ISOLATION AND CHARACTERIZATION OF mer MERCURY … and characterization... · menabjukkan terhadap persekitaran tercemar. Operon rintang merkuri (mer operon) adalah sistem biologi

ISOLATION AND CHARACTERIZATION OF mer GENE FROM MERCURY-RESISTANT BACTERIA ISOLATED FROM

POLLUTED SOIL

Tan Boon Khai

Bachelor of Science with HonoursQR 201 (Resource Biotechnology) A6 2009 T161 2009

Puut IQfdmat Maldut Akademnmiddot UNlVEKSm MALAYSIA SARAWAK

ISOLATION AND CHARACTERIZATION OF mer

GENE FROM MERCURY-RESISTANT BACTERIA

ISOLATED FROM POLLUTED SOIL

TAN BOON KHAI

This report is submitted in partial fulfillment of requirements for the degree

of Bachelor of Science with Honours (Resource Biotechnology)

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARA W AK

2009

ACKNOWLEDGEMENT

Foremost I would like record my utmost sincere gratitude towards my supervisor Dr Awang

Ahmad Sallehin Awang Husaini for providing me the opportunity to carry out this project in

Genetic Molecular Lab (GML) I would like to thank for all his constructive comments

encouragement and understanding which serve him as a good host despite his warm and

humorous personality

Special thanks to Frazer Midot Nur Hafizah George Deng Kolly Lee the postshy

graduates from GML I am indebted to you all for your willingness to share your expertise

experiences and advice all the time You all have been being the ultimate lab-neighbours

providing a great research environment And hereby I wish all the best to you all and I know

a note ofThank you doesnt seem sufficient but it is said with appreciation and truly respect

To all the members in GML thanks again for this too short but really wonderful time spending

together I am glad to bear chance to get to knowing you all

During this project I have collaborated with other lab members for whom I have great

regard and I wish to extend my warmest thanks to all those who have helped given valuable

advice and sacrificed their precious time to guide me through

I would like to gratefully acknowledge the support of this special person Mr Kuah

Meng Kiat who has been actively and always been available to advise me giving me his

untiring help His wide knowledge and logical way of thinking have been of great value for

11

me I believe that his personal guidance for me through this project will leave a remarkable

influence on my future work for the time to come

I have not travelled in a vacuum in this journey There are some people who made this

journey easier with words of encouragement and moral supports thanks for being there for me

and I am truly lucky to have you all Needless to say I would like to dedicate my love and

