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Prokaryotic promoters in the test tube and in the environment. Pseudomonas putida. Víctor de Lorenzo Centro Nacional de Biotecnología CSIC. Enrichment cultures. Strain (Plasmid)Pollutant P. putida mt-2 (pWW0)Toluene Pseudomonas sp, CF600 (pVI150)Phenol - PowerPoint PPT Presentation
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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