on this information it was hypothesized that a regulator if it exists in Pseudomonas

should bind upstream of the fabAB start sites and downstream or overlapping with the

fadB5 and fadE transcription start sites. Enmination of the available mapped promoter

regions revealed a possible consensus sequence. The promoter region of fabA, which

actually has two transcript start sites contained consensus sequences at expected locations,

both were found upstream of the transcript start sites. Similarly, for bothfodB5 andfadE,

the consensus sequence lie either downstream or overlap with the transcript start sites as

predicted (Figure 22). Alignment of the possible consensus sequences shows fairly

strong conservation, which is comparable to that observed in E. coli (Figure 21).

5.2.2 Protein Purification using Streptavidin Magnetic Particles

Since the interaction between a regulator and its consensus sequence is fairly

strong, it was hypothesized that it may be possible to isolate the FadR-like protein from P.

aeruginosa lysate using the fadB5 consensus sequence. Briefly, the strategy involved

concatamerizing the fadB5 consensus sequence by peR using biotinylated primers and

then attaching the conca tamers to streptavidin magnetic particles via biotin-streptavidin

interactions. In theory, incubation of these concatamer-bound-streptavidin magnetic

particles with P. aeruginosa lysate, should allow the separation of proteins that interact

80

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PfadBA5 TC<XlGATGGCTGATTGACAATTa:cooc I I I IGCCGGAATGTGTGCGCACCCAAGTCAAAc=GTATGAATCGAGCGTTTGCCT

CGalCAGGCCTCGI\CAATAGAGACCCGGTTATCGCGTCGGCalGTGTGCCGAAGGGTTTGGGO.CTATGCTCG3CGG1 I GCCGI\<\. •• G

GGTCCCGACACGCI:GTCCGATGTGTAAGTTCMGCTTCCATAATAGCGTGGAGATCAGTTGATGATTTACCMGGTAAAGCQt\TCA

.MIYQGKAI

CGGfTAAGCCTCTTGAGGGCGGCATCGTCGAGrTGAATTTCGI\TCTCAAGGG::GAGTCCGTCAACAAGTTCMCCGTCTCA=rC

.T v K P LEG G I VEL N F 0 L K G E S V N K F N R L T L

AGTGAGTTGCGTa:GGCAGTCGATGCGATG.6AGGCCGATGCATCGGTCA

.SELRAAVDA I KADASV

PfadE ACCACCACCG:lGTGGrCACTATCCACGAAG::GGCCXlGATTGTAGGrAGGACTGCATGGCOOGCTo:;cGi'IXlGGCAATTTGrGCCAGT

CTAAACGGGAAGTTTTCGCCTAT AACGTGACAAAa;COOGCCAA~TGA=rGCXX'J;;cAAA(X'llCl'eCAAA=AAG:JI'

CGA~=CTTCATOOAGGATT~TGTTGTT~TCTOOTTAGrCGTACTGGTACTCOOTGT~ACCT=

.ML LLWLVVLVLGVAYLA

CATCOOCGTACCCCACCCGCr=DGGCATCA=ACCTGATCCTGATOOGCGTGTTCAGCCACXlCGCOCGGCTGG

.HRRTPPAPALGI SAAYLI LMGVFSHAPGW

CTGCTGCTGGrCTTCTGGCT

.LLLVFW

PfabAB GGGCGITCGOOAGAACTGCCTGCAOOCGGG>.ATGAACGATTACCTGGCCMGCCATTCAAACGGOCGGAATTGCAACGC'ATACTGC

AACGCfGGATCGGCTCGCA~CT=GACGTOOAACGAAAC<XlGACGI\GGOOAGCCXlGAATGATCTACGACAA=

GCAA~CXlCGGGAATAAAGTGAACATcrGTT=GG6.CACTGTGAcrTTCACCGC'AACGCAACAGrCTATGACTAGGCTOOC

GCTGCGACGa;GATACAATAACCCOOCGCGACGGa;GCTOOACGAACCGCCACAACCCT~GTTCAGGGO.TTTTTGAOOAGCTCG

CATGfJCG.6AlCAACACGCCTTCAca;GAGAAGAccrGCT=T~GTOGCGGCGAGCTGTT=AACGCGCAACTTC

.M T K Q H AFT RED L L R C 5 R GEL F G P G N A Q L

CCGCCX;CCAAC .p A P N

Figure 22. Promoter region of fadBA5, fadE, and fabAB. Indicated are the putatative consensus sequences (underlined) and transcript start sites (bold) upstream of the start codon. Consensus sequences are found either overlapping with (fadBA5) or downstream of (fadE) the transcript start site, while the consensus sequence is upstream of both transcript start site of fadAB. Transcript starts sites were previosly determined by primer extension in our laboratory, and the transcript start site of fadBA5 substantiated using a promoter mapping prediction program from http://mendel.cs.rhul.ac.ukImendel.php (37).

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with the consensus sequence from those that do not. Unbound proteins can later be

separated from the bound protein with the use of a magnetic particle separator (Figure

23). Elution of the bound protein revealed some degree of purification (Figure 24). The

FadR protein in E. coli is approximately 27 kDa, a few major bands corresponding to that

approximate molecular weight was partially purified and may have the potential to

regulate the JadBA5 operon.

5.2.3 DNA-binding studies using consensus sequence-purified extract

To verify that the partially purified protein extract is able to bind to the JadB5

promoter region, as well as test its ability to bind to the JabA promoter, EMSA studies

were conducted. A -120 bp sequence encompassing the promoter region ofJadB5 was

incubated with the purified protein extract and resulted in a clear shift (Figure 25). To

determine if this protein-DNA interaction is limited to the JadB5 promoter region the

protein extract was also tested with the JabA promoter region. In the presence of the

partially purified protein extract, the amplified JabA promoter region, which contains two

consensus sequences, also resulted in a shift (Figures 22 and 25). In order to rule out any

sort of non-specific DNA-binding interactions, a competitor assay was also conducted

where increasing concentrations of unlabelled PlabA was added to the binding reactions to

compete with the biotin-labeled PlabA. In theory, by increasing the concentration of the

unlabeled PlabA, the DNA-binding protein is titrated away from the labeled PlabA, resulting

in the labeled P fabA migrating through the gel as it normally would in the absence of

DNA-binding proteins. The results of these experiments are shown in Figure 25. As the

concentration of unlabeled P labA is added to the reaction, the shifted band gradually

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M"llJl'lIio P"IIicIe

! S treptov;din

d+ • III III II

t PCA ampIlied bictinyJated oligo

1 , ..... -~-"'" o

1'-~~'--I .. I III II

I III III '1 ~088~ DNA·binding protein I--a a a =ONA.binding

Figure 23. Principle of the purification of DNA-binding protein. Consensus sequence of fadBA5 promoter region is concatmerized using PCR with a biotinylated primer. Amplified PCR product is then incubated with streptavidin magnetic particles. When PAO I clarified extract is applied to the magnetic particles, the DNA-binding protein is captured by the oligo-particle complex due its affinity to the consensus sequence concatamers whereas non-specific proteins do not bind. Application of a magnetic particle separator and several washing steps is used to separate the bound protein from the supernatant. The specific DNA-binding protein is then eluted from the immobilized particles with a high salt buffer.

83

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kDA

97.2 -

66.4

42.7

36.5

26.6

20.0

14.3

----

--

1 2

Figure 24. Streptavidin magnetic particle-purified extract. PAO 1 clarified extract was purified using concatamerized j ildB5 consensus sequence amplified using oligos 499 and 504 (biotinylated at 5' -end). Concatamerized sequence was attached to magnetic beads via biotin-streptavidin interactions. Lane 1, protein marker, and lane 2, purified extract.

84

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DNA • • • • • •

~ ( unlabelled)

DNA + • + + + • + + + + (labeled)

EXTRACT • + + • + + + + + +

1 2 3 4 5 6 7 8 9 10

Figure 25. Gel shift assay experiment performed with 120 bp PjadB5 and 320 bp PjabA

fragments labeled with biotin at the 3' -end. Streptavidin magnetic particle-purified extracts were incubated with the DNA probes. Lane I, labeled Pjad8J alone; lane 2, purified extract; lane 3, labeled PjadBJ incubated with extract; lane 4, labeled PjabA alone; lane 5, labeled PjabA incubated with extract; lane 6, purified extract alone; lanes 7-1 0, labeled PjabA incubated with extract with increasing concentration of unlabeled PjabA.

85

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decreases as the DNA-binding protein is titrated away from the labeled PpM sequence.

At the highest concentration of unlabeled P JabA, the labeled P JabA fragment migrates

similar to the DNA alone control lane.

5.2.4 Identification of DNA-binding Protein by LC-MS

To identify the proteins that were partially purified using the fadE5 consensus

sequence, liquid-chromatography mass spectroscopy (LC-MS) was applied. Initial

identification attempts were made using N-terminal sequencing by Edman degradation

but due to the small quantities of protein obtained and purity issues, this method did not

prove feasible. Rather, collaborative efforts were made to the protein through LC-MS

which basically involved running the partially purified protein extract through a 10%

SDS-PAGE gel and excising the band of interest. The excised band was then extracted

and treated with trypsin prior to the LC-MS run. The software used had limitations in that

a P. aeruginosa database was not available, and only protein sequences in E. coli were

generated. Attempts were made to BLAST those protein sequences to the P. aeruginosa

database to identify potential homologues in P. aeruginosa but no similarities were found.

Alternatively, peptide fragment sequences that were assigned high scores were used to

BLAST directly against the P. aeruginosa genome and a few transcriptional regulators

were identified. The highest scored protein that corresponded to a transcriptional

regulator was ORF PA5525.

5.2.5 DNA-binding studies using E. coli Lysate

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Since the identity of the potential transcriptional regulator P A5525 was initially

determined by protein purification and DNA-binding assays, to verify this DNA-binding

interaction electrophoresis mobility shift assays were performed as described above.