thanks to my beloved family

III

middot If JChfdmlt Mlkluf 4kd8 UNJVERSrrf MALAYSIA SARAWAK

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENT 11

LIST OF TABLES Vlll

ABSTRACT

A BSTRAK

TABLE OF CONTENTS IV

LIST OF FIGURES IX

LIST OF ABBREVIA nONS XI

10 INTRODUCTION 2

20 LITERATURE REVIEW

21 Mercury 5

22 Toxicity of Mercury 5

23 The mer Operon 7

24 merA 13

25 Application ofmer Operon and Future Prospect 14

I

IV

30 MATERIALS AND METHODS

31 Mercury-resistant Bacteria Strains 17

32 Working Culture

321 LB Agar Plate 17

322 LB Agar Broth 17

33 Preservation of Bacteria Culture 18

34 Isolation of Bacteria Total DNA and Plasmid DNA 18

35 Polymerase Chain Reaction (PCR) Amplification of Putative merA Gene 19

36 DNA Manipulation 20

361 Cloning of Putative merA Amplicon 20

362 Competent Cell 20

363 Heat Shock Transformation 21

364 BluelWhite Colony Screening 22

365 Colony-PCR 22

37 Sequencing ofDNA 22

38 Development ofMercury-resistant Bacteria Pure Culture 23

39 Preliminary Characterization of Isolated MRB 24

391 Colony Morphology Characterization 24

392 Gram Staining 24

393 Biochemical Test 25

3931 Methyl Red Voges-Proskauer (MRVP) Test 25

253932 Citrate Utilization Test

3933 Motility Test 26

310 16S rDNA Sequencing 26

v

j

40 RESULTS

41 Mercury-resistant Bacteria Culture 28

42 Polymerase Chain Reaction (PCR) Amplification of Putative merA Gene 29

43 BluelWhite Colony Screening 31

44 Colony-PCR 32

45 Sequencing Result of Putative merA Gene 34

46 Isolation of Mercury-Resistant Bacteria from Polluted Soils Sampled at Miri 37

47 Preliminary Characterization of Isolated MRB 37

471 Colony Morphology Characterization 37

48 Biochemical Test 39

49 16S rDNA Sequencing 39

50 DISCUSSION

51 Mercury-resistant Bacteria Culture 46

52 Genomic and Plasmid DNA Extraction 46

53 Primer Pair ofmerAlImerA5 47

54 Polymerase Chain Reaction (PCR) Amplification ofPutative merA Gene 47

55 Sequencing Result ofPutative merA Gene 49

56 Isolation ofMercury-Resistant Bacteria from Polluted Soils Sampled at Miri 53

57 16S rDNA Sequencing 53

60 CONCLUSION AND RECOMMENDA nON 55

VI

I

REFERENCES 57

APPENDICES 66

Vll

LIST OF TABLES

Table Descriptions Page

Table 1 Primer description of merAl and merA5 19

Table 2 Primer description ofpA and pH 27

Table 3 Isolates ofmercury-resistant bacteria sampled from polluted soil at Miri with the characteristic of colony morphologies 38

Table 4 Biochemical test result ofIsolate 1 Isolate 3 and Isolate 4 39

TableS Identities of isolates after 16S rONA sequencing 45

viii

LIST OF FIGURES

Figure Descriptions Page

Figure 1 Diversity ofmer operons Sequenced mer operons from Gram-positive and Gram-negative bacteria 10

Figure 1 Model of a typical Gram-negative mercury resistance (mer) operon 11

Figure 3 Bacterial colonies ofKlebsiella pnellmoniae formed on LB agar containing 10 ppm HgCh 28

Figure 4 PCR product ofputative positive merA gene from Klebsiella pneumoniae genomic and plasmid DNA 29

Figure 5 Purified PCR product ofputative positive merA gene from Klebsiella pnellmoniae genomic DNA 30

Figure 6 Purified PCR product ofputative positive merA gene from Klebsiella pneumoniae plasmid DNA 31

Figure 7 Bluewhite screening onto the transformed E coli XL I Blu cells with pGEMT -Easy vector with inserts 31

Figure 8 Colony-PCR onto the transformed E coli XLI Blue cells containing pGEMT -Easy vector with inserts 32

Figure 9 Plasmid Mini-preps from the transformed E coli XL I Blue cells containing pGEMT -Easy vector with inserts 33

Figure 10 BLASTN nucleotide search result of partially sequenced clone fragment amplified from Klebsiella pnellmoniae genomic DNA 35

Figure 11 BLASTN nucleotide search result of partially sequenced clone fragment amplified from Klebsiella pneumoniae plasmid DNA 36

Figure 12 LB agar (supplemented 10 ppm HgCh) with dilution factor 10-2 harbouring the MRB isolated from polluted soil sampled at Miri 37

IX

I

Figure Descriptions Page

Figure 13 Bacterial colonies formed on LB agar containing 10 ppm HgCh for pure cultures of mercury-resistant bacteria 38

Figure 14 16S rDNA PCR product amplified from Isolate I Isolate 3 and Isolate 4 40

Figure 15 Purified 16S rDNA PCR product from Isolate I Isolate 3 and Isolate 4 41

Figure 16 BLASTN nucleotide search result of partially sequenced 16S rDNA amplified fragment oflsolate 1 42

Figure 17 BLASTN nucleotide search result ofpartially sequenced 16S rDNA amplified fragment oflsolate 3 43

Figure 18 BLASTN nucleotide search result ofpartially sequenced 16S rDNA amplified fragment oflsolate 4 44

x

A

C

C

dNTPs

G

H20

H2S

Hg+

HgO

Hg2+

HgCh

IPTG

L

LB

mg

MgCh

min(s)

mL

MRB

NADH

NADPH

NB

LIST OF ABBREVIATIONS

Adenosine (DNA base)

Carbon

Cytosine (DNA base)

deoxynucleoside-5 -triphosphates

Guanosine (DNA base)

Water

Hydrogen sulfide

Mercurous mercury

Elemental mercury

Mercuricionic mercury

Mercury (II) Chloride

Isopropyl-(3-D-thioglactopyranoside

Liter

Luria Bertani

Milligram

Magnesium (II) Chloride

minute(s)

