RIP Proteases, small RNA and porins

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RIP Proteases, small RNA and porins

• bouloc_at_infobiogen.fr
• Signalisation et
• Réseaux de Régulations Bactériens 
• Institut de Génétique et Microbiologie,
• Université Paris-Sud - CNRS, UMR8621, 
• bâtiment 400, F-91405
• Orsay Cedex France

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(No Transcript)
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Membrane associated proteases
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Regulated intra-membrane proteolysis (RIP)

• Processing of membrane proteins by embedded
  proteases,
• releasing an active fragment.
• Characteristics of S2P-like proteases
• - several trans-membrane domains
• - HExxH motif -gt metalloproteases
• - LDG motif
• Active sites are located within the membrane.
• - Conserved from bacteria to eukaryotes

Enterococcus faecalis Eep cAD1 sex pheromone
Bacillus subtilis SpoIVFB pro-sk
sporulation Caulobacter cresentus MmpA PodJL
polar localization Vibrio cholerae
YaeL TcpP virulence
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Putative RseP topology
PDZ
periplasm
Signal peptide
LDG
HExxH
cytoplasm
PhoA insertion
LacZ insertion
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Genetic organizationaround rseP (yaeL)
Porin synthesis
fabZ
uppS
skp
lpxD
lpxA
lpxB
rnhB
LPS
Phospholipids
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Conditional rseP expression
Km
rseP
tetR lacI
attl
aTc
PLTetRseP
DrsePKm pPLtetRseP
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RseP is essential
?rseP pPLtetRseP
aTc
- aTc
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System to test rseP mutations
Km
PLtetRRseP
rseP
aTc
tetR lacI
attl
IPTG arabinose
attHK
Plac/ararseP
Plac/ararseP DrsePKm pPLtetRseP
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Complementation of RseP depletion
Plac/ararseP DrsePKm pPLtetRseP
Vector
RseP
RseP(E23A)
RseP(E23Q)
RseP(A235E)
RseP(D235A)
RsePDPDZ
RseP(D402A)
RseP(D402N)
RseP(E23A/D402N)
RseP(E23Q/D402N)
IPTG/Ara
aTc
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RpoE regulon activation
LPS
Porin
OM
Periplasm
DegS
RseP
IM
RseA
sE
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RpoE regulon activation
LPS
Porin
OM
Periplasm
DegS
RseP
IM
RseA
sE
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RpoE regulon activation
LPS
Porin
OM
Periplasm
DegS
RseP
IM
RseA
sE
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RpoE regulon activation
LPS
Porin
OM
PDZ
Periplasm
DegS
PDZ
RseP
IM
RseA
sE
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Degradation of RseA by RsePDPDZ
 
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Expression from a sE regulated promotor
pControl
pRseP
pRseP DPDZ
DrseP
degS pControl
degS pRseP
degS pRseP DPDZ
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RpoE regulon activation
LPS
Porin
OM
?
PDZ
Periplasm
DegS
PDZ
RseP
IM
RseA
sE
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Multicopy suppressor of RseP depletion
LPS
Porin
OM
Periplasm
DegS
RseP
IM
RseA
x
sE
sE
x
x
x
X
ori
ori
ori
ori
ori
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RseX is a suppressor of DdegS
LPS
Porin
OM
Periplasm
DegS
RseP
IM
RseA
x
sE
sE
x
x
x
X
ori
ori
ori
ori
ori
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Effect on pSUP on sigmaE activity
100
50
Miller Units
0
pUC18
pSUP
DdegS pSUP
DrseP pSUP
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Suppressor?
4 clones
fabZ
uppS
hlpA
lpxD
lpxA
lpxB
rnhB
8 clones
yedQ
yodC
yedI
yedA
vsr
dcm
yedJ
yedR
yedR
yedJ
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Suppressor?
yedR
yedJ
yedJ
yedR
yedR
yedJ
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Suppressor
yedS  ompS1 
yedR
170bp
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 conventional  and  non-conventional  RNAs

