Download P. putida - Instituto de Biotecnología

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)
Pseudomonas sp, CF600 (pVI150)
Burkholderia sp. RP007
P. putida TMB
P. putida NAH (NAH7)
Acinetobacter sp. ADP1
R. eutropha (pJP4)
Pseudomonas sp. (pP51)
P. putida UCC22 (pTDN1)
P. oleovorans (pOCT)
Burkholderia cepacia LB400
Pseudomonas sp. VLB120
Toluene
Phenol
Phenantrene
Trimethyl benzene
Naphthalene
Aryl esters
2,4 D
Trichlorobenzene
Aniline
n-alkanes
Biphenyl, PCBs
Styrene
Evolution of biodegradative capabilities
1. Genes encoding catabolic enzymes
2. Regulatory systems responding to
substrates
3. Coupling transcription to
Physiological signals
Expression of
catabiolic genes
Stages in the regulation of gene
expression systems
1
inducer
addition
+C
2
3
4
Growth
Evolutionary gravitation towards
an optimal regulation
Gain / capacity
Window of
activity
Specificity
Metabolic coupling
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 twocomponent regulatory systems
Regulation of transcription by quorumsensing systems
Structures of
some autoinducers
in quorum-sensing
systems
s factors determine promoter
specificity
Sigma 70 family
Sigma 70-like
Ec rpoD
Ec rpoS
Ec rpoF
ECF Family
Ec rpoE
Ec rpoH
Ec fecI
Sigma 54
A class in
its own!
Sigma 54 promoters
• -12/-24 consensus sequence
• Require an activator that
binds far from +1
GG-10-GC
The -12/-24 site
• Small compared to -10/-35
• GC rich (frequent in Pseudomonas)
Reporter lacZ gene fusions
Monitoring promoter activity… once at a time
Accumulation of beta-galactosidase
The big
leap
forward:
from
genetics
to
genomics
Computer-assisted
search for genes and promoters
Probing global gene expression
with Gene arrays
Condition
A
Condition
B
Differential gene
Expression (DGE)
reveals signals and
promoters
meslo
paer
ecoli
bsub
bhal
vcho
mtub
ccre
llact
staphyN
spyo
pmul
dra1
hinf
xfas
synecho
tmar
mlep
nmen
aquae
cjej
hpyl
tpal
rpxx
bbur
buch
cpneu
ctra
ureur
mycpn
mycge
Number of Regulators
450
400
350
300
250
200
150
100
50
0
700
7000
Structural genes
Enzymes
Membrane proteins
600
6000
5000
2
400
4000
R = 0,9668
300
3000
2
R = 0,8842
200
2000
Transcriptional
regulators
100
1000
0
0
0
1
2
3
4
Genome Size (Mb)
5
6
7
8
Num. ORFs
Num. Regulators
500
Regulation of translation
Example of feedback inhibition
Example of feedback inhibition