Milliliter

Mercury-resistant Bacteria

Nicotinamide adenine dinucleotide

Nicotinamide adenine dinucleotide phosphate

Nutrient Broth

Xl

OC Degree Celcius

PBS Phosphate-buffered Saline

PCR Polymerase Chain Reaction

ppm Parts per Million

RT Room temperature

sec(s) Second(s)

SH Thiol

T Tyrosine (DNA base)

UV Ultra Violet

V Volts

vv Volume over volume

X-gal 5-bromo-4-chloro-3-indoly-(j-D-galactoside

xu

Isolation and Characterization of mer Gene from Mercury-Resistant Bacteria Isolated from Polluted Soil

Tan Boon Khai

Resource Biotechnology Programme Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT The anthropogenic activities have subsequently caused the arisen levels of mercury in the environment Mercury pollution has caused severe problem to human due to its toxicity It was discovered that the prokaryotes have developed the astonishing arrays of resistance system to defend against the polluted environments Mercury resistance operon (mer operon) is one of the best understood biological systems to date for detoxifying organometallic or inorganic compounds where the mercury reductase enzyme that encoded by merA gene mediated the reduction of highly toxic ionic mercury Hg2+ to relatively less toxic and volatile elemental mercury Hgo Through the study of mer operon it can be utilized for bioremediation purpose global recycling of mercury and also allow the study of horizontal gene transfer in the natural population In this study Klebsiella pneumoniae which had been isolated from Sungai Bera Brunei Darussalam was screened for presence of merA gene using Polymerase Chain Reaction (PCR) The putative merA gene was cloned and sequenced -- where the nucleotide sequence was revealed to show 77 similarity to the polysaccharide deacetylase domain protein of Klebsiella pneumoniae Three additional mercury-resistant bacteria were successfully isolated from polluted soil sampled at Miri The three isolates were subjected to 16S rONA sequencing and successfully identified as Bacillus pumilus Bacillus thuringiensis and Bacillus aquimaris respectively

Key words mercury-resistant bacteria Klebsiella pnetmon iae merA gene mercury Bacillus

ABSTRAK Aktiviti-aktiviti antropogenik telah menyebabkan peningkatan paras merkuri dalam alam persekitaran Pencemaran merkuri telah menyebabkan masalah rumit terhadap mantsia akibat ketoksikannya Penemuan lelah dijllmpai bahawa prokariot telah memperkembangkan sistem pertahanan yang menabjukkan terhadap persekitaran tercemar Operon rintang merkuri (mer operon) adalah sistem biologi yang paling difahami sampai kini dalam penyahtoksikan sebatian merkllri di mana enzim merkuri reduktase yang dikodkan oleh gen merA terlibat dalam tindakbalas menllrunkan ion merkllri Hi+ yang amat toksik kepada unsllr merkuri Hl yang kurang toksik dan bersifat mentap Melalui kajian mer operon ia boleh digunakan untuk tujuan bioremediasi kitar semula merkuri dan juga membenarkan kajian pemindahan gen secara mengufuk dalam populasi Dalam kajian ini kehadiran gen merA dalam Klebsiella pneumoniae yand dipencilkan dari sampel tanah Sungai Bera Bnmei Darussalam telah dikaji dengan Tindakbalas Polimerasi Berantai (peR) Gen merA secara putatif lelah diklon dan dijujukkan - di mana jujukan nllkleotida telah menunjllkkan persamaan sebanyak 77 dengan domain protein polisakarida deacetilase dalam Klebsiella pneumoniae Selain illl tiga jenis bakteria tahan merkurijllga telah berjaya dipencilkan dari sampel tanah tercemar di Miri Ketiga-tiga pendlan bakteria tersebut telah tertakluk kepada penjujllkan 16S rDNA dan identitinya telah dikenalpastikan sebagai Bacillus pumilus Bacillus thuringiensis and Bacillus aqllimaris secara

ing-masing

Kat nei bakteria tahan merkuri Klebsiella onellmoniae gen merA merkuri Bacillus

CHAPTER 10

INTRODUCTION

enury is present in the environment as a result of natural processes and from anthropogenic

aources (Nascimento and Chartone-Souza 2003) The introduction of metallic mercury into