Briefly, the PA5525 region was amplified from P. aeruginosa chromosomal DNA, to

include the upstream region and cloned into the cloning vector pUC18. The vector

pUC18-PA5525 was then introduced into the E. coli transcriptional fusion strain HPSl­

I..attB::pCD13PSK-P/adBs-lacZ. The lysate from the E. coli transcriptional fusion strain

was used for EMSA studies (Section 2.10.1).

The lysate from pUC18-PA5525 as well as pUC18 harboring cultures were

incubated with labeled PfadB5 and the migration of the DNA bands was compared. The

results from the electrophoresis mobility shift assays are shown in Fignre 26. The

addition ofpUC18-PA5525 lysate caused a clear shift in the PfadB5 band as expected. The

pUC18 control vector did not exhibit a similar banding pattern as its PA5525 derivative

and the PfadB5 band is clearly not affected by the presence of the pUCl8 vector. As shown

in Figure 26, the position of the PfadB5 band does not shift and remained at the same

position as the DNA alone control.

To establish whether the shift in the PfadB5 fragment was due to specific or non­

specific interactions, the DNA-binding studies were repeated with labeled PfabA and glpD

fragments. The results from the experiments are shown in Figure 27. As observed

previously the addition ofpUC18-PA5525 lysate caused the PfadB5 fragment to disappear

but a discrete shifted band was not observed. Likewise, the lysate appears to be able to

interact with both PfabA and glpD fragments since the addition of the pUC18-PA5525

87

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DNA + • + • + Extract

• + + + +

1 2 3 4 5

Figure 26. Gel shift assay experiment performed with 120 bp PjadB5 labeled with biotin at the 3'-end (see Section 2.10.5). Cmde extract was obtained from E. coli transcriptional fusion (HPS l-},attB::pCD 13PSK-PjadB5-lacZ) cultures harboring pUC18 or pUC18-PA5525 during stationary phase and incubated with labeled probes. Lane I, biotin­labeled PjadB5 alone; lane 2, pUC 18 extTact alone; lane 3, biotin-labeled PjadB5 incubated with pUC 18 extract; lane 4, pUC18-PA5525 extract alone; lane 5, biotin-labeled PjadB5 incubated with pUC 18-PA5525 extract.

88

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DNA - + + + + + +

Extract + - + - + - +

1 2 3 4 5 6 7

Figure 27. Gel shift assay experiment performed with 120 bp PladB5, 320 bp PlabAD. or 400 bp glpD fragment labeled with biotin at the 3' -end (see Section 2.10.5). Crude extract was obtained from E. coli transcriptional fusion (HPSI-AatlB::pCDI3PSK-PladD5-lacZ) cultures harboring pUC I 8-PA5525 during stationary phase and incubated with labeled probes. Lane I , pUC I 8-PA5525 extract alone; lane 2, biotin-labeled PladB5 alone; lane 3, biotin-labeled PladB5 incubated with pUC18-PA5525 extract; lane 4, biotin-labeled PlabAD

alone; lane 5, biotin-labeled PlabAD incubated with pUCI 8-PA5525 extract; lane 6, biotin­labeled glpD alone; lane 7, biotin-labeled glpD fragment incubated with pUC I 8-PA5525 extract.

89

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lysate resulted in the disappearance of the respective bands, although nuclease activity

may have contributed to the results.

5.2.6 Assessing the regulatory potential of P A5525 by expressing the respective gene

products in the E. coli PfadBS-lacZ transcriptional fusion

To address the issue of nuclease activity and provide further insight into the

potential interaction between the PA5525 gene product and the promoter region offadB5,

the effect of expressing PA5525 on the fadB5 promoter in the E. coli transcriptional

fusion strain (HPSI-I..attB::pCD13PSK-PfadBS-lacZ) was studied. Using the same E. coli

transcriptional fusion strain from which lysate was obtained for gel shift experiments

(Section 5.2.5) 13-galactosidase assays were performed at various points throughout

growth (Figures 28 and 29). Comparison of the growth rate and final cell density of

strains harboring pUC18 and its PA5525 derivative show no significant differences

through out the growth phases (Figure 28). Measurements were taken during mid-log,

early stationary, and late stationary phases for pUC18 and pUC18-PA5525 harboring

strains. Comparisons of 13-galactosidase activity in pUC18 harboring strains to pUC18-

PA5525 harboring strains showed no significant differences. The promoter activity of

PfadBS increased slightly throughout the growth phase in the presence of the pUC18 vector

(Figure 29). A similar trend was observed with pUC18-PA5525, although an increase in

promoter activity was not detected during the transition from early- to late-stationary

phase. The results from this experiment show the expression ofPA5525 appears to affect

PfadBS during the later phases of growth in particular during the late-stationary phase.

90

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3 LS

! M

2 l

O~------r-----~------~----~ o 10 20 30 40

Time (h)

---0-- pUC18

~ pUC18-PA5525

Figure 28. Growth curves of E. coli transcriptional fusion strains HPS 1-AattB::pCD13PSK-PjadB5-lacZ harboring pUC18 or derivative encoding PA5525, grown in LB + Ap media Cultures were induced with 1 roM IPTG at time = 1 h. Activities were measured at three time points during the course of growth, ML, mid-log; ES, early­stationary, and LS, late-stationary.

91

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30 00

25 00 f:h

200 0

15 00

10 00

500

co :Q co

i ..

~ ... ... ... u

~ u u

;:) ;:) ;:) .,

a. D. a. a. f , , , co co co ... ... ... u u u ;:) ;:) ;:) a. a. a.

Mid-iog Early Late Stationary stationary

Phase/Strain

Figure 29. Transcriptional regulation of PfadBS -lacZ fusion in the E. coli transcriptional fusion strain HPSl-AattB::pCD13PSK-P.fadJIs-lacZ in the presence of pUC18 encoding PA5525 or the empty vector, grown in LB + Ap media. Cultures were induced with I mM IPTG 1 h following inoculation. Activities were measured at three time points during the course of growth, mid-log, early-stationary, and late-stationary. The values represent the mean ± the standard deviation (n = 3).

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5.2.7 Ammonium sulfate precipitation

In an attempt to optimize the protein purification protocol, P. aeruginosa PAD 1

clarified lysate was subjected to ammonium sulfate precipitation. Using increasing

concentrations of ammonium sulfate, proteins in the crude extract were gradually

precipitated out and collected as fractions for DNA-binding studies. Ammonium sulfate

precipitation is a widely use protocol as an initial step in protein purification and rarely

affects protein activity. These attributes made this protein purification protocol an ideal

extension to the previous protein purification scheme. The rationale was to fractionate the

crude P. aeruginosa PAOllysate and test whether each fraction is capable of interacting

with the promoter region offadB5. This interaction would be determined by performing

gel shift assays using the various fractions collected. The intent was to further process

any fraction that was capable of causing the PjadBS fragment to shift by ion-exchange

chromatography followed by a final streptavidin magnetic particle separation step (as

described in Section 2.10.4). The results from the DNA-binding studies using ammonium

sulfate precipitate lysate are shown in Figure 30. Briefly the experiment involved adding

ammonium sulfate to 30% and then 5% increments thereafter to 60%, followed by a final

precipitation step at 80%. Precipitated proteins at each step was collected by

centrifugation then resuspended and dialyzed against 10 roM Tris-HCI prior to DNA

binding studies. Although the addition of lysate corresponding to 50% and 80%

precipitation cuts caused the disappearance of the PjadBS band, a discrete shifted band was

not observed in either case. This suggested non-specific interactions or nuclease activity

rather than isolation of a DNA-binding protein. Unexpectedly, the banding pattern of the

precipitated proteins remained relatively constant throughout the ammonium sulfate

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precipitation steps. A difference was only observed after the addition of 80% ammonium

sulfate.

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DNA - 1+ - 1+ - 1+ - 1+ % Ammonium Sulfate 35 40 45 50

123 4 5 6 7 8 9

D A - 1+ % Ammonium Sulfate 80+

1 2 3 4 5 6 7 8 9

Figure 30. Gel shift assay experiment performed with 120 bp PladB5 labeled with biotin at the 3' -end (see Section 2.10.5). Crude extract from P. aeruginosa P AO I was subjected to step-wise ammonium sulfate precipitation. Labeled PjadB5 was incubated with resolubilized and dialyzed protein precipitate. Lane I, labeled PjadB5; alone, lanes 2, 4, 6, 8; labeled Plad!]) incubated with extract (% refers to ammonium sulfate concentration used to precipitate protein); and lanes 3, 5, 7, 9; respective extract alone.

95

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Chapter Six: Transposon mutagenesis

6.1 Introduction

Transposon-based mutagenesis approaches have been successfully used in the

identification of a variety of genes in a range of microorganisms. In P. aeruginosa,

regulators involved in adhesin expression have been recently identified using transposon­

based mutagenesis (69). Several transposon mutagenesis tools are available and have

been demonstrated to be functional in P. aeruginosa including pBT20 (69) and pTnMod­

O-Gm (20). The combination of transposon mutagenesis and fatty acid metabolism gene

fusions should allow for the development of a screen to identify possible fatty acid

metabolism regulators. In the absence of an exogenous source of long-chain fatty acids,

Jad genes in E. coli are repressed since FadR is bound to its cognate Jad operators.

