Prokaryotic promoters in the test tube and in the environment Víctor de Lorenzo Centro Nacional de Biotecnología CSIC Pseudomonas putida.

Prokaryotic promoters in the test tube and in the environment

Víctor de LorenzoCentro Nacional de Biotecnología CSIC

Pseudomonas putida

Enrichment cultures

Strain (Plasmid) Pollutant

P. putida mt-2 (pWW0) ToluenePseudomonas sp, CF600 (pVI150) PhenolBurkholderia sp. RP007 PhenantreneP. putida TMB Trimethyl benzeneP. putida NAH (NAH7) NaphthaleneAcinetobacter sp. ADP1 Aryl estersR. eutropha (pJP4) 2,4 DPseudomonas sp. (pP51) TrichlorobenzeneP. putida UCC22 (pTDN1) AnilineP. oleovorans (pOCT) n-alkanesBurkholderia cepacia LB400 Biphenyl, PCBsPseudomonas sp. VLB120 Styrene

Evolution of biodegradative capabilities

1. Genes encoding catabolic enzymes

2. Regulatory systems responding to substrates

3. Coupling transcription to Physiological signals

Expre

ssio

n o

f ca

tabio

lic g

enes

Growth

inducer addition

1

2

3 4

+ C

Stages in the regulation of gene expression systems

Gain / capacity

Specificity

Metabolic coupling

Window of activity

Evolutionary gravitation towards an optimal regulation

And now a reminder on promoters and gene

expression in bacteria...

Gene expression is a multi-step process

Transcription

mRNA stability

Translation

Overview of transcription and translation

Transcription is in itself a multi-step process

Stem-loop structure of some terminators

Bacterial genes are frequently clustered in operons

What is a promoter?

Structure of rifampin

Positive and negative regulation

Regulation of transcription by two-component regulatory systems

Regulation of transcription by quorum-sensing systems

Structures of some

autoinducers in quorum-sensing

systems

Sigma 54

ECF FamilyEc rpoEEc rpoHEc fecI

Sigma 70-likeEc rpoDEc rpoSEc rpoF

Sigma 70 family

A class in its own!

factors determine promoter specificity

Sigma 54 promoters

• Require an activator that binds far from +1

• -12/-24 consensus sequence

GG-10-GC

• Small compared to -10/-35• GC rich (frequent in Pseudomonas)

The -12/-24 site

Reporter lacZ gene fusions

Accumulation of beta-galactosidase

Monitoring promoter activity… once at a time

The big leap

forward: from

genetics to

genomics

Computer-assistedsearch for genes and promoters

Probing global gene expression with Gene arrays

Differential gene Expression (DGE) reveals signals and promoters

Condition A

Condition B

0

50

100

150

200

250

300

350

400

450

mycge

mycpn

ureur

ctra

cpneu

buch

bbur

rpxx

tpal

hpyl

cjej

aquae

nmen

mlep

tmar

synecho

xfas

hinf

dra1

pmul

spyo

staphyN

llact

ccre

mtub

vcho

bhal

bsub

ecoli

paer

meslo

Number of Regulators

R2 = 0,9668

R2 = 0,8842

0

100

200

300

400

500

600

700

0 1 2 3 4 5 6 7 8

Genome Size (Mb)

Num

. R

eg

ula

tors

0

1000

2000

3000

4000

5000

6000

7000

Num

. OR

Fs

Structural genes EnzymesMembrane proteins

Transcriptionalregulators

Regulation of translation

Example of feedback inhibition

Example of feedback inhibition