Ibe environment is one of the major aggressions against man and environment (Nascimento

and Chartone-Souza 2003) due to its toxicity For example Minamata disease which was

discovered in 1956 around Minamata Bay Japan is the first instance on record of severe

methylmercury poisoning having affected thousands of people 887 of whom were killed

(Daher 1999)

It was discovered that the prokaryotes have developed the astonishing arrays of

resistance system to defense against the polluted environments (Huang et at 1999) Mercury

resistance operon (mer operon) is one of the best understood biological systems to date for

detoxifying organometallic or inorganic compounds (Nascimento and Chartone-Souza 2003)

The reduction of highly toxic ionic mercury Hg2+ to relatively less toxic and volatile

elemental mercury HgO is mediated by mercury reductase enzyme which is encoded by merA

gene in the mer operon (Jaysankar 2004 Barkay and Wagner-Dobler 2005 Chadhain et at

2006) Through the study of mer operon it can be utilized for bioremediation purpose global

ncycling of mercury and also allow the study of horizontal gene transfer in the natural

population The use of bacteria in remediating the polluted environment is a promising

2

technology (Nascimento and Chartone-Souza 2003) as they are at a lower cost and higher

efficiency and could be promoted to pilot scale operation in future (Barkay et al 2003)

In this study the mercury resistant bacteria (MRB) Klebsiella pneumoniae which had

been successfully isolated from the polluted soil sampled at outflow of Seria crude oil tenninal

plant Sungai Bera Bnmei Darussalam in the previously done work will be screened for the

presence of merA gene using Polymerase Chain Reaction (PCR) amplification using the

sequence specific merAlImerA5 primer pair The putative merA gene amplicons will be

cloned and sequenced The sequence will be used as query to compare to known sequences in

NCBI database (httpwwwncbinlmnihgov) using BLASTN nucleotide search There will

be the attempts onto the isolation of mercury resistant bacteria from the polluted soils sampled

at Miri as well Despite the Gram staining biochemical testing and microscopic examination

the 16S rDNA sequencing method will be conducted to identity the MRB isolates at molecular

level

The Research Rationale

Is the presence ofme rA gene detected in the mercury-resistant bacteria that had been isolated

Hypothesis

Bacteria that able to colonize in polluted soil (such as oil sludge) should have possessed the

mercury resistance ability which is rendered by mercury reductase which is encoded by mer

gene Thus it is possible to isolate and characterize the merA gene from the isolated mercuryshy

tant bacteria

3

1_W4~b Objectives

To identify the mercury-resistant bacteria morphologically and molecularly

To isolate and characterize merA gene from the mercury-resistant bacteria

4

t mt MJklrma Akaemf~ UNlVERSm MALAYSIA SARAWAK

CHAPTER 20

LITERATURE REVIEW

21Mercury

Mercury its chemical symbol Hg derived from the word Hydragyrum which means liquid

silver or quick silver in Greek (Tekranreg Instrument Corporation 2006) Mercury can exist

as a metallic liquid or vapor (Summers 1986) and exist as liquid at room temperature

(Michael 2005) The Elemental form of mercury Hgo has high vapor pressure (Henrys

constant of 03 very low aqueous solubility (6 fJg per 100 mL of water at 25degC) and it is

volatile at a Iiquidair interface but may coalesce into a liquid in a closed system (Barkay et al

2003) Due to its unique properties mercury use is widespread particularly in the production

of gold vaccines antimicrobials amalgams in dentistry and electronics (Schelert et al 2004)

As a consequent of anthropogenic activities the release of mercury into the air water and on

the land leads to environment pollution and it is an increasing problem both for developing

and developed countries (Nascimento and Chartone-Souza 2003)

11 Toxicity of Mercury

Mercury the 6th most toxic in a universe of 6 million substances exists naturally in small

amounts in the environment being the 16th most rare element on Earth (Nascimento and

Chartone-Souza 2003) Mercury can exist in three oxidation states namely elemental mercury

0 mercurous mercury Hg+ or mercuric mercury Hg2+ The latter two can combine with

5

other elements to fonn either organic or inorganic mercury compounds (Hu 1998) Among

those the organic mercury compound in fonn of methylmercury (palmer 2001) is the most

toxic (Goldwater amp Clarkson 1972 Hu 1998)