Therefore, the disruption of the JadR gene should theoretically relieve repression of these

Jad genes. Based on the assumption that the FadR homologue acts as a repressor ofJad­

genes in P. aeruginosa, subjecting aJad-reporter strain to transposon mutagenesis should

allow for the identification of FadR-like protein(s). The promoter region ofprobableJad­

gene fused to the lacZ-reporter gene can be integrated in the chromosome of P.

aeruginosa and this strain subjected to transposon mutagenesis grown under repressive

conditions. Disruption of the JadR gene should relieve repression and result in an

apparent 'upregulation' oftheJad gene that would appear as more intensely blue colonies

on an appropriate indicator medium (Figure 31). The target of this transposon-based

mutagenesis strategy will be the P. aeruginosa transcriptional fusion strain PAOl­

attB::P/adB5-1acZ. The basis of this approach is similar to the bioinformatic approach

discussed in Chapter 4, but rather than constructing

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Under non-;nducing condmons - absence 171' exugenous l'alty adds ;acyI-CoA)

WTcell

RNA polymerase ,... 0 = FadR

~ Gr

Tmngposun mutant ,...8 x

I'adR.:"Tn Pl'ad&45-1aaZ"

Figure 31. Principle of the transposon mutagenesis screen used to find the FadR-like protein in P. aeruginosa. Upper panel depicts wild-type PAOI-attB::PjadBAs-lacZ cell in the absence exogenous fatty acid. FadR is expressed from the fadR loci and binds to the promoter region of the fadBA5-1acZ fusion repressing transcription of lacZ. The absence of ~-galactosidase leads to the formation of white colonies on media containing X-gal. Bottom panel shows the disruption of the fadR loci by transposon insertion under the same conditions. Inactivation of fadR relieves repression of fadBA5-1acZ, allowing for the expression of ~-galactosidase, which leads to the formation of blue colonies.

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particular mutants of interests based on bioinformatics, a screen was used to identifY

other potential transcriptional regulators. The location of the transposon integration sites

will be determined by low-stringency PCR followed by sequencing and any probable

transcriptional regulators identified will be characterized using a variety of approaches.

These approaches, similar to experiments performed previously on other potential FadR

candidates include investigating the effect on fadB5 in P. aeruginosa and E. coli

transcriptional fusion strains and electrophoresis mobility shift assays with P fodBj.

6.2 Results

6.2.1 Transposon mutagenesis and insertion sites

Transposon mutants in strain PAOI-attB::PjadBs-1acZ were obtained using the

vector pBT20. Colonies that appeared more intensely blue on PIA + Om + X-Gal were

selected and the insertion sites detennined by low-stringency PCR as described in

Section 2.S. Table 7 lists the location of the insertion sites and the corresponding

functional class of the ORF disrupted. The apparent upregulation ofPjadBj resulted from

the inactivation of various types of genes including those involved in fatty acid

metabolism, two-component regulatory system and transport molecules. A significant

proportion of the transposon insertions occurred in genes involved in LPS biosynthesis

(Figure 32). Most importantly, among the inactivated genes were a few probable

transcriptional regulators PA2601, PA3006 and PA3508. The gene products ofPA2601,

PA3006, and PA3508 are classifed as LysR-, TetR-, and IclR-type regulators respectively.

Various studies were performed to assess the regulatory potential of these probable

transcriptional regulators.

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Table 7. Transposon Insertion Sites

Accession F ..... oo .... on Number PA0005 probable acyltransferase PA0257 hypothetical protein PA0296 probable glutamine synthetase PA0413-0414 still frameshift probable component of chemotactic signal

transduction system PA0910 hypothetical protein PA0938 hypothetical protein PAI505 molybdopterin biosynthetic protein A2 PAI580 citrate synthase PAI833 probable oxidoreductase PA2601 probable transcriptional regulator P A2705 hypothetical protein P A3006 probable transcription regulator P A3141 nucleotide sugar epimerase/dehydratase WbpM PA3145 glycosyltransferase WbpL P A3146 probable NAD-dependent epimerase!dehyratase WbpK PA3147 probable glycosyl transferase WbpJ P A3 148-3 149 probable UDP-N-acetylglucosamine 2-epimerse WbpI PA3149 probable glycosyltransferase WbpH PA3155 probable aminotransferase WbpE PA3155-3156 probable aminotransferase WbpE P A3156 probable acetyltransferase WbpD P A3158 probable oxidoreductase WpbB P A3159 probable UDP-glucose/GDP-mannose dehydrogenase WbpA PA3219 hypothetical protein PA3238 hypothetical protein P A3414 nucleotide sugar epimerase!dehydratase WbpM P A3508 probable transcriptional regulator PA3577 hypothetical protein PA3716 hypothetical protein PA3798 probable aminotransferase PA3835-3836 hypothetical protein PA3865-3866 probable amino acid binding protein - pyocin protein PA3868 hypothetical protein PA4454-4455 hypothetical protein PA4455 probable permease of ABC transporter PA4696 acetolactate synthase ill large subunit

99

# of times inactivated

2 I I I

2 I I I I I I 3 2 2 2 I I 2 1 1 1 8 4 1 1 I 2 I I 1 1 1 I 1 1 1

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Accession Fnnction

Number PA4852 hypothetical protein PA4999 hypothetical protein PA4999-5000 hypothetical protein-probable glycosyl transferase PASOOO probable glycosyl transferase PAS022 hypothetical protein PA5162-5163 dIDP-4-dehydrorhamnose reductase P A5163 glucose-I-phosphate thymidylyltransferase PA5448 glycosyltransferase WbpY PAS454 oxidoreductase Rmd PAS474 probable metalloprotease PA5529 probable sodium/proton antiporter PAS563 chromosome partitioning protein Soj

# of times inactivated

I I I I 1 1 1 1 1 1 1 1

6.2.2 Characterizing PA01-attB::PfadBs-lacZl/ifadR::Tn (pA2601, PA3006

and P A3508) by /I-galactosidase assays

To investigate the effect of transposon insertions in PA2601, PA3006, and

PA3508, the mutated fusion strains were grown in media in which the P/adBSpromoter has

been previously shown to be repressed (see Section 3.2.2). Similar to the rationale

described in Section 4.2.2, if one of these genes encodes a repressor of the fadBA5

operon, an insertion mutation would prevent the gene product from being expressed thus

relieving repression (see Figure 31). This de-repression can be directly observed through

~-galactosidase assays. Briefly, the Pseudomonas transcriptional fusion strain PAOI-

attB::P/adBs-1acZ along with the transposon mutated derivatives (M'A2601::Tn,

M'A3006::Tn, and M'A3508::Tn) were grown in LB, media in which the PfadBS promoter

was shown to be repressed and ~-galactosidase activities measured at various growth

phases and compared. The results from these experiments are shown in Figures 33 and

100

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Related to phage, transposon,or

plasmid 1

Putative enzymes 5

Cell division

Transcriptional regulators

6

1 --------------

Hypothetical, unclassified, _------­

unknown 14

Transport of small molecules

1

Translation, post· translational modification, degradation

1

Amino acid biosynthesis and

metabolism 1

Biosynthesis of cofactors,

::::~~_------Pro:.~~:groups '" and carriers

1

Membrane proteins 6

Cell wall

26

Fatty acid and phospholipid metaboliem

2

Energy metabolism 1

ChemotaxisfTwo­component

regulatory systems 1

Figure 32. Transposon insertion sites grouped according to functional class, The transcriptional fusion strain PA01·attB::PjadBS-1acZ was subjected to transposon mutagenesis using the mini-transposon vector pBTIO and blue-white selection performed on PIA + Om + Xgal, Blue colonies were grown and the transposon insertion sites determined by low-stringency peR and sequencing. Shown are the number of transposon insertion mutants grouped according to different functional classes.

101

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34. Figure 33 shows the growth curve of PAOl-attB::PjadBs-lacZ along with the

transposon mutated derivatives grown in LB media. The growth rate of all the transposon

mutants were comparable to that of the control strain PAOl-attB::PjadBs-1acZ with the

exception of M>A3508::Tn, which started to deviate slightly during late log phase. The

fina1 cell density of M>A3508::Tn was also different, reaching a density of approximately

65% of the control strain. To obtain an accurate assessment offadE5 promoter activity, p­

galactosidase activities were measured at early-log, twice during mid-log, late-log, and

early-stationary phases. Since these ORFs represent potential repressors of the fadB5

operon, and increase in p-galactosidase activity was expected compared to the control

strain. The results from the f3-galactosidase experiments are shown in Figure 34. As

expected the P-galactosidase activity of the control strain PAO l-attB::PjadBs-lacZ was low

and remained low throughout all growth phases. Inactivation ofPA2601 and PA3006 by

transposon mutagenesis both caused a de-repression of the fadE5 promoter and a

significant increase in f3-galactosidase activity was observed. Activity of the fadE5

promoter in the M> A260 I ::Tn strain was approximately five-fold greater than observed in

the control strain and gradually decreased to approximately two-fold at early stationary

phase. The PjadBS promoter activity in the M>A3006::Tn strain was approximately three­

fold greater during early-log phase and increased to approximately seven-fold and

remained relatively constant throughout the remainder of the growth phases. Surprisingly,

disruption of PA3508 did not appear to affect the promoter activity of fadB5 to a

significant extent, with f3-galactosidase activity measurements remaining close to that of

the control strain throughout all growth phases.

102

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6~----____________________ ~

4

---0-- PA01-attB::PradBl>lacZ

= = ~ 2601::Tn 'CI Q 0 --0-- 3006::Tn

to 3508::Tn 2

o~~ __ ~ __ ~ ____ ~ ____ ~ __ ~

o 10 20 30 40 50

Time (h)

Figure 33. Growth curves of P. aeruginosa transcriptional fusion strains PAOI­attB::PfodBS-1acZ and transposon mutant derivatives (M'A2601::Tn, M'A3006::Tn and M'A3508::Tn) grown in LB media. Indicated are the time points at which ~-ga1actosidase activities were measured, EL, early-log; MLl, mid-log I; ML2, mid-log 2; LL, late-log, and ES, early-stationary.