Mercury has no known biological function (Wagner-Dobler 2003) and it has very high

affinity to thiol (SH) groups in proteins (Glendinning and Brown 2005) Mercury toxicity is

mainly due to the fonnation of covalent bonds of both ionic and organic mercury with sulfur

atoms in cysteine residues of target proteins (Sche1ert et ai 2004) causing disruption of metal

thiolate bonds ofproteins and alters the protein structure the change in redox status of the cell

and the interference with essential metal uptake (Sigaud-Kutner et al 2003)

Organic mercury is able to reach the Central Nervous System (CNS) where it is

oxidized to Hg2+ and leads to neurological damage (Taylor amp Francis 1995) Several years

ago there has been some concern that mercury contained in dental amalgams adversely affects

human health produces illnesses including multiple sclerosis and Alzheimers disease but this

conjecture has not been conclusively proven (Hu 1998 Baldwin amp Marshall 1999) Mercury

bull also genotoxic the inorganic mercury is capable of strong reversible interactions with the

nitrogen in purines and pyrimidines while the organic mercury compounds such as

methylmercury can cause irreversible damage to nucleic acids (Sletten and Nerdal 1997)

ercury poisonings have been reported from ingestion of mercuric chloride (an inorganic

compound which is used as a disinfectant) and also from contaminated illegal drugs such as

amphetamines or from the exposure to fungicides containing organic mercury compounds and

industrial accidents in which mercury vapour was inhaled (Jaysankar 2004)

6

Mercury is primarily deposited in the environment as ionic mercury Hg2+ and it may

to the neurotoxic substance methylmercury Following methylmercury

blOlICClllDl~lallon and biomagnifications in food chains it poses a risk to consumers at the

upper trophic levels (Barkay et ai 2003 Barkay and Wagner-Dobler 2005) for having higher

mercury concentrations accumulated within bodies (Nascimento and Chartone-Souza 2003)

Minamata disease was discovered in 1956 around Minamata Bay Japan It is the first instance

on record ofsevere methylmercury poisoning affected thousands ofpeople 887 of whom were

killed (Daher 1999) due the consumption mainly by fishermen and their families of large

amounts of fish and shellfish which had been contaminated with methylmercury and the

methylmercury was resulted resulting from the transformation of the HgCb discharged from a

chemical plant (Nascimento and Chartone-Souza 2003)

23 The mer Operon

Mercury resistance to inorganic and orgamc mercury compounds was first reported in

SlIlphylococcus aureus by Moore (1960) and this mercury resistance mediated by the

microbial mer operon was discovered in the early 1970s (Summers amp Lewis 1973)

Detoxification of mercury by enzymatic reduction was proposed more than three decades ago

(Summers amp Silver 1972) and it has been realized later that the mer operon which confers

both resistance and detoxification capabilities to its possessor is almost universally distributed

in resistant bacteria populations (Okino et al 2002 Barkay et al 2003 Jaysankar 2004) and

the mer operon is fairly highly conselVed (Jaysankar et al 2008) The reported genera to

posses resistance to mercury are Acinetobacter Aeromonas Alcaligenes Azotobacter

Bacteriodes Be ijerinckia Chromobacterium Citrobacter Clostridium

sporium Deinococcus Desulfovibrio Enterobacter Escherichia Erwinia

7

Klebsiella Micrococcus Moraxella Morganella Mycobacterium

Planococcus Proteus Rhodococcus Staphylococcus Streptococcus

_iPlOntyceS Xanthomonas Hyphomonas Thiobacillus Vibrio and Yersinia (Robinson and

~lVi1lIflll 1984 Baldi et at 1989 Osborn et al 1997 Nascimento and Chartone-Souza

Bacteria may respond to mercury exposure usmg several strategies however

fllUmllllism involving enzymatic reduction of mercuric ion Hg2+ to elemental mercury HgO

11U1ialYzed by products of the mer operon is the only resistance mechanism that has been

damb4~ (Schelert et ai 2004)

The operons designated mer operons consist of a cluster of linked genes in an operon

most known naturally occurring systems (Silver amp Phung 1996 Barkay et aI 2003) Most

operons contain at least the mercury-resistance genes merR merD merT merP and merA

1oi1lVII amp Phung 1996 Osborn et ai 1997)

The mer resistance components can be sub-grouped into three categories based on the

constituted that encodes for the functional protein ie merR (regulators of operon