103

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800

r

T

n ~] m ,

i f. E E ! E E f. i r: E c ! f. E E j E {:. c J;: J;:

"" I • i .;;

• i :li .;; "" • • i z .;; ~

.. !i! ~ !i! I ~ .. ! ~ OJ l i , i i , ~ ~ ~ ~ p

0 0 0 ~

0 f f f f

Early log Mid log Mid log Late log Early

Phase/Strain Stationary

Figure 34. Transcriptional regulation of P/adBs-lacZ fusion in the P. aeruginosa transcriptional fusion strain PAOl-attB::P/adBs-1acZ and transposon mutant derivatives (M'A2601::Tn, M'A3006::Tn and M'A3S08::Tn) grown in LB. /3-galactosidase activities were measured at five time points, EL, early-log; MLI, mid-log I; ML2, mid-log 2; LL, late-log, and ES, early-stationary. The values represent the mean ± the standard deviation (n = 3).

104

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6.2.3 Assessing the regulatory potential of P A2601, P AJ006 and P AJ508 by

expressing the respective gene products in the E. coli p/adBS-lacZ

transcriptional fusion strain

To elucidate the potential interactions between the PA2601, PA3006, and PA350S

gene products and thefadB5 promoter region, the respective ORFs were cloned into the

cloning vector pUC18 and transformed into the E. coli P/adBj-lacZ transcriptional fusion

strain HPSl-AattB::pCD13PSK-P/adBs-lacZ. Similar to the experiments described in

Seetions 4.2.4 and 5.2.6, the E. coli transcriptional fusion strains harboring pUC1S and

the derivatives were grown in LB + Ap and li-galactosidase assays performed during

mid-log, early-stationary and late-stationary phases following induction with 1 mM

lPTG. If the gene products of any of these genes bind to the promoter region of fadB5

then expression of these genes in the transcriptional fusion strain should repress the

fadB5 promoter causing a decrease in p-galactosidase activity compared to the control

strain (harboring pUC1S). The results from these experiments are shown in Figures 35

and 36. The growth curve of the various transcriptional fusion strains are shown in

Figure 35. Comparison of the growth rate and final cell densities show no significant

differences between the control strain harboring pUC1S and its derivatives. The results

from the P-galactosidase assays are shown in Figure 36. The activity observed in the

pUC18 strain was relatively high and increased slightly throughout the growth phases.

Both PA3006 and PA350S derivatives showed consistent reduction in activities of

approximately 35% compared to the control strain throughout growth whereas a decrease

in activity in the P A260 1 derivative was not observed until late-stationary phase. The

results from these experiments show that the gene product ofPA3006 and PA350S affect

105

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3

ML -----fr-- pUC18

2 ~ --<>-- pUC18-PA2601

~ pUC18-PA3006

= = :6 pUC18-PA3508 \C> = 0

O~~----r-----~-------r----~ o 10 20 30 40

Time (h)

Figure 35. Growth curves of E. coli transcriptional fusion strains HPS 1-AattB::pCD13PSK-PfadBs-1acZ harboring pUC18 or derivatives encoding PA2601, PA3006 or PA3508, grown in LB + Ap media Cultures were induced with 1 mM IPTG at time = 1 h. Activities were measured at three time points during the course of growth, ML, mid-log; ES, early-stationary, and LS, late-stationary.

106

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3000,-____________________________________ ~

T

2500.

500

o· • • CO ~ co co co ~ ... co CO ~ CD co ~ co co 51 ~ co .. co ~ CO co

~ U CD co U CD co .. U

~ ~ ~ ~ .. f ~ ~ ::> ~

::> ::>

'" '" '" , , CD CD CD co CD co

~ ~ ~ ~ ~ ~ ~ ~ ~ u u u u u u u u u ::> ::> ::> ::> ::> ::> ::> ::> ::> '" '" '" '" '" '" '" '" '" Mid-log Early Late

Stationary Stationary

Phase/Strain

Figure 36. Transcriptional regulation of p/adBS-1acZ fusion in the E. coli transcriptional fusion strain HPSl-AattB::pCD13PSK-P/adBs-1acZ in the presence of pUC 1 8 encoding either PA2601, PA3006, PA3508 or the empty vector, grown in LB + Ap media. Cultures were induced with 1 mM lPTG 1 h following inoculation. Activities were measured at three time points during the course of growth, mid-log, early-stationary, and late­stationary. The values represent the mean ± the standard deviation (n = 3).

107

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the promoter region of PfadBsthroughout all growth phases whereas the effect ofPA260l

was delayed and was only observed during later phase of growth.

6.2.4 DNA-binding studies using E. coU Lysate (pUC18 and PA2601, PA3006, and

PA3508 derivatives)

To obtain more insight into the potential interactions between the gene products

of PA260l, PA3006, and P3508 and the fadE5 promoter, DNA binding studies were

included. Using lysate from the E. coli transcriptional fusion strains used in the p..

galactosidase assays experiments described above in Section 6.2.3, DNA-binding studies

were performed on PfadBS. The results from these DNA-binding studies are shown in

Figures 37 and 38. Figure 37 shows the results from the experiments with PA3006 and

PA3508. Although the PfadE5 band disappears upon addition of the PA3006 lysate the

banding pattern observed is comparable to that seen with the pUC18 control lysate. On

the other hand, the addition ofPA3508lysate caused a clear shift in the PfadB5 band, and

a discrete shifted band was observed producing a banding pattern quite distinct from the

control. As shown in Figure 38, PA2601 did not appear to interact with PfadBS as the

PfadBS band remained unshifted and the banding pattern ofPA260l and the control were

relatively similar. The results from these experiments show that when the DNA and

protein elements are isolated from a system, PA3508 appears to be the only protein

capable of interacting with PfadBs.

6.2.5 A closer look at P A3508

Although inactivation ofPA3508 by transposon mutagenesis in the P. aeruginosa

108

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D A + • + • + • +

Extract • + + + + + +

1 2 3 4 5 6 7

Figure 37. Gel shift assay experiment performed with 120 bp PladB5 labeled with biotin at the 3'-end (see Section 2.10.5). Crude extract was obtained from E. coli transcriptional fusion (HPS 1-J.aIlB :: pCDl3PSK-PladBylacZ) cultures harboring pUC IS, pUCIS-PA3006 or pUCIS-PA350S during stationary phase and incubated with labeled probes. Lane I , biotin-labeled PladB5 alone; lane 2, pUC 18 extract alone; lane 3, biotin-labeled PladB5 incubated with pUCIS extract; lane 4, pUCIS-PA3006 extract alone; lane 5, biotin­labeled PladB5 incubated with p CIS-PA3006 extract; lane 6, pUC18-PA3508 extract alone; lane 7, biotin-labeled PladB5 incubated with pUCIS-PA350S extract.

109

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DNA + - + - +

Extract - + + + +

1 2 3 4 5

Figure 38. Gel shift assay experiment perfonned with 120 bp PlodBs labeled with biotin at the 3' -end (see Section 2.10.5). Crude extract was obtained from E. coli transcriptional fusion (HPS l -AnIlB: :pCD 13PSK-P/adBS-lacZ) cultures harboring pUC18 or pUC 18-PA2601 during stationary phase and incubated with labeled probes. Lane I, biotin­labeled P/odBS fadB5 alone; lane 2, pUC 18 extract alone; lane 3, biotin-labeled PlodBS incubated with pUCl8 extract; lane 4, pUC18-PA2601 extract alone; lane 5, biotin­labeled PlodBS incubated with pUC 18-PA260 1 extract.

110

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transcriptional fusion strain did not produce an increase in f3-galactosidase activity as

expected, the gene product does appear to interact with the promoter region of fadB5.

Evidence of this is based on data obtained from the E. coli transcriptional fusion studies

and in particular the DNA-binding experiments. A closer examination of genes

surrounding PA3508 also strengthens the case that PA3508 may be involved in regulating

fatty acid metabolism. In P. aeruginosa, transcriptional regulators are usually clustered

around genes that they control and analysis of regions up and downstream of P A3508

reveals a number of probable fatty acid metabolism related-genes (Figure 39).

Collectively, the results from the experiments performed suggest that PA3508 is an ideal

candidate for further studies.

To address the issue of non-specific interaction several approaches were taken.

Since it is assumed that the gene product of P A3508 is responsible for the shift in PjadBj

that was observed, decreasing the concentration of PA3508 should cause the PjadBS band

to migrate norma1ly. Therefore to test this hypothesis the gel shift were repeated across a

gradient ofPA3508 lysate concentrations. The results from the experiments are shown in

Figure 40. It is apparent from the figure that at higher concentrations, the PjadBS fragment

is shifted whereas a gradual decrease in P A3508 results in the disappearance of the

shifted band and the re-appearance of the PjadBS at its normal position. Thus it would

appear that the gene product of PA3508 is responsible for the PjadBs shift. Since FadR in

E. coli is capable of interacting with both fad genes and the fabAB operon it would be

interesting to determine whether PA3508 has the same ability. Consequently, to test

whether the gene product of PA3508 is capable of binding to the promoter region of

fabA, the gel shift experiment was repeated across a gradient of P A3508 lysate

111

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G ~

s § IJ ~ '$ .c1SiJ

1. ~ ..§>

I ~ 'i -8 '5 .~

.~ ~ ·fi .S; .~ ..... I .g ~

<t1 .e-Ii ~ ~ ~ -e 9 ?} ~ ~ ';'Q, tJ ~ -g

<t1 -& ~ ,g ~ ~ .s: ..:::t ;& ..,'!:.> ..... -2 ..,'!:.> ~ ~ .Jj -c) <i' .1 <i' -h I ~ ~ '8 '8 0

.$ ~ ~ G. ~ ~ Q,

PA3505 PA3506 PA3507 PA3508 PA3509 PA3510 PA3511

< < < < < < < I

Figure 39. Organization of genes surrounding the PA3508 locus. PA3508 encodes a probable transcriptional regulator that belongs to the IclR-type family of regulators. Found adjacent to the PA3508 ORF are probable short-chain dehydrogenases, genes that are potentially involved in fatty acid metabolism.

112

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DNA + - + + + + + + +

Extract - +

1 2 3 4 5 6 7 8 9

Figure 40. Gel shift assay experiment performed with 120 bp PjadBs labeled with biotin at the 3' -end (see Section 2.10.5). Crude extract was obtained from E. coli transcriptional fusion (BPS 1-2atlB::pCD13PSK-PjadBs-lacZ) cultures harboring pUC18-PA3508 during stationary phase and incubated with labeled probes. Lane I, biotin-labeled PjadBS alone; lane 2, pUC18-PA3508 extract alone; lane 3-9, biotin-labeled PladB5 incubated with gradual decreasing concentrations of pUC18-PA3508 extract.

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concentrations using the promoter region of JabA as the probe. The results from the

experiments are shown in Figure 41. It is evident from the results that the product of

PA3S08 is capable of causing a definite shift in the PfabA fragment. The pUC18 extract

also appeared to cause a shift in the P fabA fragment; however, the shift was not as distinct

as the band observed with the PA3S08 extract suggesting non-specfic rather than specific

interactions. Figure 41 also shows that as the PA3S0S concentration is reduced the

shifted band appears at lower position in the gel and finally at the lowest concentration

re-appears as a faint band at its normal position. This observation may give insight into

the structural nature of the PA3S0S gene product when it binds to its target DNA

sequences. Based on these observations there is strong evidence that P A3S08 does indeed

interact with the promoter regions of both JadB5 and JabA. strengthening the hypothesis

that fatty acid degradation and biosynthesis are coordinately regulated in P. aeruginosa.

As a final examination, the PA3S08 gene product was expressed and isolated as a His­

tagged protein, and the next objective was to subject the purified protein extract to gel

shift assays to demonstrate a shift in bothJadB5 andJabA fragments.

Briefly, the P A350S ORF was cloned in-frame into the expression vector pET2Sa

and expressed in the E. coli strain ER2566. Figure 42 shows the clarified lysate of the

expression strain prior to and after induction with I mM IPTG. The induced protein is

approximately 30 kDa agreeing with the predicted molecular weight of PA350S. The

induced lysate preparation was then purified on a Ni+-NTA column and eluted with high

salt buffer (as described in Section 2.10.2). The PA350S gene product was purified to

near homogeneity shown in Figure 43 and was then used in subsequent DNA-binding

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DNA + • + • + + + + + +

Extract • + + [

1 2 3 4 5 6 7 8 9 10

Figure 41. Gel shift assay experiment performed with 320 bp PlabAH labeled with biotin at the 3'-end (see Section 2.10.5). Crude extract was obtained from E. coli transcriptional fusion (HPS 1-}.attB::pCD13PSK-PladH5-lacZ) cultures harboring pUC 18 or pUCI8-PA3508 during stationary phase and incubated with labeled probes. Lane I, biotin­labeled PlabAH alone; lane 2, pUCI8 extract alone; lane 3, biotin-labeled PlabAH incubated with pUC 18 extract; lane 4, pUC18-PA3508 extract alone; lane 5-10, biotin-labeled PlabAH incubated with gradual decreasing concentration of pUC 18-PA3508 extract.

liS

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kDA

97.2 -66.4 -55.6 -42.7 -36.5 -26.6 -

20.0 -

14.3 -

1 2 3

Figure 42. 10% SOS-PAGE electrophoresis gel showing the overexpression of PA3508-His6 in E. coli ER2566. Lane 1, protein marker; lane 2, total protein of uninduced cultures with pET28a-PA3508, and lane 3, total protein of IPTG-induced cultures with pET28a-PA3508.

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- 97.2

66.4 -- 55.6

- 42.7

- 36.5

- 26.6

- 20.0

- 14.3

1 2 3 4 5 6

Figure 43. 10% SOS-PAGE electrophoresis gel showing PA3508-His6 purified from E. coli ER2566 using Ni+-NTA column. Lane 1, column loading filtrate ; lane 2, column washing filtrate; lanes 3, 4, and 5, are respectively, 0.5 fLg, 1.0 fLg and 1.5 fLg of PA3508-His6, and lane 6, protein marker. Protein concentrations were detennined by Bradford assays using BSA as the standard (8).

117

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studies. Incubation of the purified PA3S08 preparation with the promoter regions of

JadB5 and JahA resulted in a shift of both fragments, although unlike the transcriptional

fusion extract, a defined band was not clearly observed as shown in Figure 44. The fact

that a defined shifted band was not observed raised some concern as to the nature of the

interaction between PA3S08 gene product and the promoter regions ofJahA andJadB5

since the absence of defined band is often indicative of non-specific interactions. To

address these concerns the gel shift experiment was modified to include an internal

region of the glpD gene as a probe. As mentioned earlier, glpD is a glycerol metabolism

gene and although the amplified peR product has some sequence similarity to the PfadB5

sequence there is enough differences to allow the two sequences to be differentiated

while maintaining a degree of stringency. Incubation of purified PA3S08 gene product

with PfabA which serve as a control and glpD caused a shift in both fragments (Figure 45).

In both experiments, the DNA band intensity at its normal position is reduced, and

appears at a higher position in the gel. The cumulative results suggest that the gene

product ofPA3S08 is capable of binding to PfadB5, PfabA and glpD.

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DNA - + + + + + + + +

Protein + - -

J 2 3 4 5 6 7 8 9

Figure 44. Gel shift assay experiment performed with 120 bp PjadB5 and 320 bp PjabA

labeled with biotin at the 3' -end (see Section 2.10.5). PA3508-His6 was overexpressed in E. coli strain ER2566 as described in Section 2.10.2. The labeled DNA fragments were incubated with the purified protein and the mobility assayed. Lane I, purified PA3508-His6 alone; lane 2, biotin-labeled Pjad85 alone; lanes 3-5 , biotin-labeled PjadB5 incubated with decreasing concentration of purified PA3508-His6, lane 6, biotin-labeled PjabA alone; lanes 7-9, biotin-labeled PjabA incubated with decreasing concentration of purified PA3508-His6.

119

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DNA + + + +

Protein • + • +

t 2 3 4

Figure 45. Gel shift assay experiment performed with 320 bp PjabAB and 400 bp glpD fragment labeled with biotin at the 3' -end (see Section 2.10.5). PA3S08-His6 was overexpressed in E. coli strain ER2566 as described in Section 2.10.2. The labeled DNA fragments were incubated with the purified protein and the mobi lity assayed. Lane I , biotin-labeled PjabAB alone; lane 2, biotin-labeled PjabAB incubated with purified PA3S08-His6; lane 3, biotin-labeled glpD alone; lane 2, biotin-labeled glpD incubated with purified PA3508-His6.

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Chapter Seven: Discussion

The purpose of this study was to investigate the regulation of fatty acid

metabolism in P. aeruginosa. Understanding the regulation of fatty acid metabolism is

important because a) fatty acid biosynthesis is involved in virulence expression and b)

fatty acid degradation has been shown in our laboratory to be involved in the degradation

of lung surfactant phosphatidylcholine. Since the regulation of fatty acid metabolism is

fairly well established in E. coli, this served as a basis for this study. Through a series of

studies, a central regulator, FadR was shown to be involved in the regulation of fatty acid

biosynthetic and degradative processes in E. coli. FadR served to balance the two

processes by activating the biosynthetic pathway in the absence of exogenous fatty acids

while concurrently repressing the degradative pathway. In contrast, the presence of an

exogenous source of fatty acid results in the de-repression of the fatty acid degradative

pathway and the repression of the fatty acid biosynthetic pathway. This mechanism of

regulation makes logical sense as fatty acid biosynthetic process should be activated in

the absence of fatty acids to synthesize fatty acid and fatty acid degradative processes

repressed to prevent the degradation of newly synthesized fatty acids and vice versa. The

first step in this study was to establish whether this form of regulation is likely to exist in

P. aeruginosa.

To investigate the regulation of fatty acid metabolism in P. aeruginosa

transcriptional fusions were constructed using the promoter regions of previously mapped

fatty acid metabolism genes. The promoter regions offadB5,fadE andfabA were fused to

the lacZ gene and the transcriptional fusion integrated into the chromosome of P.

aeruginosa PAOl. The assumption was that the transcriptional fusion should reveal when

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and if these promoter regions are responsive to exogenous sources of fatty acids. From a

clinical perspective oleic and palmitic acids were chosen in this study because there are

most representative of the fatty acid content of lung surfactant phosphatidylcholine, and

also because they have been previously demonstrated in our lab to support the growth of

P. aeruginosa. Measuring and comparing the induction of the promoters of these genes

via ~-galactosidase assays should reveal insights into the mechanism of fatty acid

metabolism regulation in P. aeruginosa. Comparison of the ~-galactosidase activity of

the fatty acid degradation-related genes, fadB5 and fadE to the fatty acid biosynthetic

gene,fabA suggested an inverse relationship between the representative genes. When the

transcriptional fusions were grown in media that lack fatty acids, one would expect that

the fatty acid biosynthetic genes would be more active to synthesize required fatty acids

while the fatty acid degradative genes are repressed to prevent the degradation of the

newly synthesized fatty acids. ~ revealed from the ~-galactosidase assays, this

relationship was observed between the PjabA-lacZ fusion compared to PjadB5-1acZ and

PjadE"lacZ. The fabA promoter activity was significantly higher compared to both the

fadB5 and fadE promoters. In contrast when the strains were grown in media that

contained fatty acids the opposite relationship was observed. Both the fadB5 and fadE

promoters were significantly upregulated compared to the fabA promoter. Again this

result was anticipated since fatty acid degradative genes would be expected to be active

in the presence of exogenous fatty acid source to degrade it into carbon and energy

sources. To strengthen these results, two different fatty acid sources, oleic and palmitic

acids were used in this study and the same observations were made regardless of which

was used. The transcriptional fusion studies corroborate earlier microarray experiments

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performed in our lab; strengthening the hypothesis that fatty acid metabolism is inversely

regulated in P. aeruginosa. Equally important was the fact that these experiments

demonstrated the utility of the transcriptional fusions, which can be exploited to further

investigate the regulation of fatty acid metabolism. Since fadB5 has been the most

characterized and studied of the fatty acid genes, and has been demonstrated to be

involved in fatty acid metabolism in our laboratory, subsequent experiments relied on the

fadB5 region.