_lSicln) merA (ezymatical converters of toxic mercuric compounds Hg2+ into a relatively

IOlll-olOXIC fonn HgO and merT with merP (transporters of Hg2+ into the cells) (Misra 1992

1993 Silver and Phung 1996 Osborn et al 1997 Jaysankar 2004)

In some cases regarded as broad-spectrum resistance by which the bacteria exhibits

to both inorganic and organic mercunc compounds despite of merA gene

8

lIkllIticaal merB genes

2003 Felske et al 2003)

~-

are required to degrade organomercurials such as phenylmercuric

I_tate (PMA) by cleaving the C-Hg bond before Hg2+ reduction by mercuric reductase

eacOCScxl by merA (Osborn et al 1997 Huang et al 1999) For the resistance only to

iIDCIlrganlc mercuric compounds is called as narrow spectrum resistance which only involves

that encodes for mercury reductase to reduce the toxic reactive ionic mercury Hg2+

to volatile relatively inert and relatively less toxic elemental form HgO vapor (Barkay et

Mercury-resistance determinants have been found in a wide range of Gram-negative

Gram-positive bacteria isolated from different environments (Nascimento and Chartoneshy

2(03) and it usually located on plasmids (Summers and Silver 1972 Brown et al

86 Griffin et aI 1987 Radstrom et al 1994) and chromosomes (Wang et al 1987 Inoue

al 1991) and are often components of transposons (Misra et al 1984 Kholodii et al

993) and integrons (Liebert et al 1999) Further suggestion is that transposable element may

involved in the horizontal dissemination of mer operons among Gram-positive bacteria

Bacterial mer operons are not all the same it may vary in the number of genes as well

their nature and organization (lohara et al 2001) Interesting findings also pointed out the

IlelatllgClllCIt) of the mer operon that (i) merB is more common in Gram-positive mer operons

~UILl~ to date than in Gram-negative operons (ii) merR in low-GC Gram-positive operons

transcribed in the same direction as the rest of the operons genes but in the high-GC Gramshy

_n Streptomyces operons and all Gram-negative operons merR is transcribed divergently

9

------------------------------------------------------------

_~rnkl~

merR mer( merT merP maC merE merA mere maD

tiIllDlmiddotmiddotne2atlve bacteria

the structural genes while the Gram-negative manne bacterium Pseudoalteromonas

is the exception with merR cotranscribed with merTPCAD (Barkay et al 2003)

Bacillus cereus Clostridium r11I1-11I7gtI

Staphylococcus aureus pl258

Streptomyces ividans

Streptomyces pRJ28

I I Exiguobacterium sp

1- Pseudomonas sp ED-23

Pseudomonas stutzeri OX pPB

_ ~ Pseudomonas sp K62 pMR26

Serratia marccens pDU 1358

Pseudomonas aeruginosa Tn50 1

Shigellaflexneri Tn21

Alcaligenes pMER610

-- t-- Pseudomonas sp ADP

Xanthomonas campestris Tn5044~--Imiddot=- Xanthomonas sp Tn5053

Pseudomonas fluorescens

Shewanella plltrejaciens pMERPH

Thiobacillus jerrooxidans

Pseudoalteromonas I Diversity ofmer operons Sequenced mer operons from Gram-positive (above line) and Gram-negative line) bacteria Arrows indicate the direction of translation of each gene product Colorless arrows indicate

with unknown functions Several variations on the structure and organization of known mer operons reflect mosaic nature of the operon (Barkay et al 2003)

Typically mer operons of Gram-negative bacteria are organized in the gene order

merT merP(then sometimes merC) merA and merD as in transposon Tn21 (lohara et

2(01) According to Nascimento and Chartone-Souza (2003) merB seldom occurs in

10

HgX2 HgO

J Merp

Periplasm

Cytosol

N-termlnus MerD

t---------______~a~c~tI~vation antagonist

~

2 Model of a typical Gram-negative mercury resistance (mer) operon The symbolmiddot indicates a cysteine X refers to a generic solvent nuceophile RSH is the low-molecular-mass cytosolic thiol redox buffer

as glutalhione Parentheses around gene or protein designations indicate proteinsgenes that do not occur in examples of the operon (Barkay et ai 2003)

reference to Figure 2 (Barkay et at 2003 page 7)

11