Several approaches were used to investigate the regulation of fatty acid

metabolism, in particular degradation in P. aeruginosa. The first approach used was

based on the relative conservation of regulatory mechanisms of fatty acid metabolism in

Gram-negative bacteria The regulation of fatty acid degradation is a fairly well

understood process in E. coli and served a basis for this study. Although differences were

expected, the fundamental regulatory mechanism was assumed to be similar, especially in

light of the data described above and considered an appropriate starting point of this

investigation. FadR homologues have been discovered in many bacteria closely related to

E. coli, including Salmonella enterica, Vibrio cholerae, Pasteurella multocida, and

Haemophilus injluenzae. The E. coli and S. enterica FadR proteins differ in only 7 of239

residues while other FadR homologue sequences are markedly different from E. coli and

from one another. For instance, P. multocida and H injluenzae, which despite both being

Pasteureliaceae, have FadRs that are only 54% identical, while H injluenzae FadR is

only 47% identical to E. coli FadR (58). In V. cholerae, the FadR protein unusually

contains 40 residues that are inserted into the center of the protein relative to the E. coli

protein. Despite these differences alignment of the residues critical for DNA binding by

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E. coli FadR are conserved in all known FadR proteins (58) suggesting that

bioinformatics may be useful in searching for the FadR homologue(s) in other bacteria

With the availability of the P. aeruginosa genome sequence, the application of

bioinformatics offered an appealing approach to search for a FadR-like protein.

Bioinformatics has been proven a useful tool in the discovery of a number of proteins

related to fatty acid metabolism. The application of bioinformatics has not only led to the

discovery of novel enzymes and synthetic mechanisms in Gram-positive pathogens but

also to the identification of new components of the FASII system in E. coli (133). Using

the FadR protein sequence of E. coli, a BLAST search was performed against the P.

aeruginosa PAOI genome. Several proteins with modest similarities to the E. coli FadR

were identified which included the ORFs PA1627, PA4769 and PA5356. All three

proteins have been assigned to the GntR-type family of transcriptional regulators, which

is the family to which the E. coli FadR belongs. The fact that these probable

transcriptional regulators displayed some similarities to the E. coli FadR along with the

fact that they belonged to the same family of transcriptional regulators made them

attractive candidates for further study. To assess their potential regulatory roles in fatty

acid degradation, three studies were performed. The first was to construct isogenic

mutations at the respective loci in a PjadBj-lacZ fusion background in P. aeruginosa using

the gene replacement system shown in Figure 4. Since the fadB5 promoter regions is

repressed in media that lacks fatty acid according to previous experiments, disruption of

the repressor in this background should result in an increase in PjadBj-lacZ activity that

can be observed via ~-galactosidase assays. Secondly, to isolate the fadE5 promoter

region and candidate transcriptional regulators to study any potential interaction between

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the two, an E. coli PfadB5-1acZtranscriptional fusion was constructed. ThefadB5 promoter

region was integrated into the chromosome of the E. coli strain HPSI as described in

Section 2.7.2.2. The resulting transcriptional fusion displayed functional (3-ga1actosidase

activity, which would allow the interaction between the probable transcriptional regulator

and the fadB5 region to be studied. Briefly, the reasoning was that if the probable

transcriptional regulator were expressed in the E. coli P fadBs-lacZ fusion strain and the

transcriptional regulator did indeed repressed the fadBA5 operon then a reduction in ~­

galactosidase activity should be observed. The third approach involved determining

whether the probable transcriptional regulators directly interacted with promoter region

offadB5 using electrophoresis mobility shift assays (EMSA). Here, the labeled promoter

region of fadB5 was incubated with lysate obtained from the E. coli transcriptional

fusions expressing the candidate transcriptional regulators. Interaction between PfadB5 and

the different candidates can then be assessed by determining whether the migration of

PfadB5 through an acrylamide gel is affected by the presence the respective candidate

transcriptional regulators.

Although there were some similarities between the probable transcriptional

regulators PA1627, PA4769, and PA5356 and the E. coli FadR, along with the fact they

are all a part of the same family of regulators, it appears from these studies that these

candidates are not involved in regulating the fadB5 promoter region. Studies from the P.

aeruginosa transcriptional fusions demonstrated that disruption of these candidate

regulators by isogenic mutations did not cause an increase in ~-galactosidase activity as

expected. These observations were consistent for all measurements made during growth,

which included measurements at early-log, mid-log, late-log and early-stationary phases.

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Expression of these candidate regulators in the E. coli transcriptional fusion did result in

a repression of ~-galactosidase activity; however, the repression was fairly modest and

was only observed during early and late stationary phases for PA1627 and PA5356. Gel

shift experiments provided additional evidence that PA4769, and PA5356 are not

involved in regulating fadB5. Incubation of the candidate regulators with labeled PfadB5

either did not cause a shift or did not significantly differ from control experiments.

Although the gene product ofPA5356 appeared to cause a shift in PfodB5. the interaction

was determined non-specific since the same lysate was capable of binding to and shifting

a random DNA sequence. Although the prospect that the mechanism of fatty acid

regulation is conserved across Gram-negative bacteria seemed appealing, data from these

experiments suggest that the regulatory mechanisms are much more complex and diverse

than initially assumed. Although, S. enterica and E. coli are closely related, and the f3-

oxidation systems of these two bacteria have long been thought to be essentially identical,

differences are emerging and it appears that the two systems are not functionally

equivalent (59). Differences in the ~-oxidations systems may translate into differences in

regulatory mechanisms in Gram-negative bacteria and may help explain why a simple

bioinformatic approach was useful in identifYing fatty acid regulatory proteins in P.

aeruginosa. Comparison of the E. coli and P. aeruginosa genomes allude to the potential

complexity of fatty acid metabolism in P. aeruginosa. For a number of genes involved in

f3-0xidation, there are many more fold genes involved in P. aeruginosa than in E. coli.

For instance, there are five potentialfadBAs in P. aeruginosa compared to one in E. coli,

and there are 4 potentialfadDs compared to one in E. coli. The fact that P. aeruginosa is

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such a nutritional versatile organism also suggest that a more complex regulatory network

is involved.

Despite the strength of bioinformatics, there are a number of instances when

identification of novel proteins is limited using a straightforward bioinformatic approach,

as exemplified above. The second approach utilized various protein purification schemes

and EMSA studies to attempt to identify a fatty acid metabolism regulator protein. Since

the interaction between a transcriptional regulator and its cognate operator site is

relatively strong it is reasonable to assume that the interaction can be exploited to isolate

and purify a transcriptional regulator using its cognate operator site sequence. Initial

alignment and analysis of the promoter regions of fadE5, fadE and fabA revealed a

probable consensus sequence that had comparable conservation to the FadR DNA­

binding elements found in E. coli. Briefly, concatamers of the putative consensus

sequence were generated by peR using biotinylated primers, which allowed for

subsequent attachment to streptavidin magnetic particles. Incubation of P. aeruginosa

clarified lysate with the streptavidin magnetic particles allowed for proteins that interact

with the consensus sequence to be separated from the bulk extract. The results suggest

that this technique can be potentially used as a means of DNA-binding protein

purification. Although 100% homogeneity was not attained the clarified extract was

purified to some degree. The major bands that were observed were approximately 36

kDa, slighltly larger than the E. coli FadR (26.6 kDa). Some minor protein bands of

lower molecular weights were also observed but the likelihood that these bands

correspond to transcriptional regulators is low since they are too small to contain

essential DNA-binding elements that exist in transcriptional regulators. Initia1ly, an

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galactosidase activity of fusion strain expressing P A5525 from the expression vector

pUC18 was not significantly different from the control strain containing the empty

pUC18 cloning vector. There was however, a slight difference in late stationary phase. If

the gene product of PA5525 does interact with the fadB5 promoter region, one would

expect a repression of ~-galactosidase activity throughout the phases. Since this was not

observed it appears that PA5525 does not regulate thefadBA5 operon. To conclusively

eliminate PA5525 as a potential regulator of the fadBA5 operon, EMSA studies were

performed as above. Lysate from the fusion strain expressing PA5525 was incubated with

labeled PfadB5 and a shift in the Pf adB5 fragment was observed as shown in Figure 26. This

result is not entirely surprising since the identity of P A5525 was determined by protein

purification experiments using the PfadB5 consensus sequence to isolate proteins that

recognize and bind to PfadB5. Therefore the gene product of PA5525 was expected to

interact to some extent with the fadB5 promoter region. The results from the EMSA

studies agree with the results acquired from the ~-galactosidase activity experiments by

demonstrating direct interaction between the PA5525 gene product and PfadB5. To

determine whether the interaction between PA5525 and PfadB5 is specific, PA5525 lysate

was tested against the fabA promoter region as well as a random DNA sequence

corresponding to an internal region of glpD, a gene involved in glycerol metabolism (see

Figure 27). From the results it appears that the interaction between the gene product of

PA5525 and thefadB5 promoter region is not specific since PA5525 was able to cause a

shift in both the fabAB promoter region and glpD fragments. Based on these findings it

appears that PA5525 is not involved in regulating thefadB5 operon. It does appear to

interact with the promoter region, though non-specifically.

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attempt was made to identifY the proteins present in the band by N-terminal sequencing

via Edman degradation but since the concentration of the protein was very low and there

were issues with purity, a useful sequence was not obtained. As an alternative, a

collaborative effort was made to identifY the proteins present in the bands by liquid

chromatography mass spectroscopy (LC-MS). Since a P. aeruginosa database was not

available, the software used was only able to identifY peptides that corresponded to

proteins found in the E. coli database. Attempts were made to BLAST those protein

sequences to the P. aeruginosa database to identifY potential homologues in P.

aeruginosa but no similarities were found. Rather than performing a BLAST search

against the P. aeruginosa database using the entire E. coli protein sequence, another

approach was taken. Peptide fragment sequences that were assigned high scores were

used to BLAST directly against the P. aeruginosa genome and a few transcriptional

regulators were identified. The highest scored protein that corresponded to a

transcriptional regulator was ORF PA5525, which coincidentally belonged to the GntR­

type family of transcriptional regulators. The molecular weight ofPA5525 is predicted to

be approximately 28 kDa, which is slightly smaller than the protein band that was

observed from purification. Despite this discrepancy in molecular weight, PA5525 cannot

be definitively ruled out since variations in salt concentration among other factors may

have affected its migration through the gel, therefore the potential regulatory role of

P A5525 was evaluated. Similar approaches were taken as above to assess the regulatory

role ofPA5525. The ORF PA5525 was expressed on a cloning vector in the E. coli lacZ

fusion strain HPSI-AattB::pCD13PSK-PjadB5-1acZ and (:I-galactosidase activity measured

throughout growth (Figures 28 and 29). Throughout log and early stationary phase the (:1-

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The success of the protein purification approach relied on the assumptions that i)

the binding interaction between the regulator and PjadB5 is strong and specific and ii) the

consensus sequence utilized in the purification scheme is used by the transcriptional

regulator to recognize and bind to PjadB5. Although a partially purified protein extract was

obtained from this experiment, 100% purification was not achieved. There appears to be

a significant amount of non-specific interaction between the putative P jadB5 consensus

sequence and cytoplasmic proteins in P. aeruginosa, and many types of interactions may

have contributed to this non-specificity including hydrophobic and electrostatic

interactions. To address these issues and improve the overall purification scheme, future

experiments could incorporate additional purification steps. Potential improvements to

the current purification scheme may include incorporation of ion-exchange

chromatography and 2D-gel analysis as part of the overall scheme. Including ion­

exchange chromatography as part of the purification scheme should eliminate some of the

non-specific binding proteins from the sample and improve the overall purification

efficiency. Another issue that was encountered during the course of these experiments

was the difficulty with identifying the protein following purification. Since purification

was not 100% the other proteins in the partially purified sample made it difficult to

identify the protein of interest using N-terminal sequencing. By running the partially

purified protein sample on a 2D gel, the resolution could be improved, addressing the

difficulties encountered with N-tenninal sequencing.

It was not immediately apparent but analysis of the promoter region of fadB5

around the putative consensus sequence revealed a dyad symmetry element that may be

involved in regulation. The presence of this dyad symmetry element, which spans 18 bp,

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is relatively strong and is an indication that a transcriptional regulator binds to this region

(Figure 48). The inability to purify a transcriptional regulator using the concatamerized

consensus sequence may be explained by the exclusion of this dyad symmetry element in

the concatamerized sequence. Future work that employs this method to isolate the

transcriptional regulator should include this regulatory element Additionally,

sequentially deleting regions of the promoter region of fadB5 in a lacZ reporter strain or

vector and monitoring the response to exogenous fatty acids can delineate the essential

regulatory elements. By narrowing the fatty acid responsive element, a more specific

binding sequence can be determined which would improve the specificity of the protein

purification scheme.

The last approach relied on mutagenizing the P. aeruginosa transcriptional fusion

strain PAOl-attB::P/adBs-1acZ by transposon mutagenesis. This approach is similar in

principle to the bioinformatic approach mentioned above. However, rather than selecting

potential transcriptional regulators based on sequence homology, a screen was developed

which would allow genes that affected the promoter activity of fadB5 to be identified.

Briefly, a mariner transposon was introduced into the fusion strain PAOl-attB::P/adBJ­

lacZ on the vector pBT20 and selected on media on which the fadB5 promoter region is

known to be repressed. The use of the chromogenic substrate X-gal (S-bromo-4-chloro-3-

indoyl-~-D-galactoside), which was incorporated into the growth media allowed for the

promoter activity ofthefadB5 regions to be visualized and quickly gauged. Mutants that

appeared blue are indicative derepression of the fadB5 promoter region corresponds to

potential transcriptional regulators of the fadB5 region. These mutants were selected and

the transposon inactivation sites determined by low-stringency peR and sequencing.

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PfadBA5 TCCXD'.TGXTGATTGACAATTa::ctn:111 IOCCOOAATGTGTOCG::ACCO\AGT~ATGAlWCGAtl:G1 IIGCCT

.. ill =CD:CTCGlCAATAGAGPCCCOOTTATCGCGTCGClOO:lG1 GTOCCGI\A=nTOOG>.CTATG:ITCli£I:lGII OCCG>A .. G

GGfCCCG\CACGG:GTCCGATGTGTAAGTTCAAGCTTCCATMTAOCGTcn<'G".TCAGTTGATGATTTACCMGGT'AAAOOCATCA

~MIYQGKAI

CXDTAAGCCTCTTGA,4,,4 'lI3CATCGTCGAGrTGAATTTCGlTCTCAAC!JJIX,(lAGTCCGTO&.ACAAGTTCAACCGTCTCAcx:CTC

~T V K P LEG G I VEL N F D L K G E S V N K F N R L T L

AGTGAGTTOCGTa:GC3CAGTCGATa:GATCAi'GClCCG'\TGCATCGGrCA

~SELRAAVDAI KADASV

Figure 46. Promoter region of PfadBA5 showing dyad symmetry element. Shown are the putative consensus sequenced (underlined), the transcript start site previously determined by primer extension and verified by promoter mapping program Cbolded and underlined), and the start codon of fadBA5 operon. Opposed arrows indicate an 18 bp inverted repeats.

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Surprisingly, a significant portion of the inactivations occurred within genes

involved in lipopolysaccharide (LPS) biosynthesis. Fatty acid biosynthesis is directly

involved in LPS biosynthesis and fatty acid degradation is related to fatty acid

biosynthesis through metabolism. Is it possible that there is some association between

fatty acid degradation and LPS biosynthesis?

Lipopolysaccharides (LPS) of Gram-negative bacteria are major components of

the cell wall. The hydrophobic lipid A component of LPS secures these molecules in the

outer membrane, while the core oligosaccharide links the lipid A region to the 0 antigen

or 0 polysaccharide (107). The lipid A region of LPS is thought to be responsible for the

toxicity of LPS and is composed of a phoshorylated diglucosamine moiety substituted

with fatty acids (115). When LPS is shed by bacteria into host tissues, it is usually bound

by LPS binding protein, which is transferred to the CD 14 receptor on macrophages,

thereby inducing the secretion of cytokines including tumor necrosis factor alpha (1NF­

a), interleukin-l (lL-l), IL-6, IL-8, and IL-IO (76). The fatty acid distribution, their

length, and the site of attachment strongly influence the toxicity properties of this

molecule (115). Previous studies have demonstrated that growth temperatures effect the

fatty acid composition of LPS. Cells grown at 1ST relative to those grown at 4S"C

contained increased levels of the fatty acid hexadecenoate and octadecenoate and reduced

levels of the corresponding saturated fatty acids. On the otherhand, lipid A fatty acids

showed decreases in dodecanoic and hexadecanoic acids and increases in the level of 3-

hydroxydecanoate and 2-hydroxydodecanoate when temperatures were decreased (68).

Modification of the fatty acid moiety of LPS also appears to have a role in pathogenicity.

P. aeruginosa in CF patients synthesize LPS with a variety of penta- and hexa-acylated

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lipid A structures under different environmental conditions. LPS with specific lipid A

structures are synthesized indicating unique recognition of the CF airway environment.

CF-specific lipid A molecules contajnjng palmitate and aminoarabinose were associated

with resistance to cationic antimicrobial peptides and increased inflammatory responses,

indicating that they are likely to be involved in airway disease (30). DiRusso and

Nystrom have hypothesized that FadR in E. coli interacts with other regulatory activities

to co-ordinate lipid biosynthesis with lipid turnover; it may be possible that these

regulatory activities involve LPS biosynthesis (25). It is evident from the above

observations that fatty acid plays an important role in LPS in response to the

environment. Modification in the fatty acid component of LPS appears to be related to

toxicity, temperature, and pathology. Whether these modifications are directly

coordinated by a regulatory such as FadR in P. aeruginosa or the result of some

secondary effect remains to be determined. The involvement of E. coli FadR in other

regulatory activities, however, suggests that this hypothesis is fairly reasonable. It is

tempting to suggest that there is a definitive relationship between LPS biosynthesis and

fatty acid degradation in P. aeruginosa, but clearly more experiments are needed and at

the moment any association is purely speculative.

A number of transcriptional regulators were identified by the transposon

mutagenesis approach. These included the ORFs PA2601, PAJ006 and PAJ508. The

three probable transcriptional regulators PA2601, PAJ006 and PAJ508 belong to

different classes of regulators, LysR, TetR, and IclR respectively. To evaluate the

potential relationship between these probable transcriptional regulators and the fadE5

promoter region similar experiments were performed as above. Initial assessment of these

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regulators involved comparing p-galactosidase activity of HPSI-A.attB::pCD13PSK­

P fodBS-1acZ harboring the various probable regulators. Derepression of p-galactosidase

activity was observed throughout the growth phase for both PA3006 and PA3508 while

derepression was only observed during the later stages of growth for P A2601 (Figure 35).

The fact that derepression was observed throughout the growth phase for PA3006 and

PA3508 showed promise and further experiments were performed to evaluate these ORFs.

In order to determine whether the gene product of these ORFs interact with the JadB5

promoter region, EMSA studies were performed as described above. Comparison of the

gel shifts revealed that PA3508 caused a distinct shift in the PfadB5 fragment whereas

PA3006 banding pattern appeared similar to the vector only control. For confirmation,

gel shift experiments were also carried out on P A2601 and as expected the lysate was

unable to cause a shift in PfadB5 fragment The fact that PA3508 was able to shift the PfadB5

promoter fragment along with the fact that a discrete shift band was observed was

encouraging therefore additional studies was performed on this transcriptional regulator

candidate.

Along with the experimental data, which supported PA3508 as potential regulator

oftheJadB5 operon, was the observation that PA3508lies in close proximity to genes are

related to fatty acid metabolism. The organization of PA3508 and adjacent genes are

shown in Figure 39. Since FadR in E. coli has the ability to regulate both fatty acid

degradative and biosynthetic genes, it would be interesting to see ifPA3508 was capable

of interacting with the PfaM promoter region. To determine whether this was the case, gel

shifts were performed across a gradient ofPA3508lysate concentration on both PfadB5 and

PfahA fragments (Figures 40 and 41). The results from the gradient gel shift experiments

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revealed that the gene product of P A3508 was capable of interacting with the promoter

region ofbothfadB5 andfabAB. At higher concentrations the lysate was able to shift the

P JodJJj and P fabA fragments while reducing the concentration caused the shifted band to

disappear and migrate normally. Thus the results from these experiments suggest that

P A3508 is a common regulator of the fad and fab pathways in P. aeruginosa behaving

similarly to the FadR protein in E. coli. In order to definitively conclude that PA3508 is

the transcriptional regulator of the fadBA5 operon, a HiS6-tagged purified PA3508 was

expressed and tested for its ability to shift the promoter regions of fadB5 and fabAB.

Similar to the results obtained with the E. coli lysate, the HiS6-tagged purified PA3508

was able to cause a shift in bothfadB5 andfabAB promoter regions (Figure 44). This is

strong evidence that PA3508 encodes a transcriptional regulator that interacts with PJadB5

and PJabA. To test the specificity of this interaction, the His6-tagged purified PA3508

incubated with a random sequence that corresponds to the internal region of the glpD

gene. The results (see Figure 45) reveal that the gene product of P A3508 is capable of

interacting with the glpD fragment as well and it appears that PA3508 does not

specifically interact with PJadB5 or PJabA.

Based on these results it appears that PA1627, PA4769, PA5356, PA5525,

PA2601, PA3006 and PA3508 are not involved in regulatingfadB5. Although the gene

products of some of these ORFs were capable of interacting with the promoter region of

fadB5, the interaction was non-specific. The gene products ofPA1627, PA4769, PA5356,

and PA5525, which represent the GntR-type regulators that are most similar to the E. coli

FadR did not appear to regulate the fadBA5 operon. These observations suggest that the

regulator offadBA5 operon is most likely not a part of this family and may use a slightly

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different mechanism of regulation. The results from the transposon mutagenesis

experiments did identify some potential regulators of the fadBA5 operon including

PA2601, PA3006, and PA3508. It was determined from the transcriptional regulation

studies that PA3006 behaved in a manner most expected of afadBA5 operon regulator.

Transposon mutagenesis of this gene caused an upregulation of PfadBs-lacZ in P.

aeruginosa while expression in an E. coli reporter system led to repression of the P fadBS­

lacZ fusion. However, direct DNA-binding assays, revealed that the gene product was

unable to bind to the PfadBs. The ORF PA3006 has recently been designated a PsrA

regulator, which is associated with rpoS and may be a part of the global regulatory

network. If this is the case, this may explain the observation that the gene product was

unable to directly bind to PfadBS in the EMSA studies and unable to cause a complete

repression in the E. coli reporter system since it may need a cofactor to function

optimally. Although inactivation of PA3508 did not cause an upregulation infadBA5 as

expected, expression of the gene product in the E. coli reporter system did cause

repression and the gene product was capable of interacting with PfadB5 as demonstrated in

the EMSA studies. The inability to cause a de-repression of the fadBA5 operon in the P.

aeruginosa transcriptional fusion strain was puzzling but based on the results from the

other experiment, PA3508 seemed like the most promising candidate and therefore

investigated further. However, additional DNA-binding studies using purified PA3508

revealed that the gene product does not interact specifically with PfadB5.

Although the experiments performed in this thesis did not identify the regulator of

the fadBA5 operon, it gave insights into the regulation mechanism of fatty acid

degradation in P. aeruginosa. It is hoped that future studies may build upon the collective

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data gathered in these experiments and elucidate the regulation of fatty acid metabolism.

Some potentially promising approaches include expression of a PAOI chromosomal

library in the E. coli PjadBS'lacZ transcriptional fusion developed in this thesis, refining

the regulatory element in PjadBJ by mutations or deletion analysis and potentially using

the refined sequence to extract the regulator as described in this thesis. Identification of

additional genes that are a part of the fad-regulon will allow a more accurale consensus

sequence to be developed and potentially generate other avenues to identifY the regulator.

Until then the regulation of fatty acid degradation in P. aeruginosa will remain a mystery.

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Appendix I

Introduction

During the course of experiments carried out as part of this thesis, genetic tools

that facilitated the study of the regulation of fatty acid metabolism in P. aeruginosa were

developed. One such tool was the development of simple method for the construction of

targeted transcriptional fusion to lacZ using Flp-mediated site-specific recombination

adapted from a previous method applied in Salmonella typhimurium (28). Briefly,

suicidal vectors containing promoterless lacZ genes and the Flp recognition target (FR1)

site in both orientatious were constructed to create transcriptional fusions. These vectors

can be transformed into strains containing a single FRT site created downstream of the

promoter of interest by Flp-mediated site-specific recombination. The Flp protein

supplied by a conditionally replicating plasmid promotes site-specific recombination

between the FRT sites, creating an integrated lacZ fusion to the gene of interest (Figure

47). Although this project was not the main focus of this thesis, it has direct application in

the study of regulation of fatty acid metabolism as well as other processes and may be

applied in future studies.

Methods

Construction of FRT-lacZ integration vectors involved the following steps.

Plasmid pTZ120, which supplies the lacZ gene, was digested with SapI and NarI, then

blunt-ended using T4 DNA polymerase. The digestion product was self-ligated, then

transformed into E. coli strain DH5a and selected on LB + Ap media Positive clones

were digested with BamHI and ligated to the Om-cassette obtained from pPS856

139

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Gm"

c

pXR6KAJlspHI-BspMI-FRT-lacZ

tHiROK

FRT /acZ --x

III •• " '11111' --FRT

+ Ap (pCD13-onT-Ap)

1 __ ,lac.?:>- ..Q. , tHiROK " .A Gm ~~::)I-----IIID'~'~IIQ~ -­FRT FRT

Figure 47. Schematic representation of FRT-lacZ integration. The vector pXR6KMspID-BspMI-FRT-lacZvector is introduced along with pCD13-oriT-Flp into a host containing an FRT-site inserted within the gene of interest. Flp-mediated recombination integrates the vector at the FRT-site creating the lacZ-transcriptional fusion. Corresponding FRT orientations lead to functional fusions.

140

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digested with the same enzyme. The ligation mixture was then transformed into DH5a

and selected on LB + Gm. Due to non-directional cloning, the Gm-cassette was inserted

in one of two different orientations. Clones corresponding to the different Gm-cassette

orientations were designated pDTNI00Gm-FRTl and pDTNI00Gm-FRT2. The different

constructs were then transformed into DH5a-.wttB::pCD13SK-Flp to remove the Gm­

cassette via Flp-mediated excision and selected on LB + Ap. Colonies were then patched

onto LB + Ap and LB + Gm media and ApRGms clones were designated as pDTNIOO­

FRTJ and pDTNI 00-FRT2. To replace the bla-colEl region, the FRT -lacZ fragment was

sub-cloned into the pXR6K vector, which carries GmR-oriR6K. This involved the

following steps: The pXR6K vector was digested with BspHl and BspMI, blunt-ended

with T4 DNA polymerase then self-ligated and transformed into DH5a-pir 116.

Transformants were selected on LB + Gm. The resulting construct was then digested with

XmnI and PvuIl, which allowed for the subsequent sub-cloning of FRTl-lacZ and FRT2-

lacZfragments from pDTNI00-FRTl and pDTNI00-FRT2 respectively. The FRT-lacZ

fragments were digested with AjlII and XhoI then blunt-ended and ligated to

pXR6KABspHl-BspMIMmnI-PvuIl. Ligation mixture was transformed into DH5a­

pirl16 and selected on LB + Gm + X-Gal. Blue colonies were verified by digestion and

designated as pXR6KABspHl-BspMI-FRTl-lacZ or pXR6K11BspHl-BspMI-FRT2-lacZ.

The constructs were then transformed into the mobilizable E. coli strain ER2566mob-

pir 116 and selected on LB + Gm. Construction of the FRT-IacZ integrated fusion

involves conjugation by triparental mating. Essentially 500 jJl of each: the recipient,

which contains a previously inserted FRT site, the FRT-lacZ integration vector donor

strain (pXR6K11BspHl-BspMI-FRT-lacZl ER2566mob-pir 116) and the helper plasmid

141

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strain (pCD 13-oriT-Flp/ ER2566mob-pir 116) are combined and selected on PIA + Om

media (Figure 47). Successful integration in the correct orientation can then confirmed

by PCR using primers that anneal to the lacZ gene or Gm-cassette in combination with a

primer that anneals to the gene of interest.

142

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