• Messenger RNAs ? express proteins
• Ribosomal RNAs ? ribosome components
• transfer RNA ? transfer amino acids for
  protein synthesis
• Small regulatory RNAs (sRNA)
• 100 to 300 nts, secondary structure
• Conditional expression
• Difficult to detect (biocomputing and genetic)

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E. coli sRNAs before 2000

• Stable and abundant
• 4.5S RNA secretion machinery
• 10Sb RNA RNase P catalytic part
• 10Sa RNA (tmRNA) protein quality control
• 6S RNA modulator of RNA Pol. activity
• Spo42 antisense regulator
• Detected by multi-copy expression
• MicF transcription divergent from ompC, acts
  on ompF mRNA
• DsrA increase in capsule synthesis
• RprA suppressor of dsrA
• Transcriptional analysis
• OxyS divergent transcription from oxyR
• GvcB divergent transcription from gvcA

Pichon C. et al. PNAS 2005
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Global searches for sRNAs in E. coli
Wassarman KM, Repoila F et al. (2001) Genes Dev
151637-1651
Rivas E, et al. (2001) Curr Biol. 111369-73.
Vogel J. et al. (2003) Nucleic Acids Res. 2003
316435-43
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Examples of sRNA activities
Storz et al. 2004 Current Opinion in Microbiology
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E. coli Hfq

• Host factor necessary for the replication of
  RNA phage Qß
• in E. coli.
• 30 00060 000 copies per cell
• Pleiotropic regulator
• Hfq inactivation generates numerous phenotypes
• Reduce growth rate and yield
• Plasmid supercoiling
• Bacterial size
• Osmosensitivity
• UV sensitivity

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Suppressor

• Physical characterization
• Identification of its targets
• Mode of action

yedS  ompS1 
yedR
170bp
RseX RNA suppressor of eXtracytoplasmic stress
protease (RseP)
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RseX
rseX
ompS1 (yedS)
yedR
zz
91
164
284
Rho-independent terminator
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rseX trancription is required for DrseP
suppression
Plac/ararseP DrsePKm pPLtetRseX
aTc
-
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Suppression requires Hfq
Plac/ara-1rseP DrsePKm
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Specific Hfq-binding to RseX
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Identification of RseX targets

• In vitro transcription of RseX

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Identification of RseX targets

• In vitro transcription of RseX
• RseX biotinylation

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Identification of RseX targets

• In vitro transcription of RseX
• RseX biotinylation
• Association of biotinylated RseX to streptavidin
  beads

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Identification of RseX targets

• In vitro transcription of RseX
• RseX biotinylation
• Association of biotinylated RseX to streptavidin
  beads
• Incubation of total RNA extracts with
  RseX-biotin-streptavidin complex

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Identification of RseX targets

• In vitro transcription of RseX
• RseX biotinylation
• Association of biotinylated RseX to streptavidin
  beads
• Incubation of total RNA extracts with
  RseX-biotin-streptavidin complex
• Purification of RNAs associated to the complex
  using a magnet
• Elution of RseX-bound RNAs

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Identification of RseX targets

• In vitro transcription of RseX
• RseX biotinylation
• Association of biotinylated RseX to streptavidin
  beads
• Incubation of total RNA extracts with
  RseX-biotin-streptavidin complex
• Purification of RNAs associated to the complex
  using a magnet
• Elution of RseX-bound RNAs
• Cy3-labelled cDNA synthesis

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Identification of RseX targets

• In vitro transcription of RseX
• RseX biotinylation
• Association of biotinylated RseX to streptavidin
  beads
• Incubation of total RNA extracts with
  RseX-biotin-streptavidin complex
• Purification of RNAs associated to the complex
  using a magnet
• Elution of RseX-bound RNAs
• Cy3-labelled cDNA synthesis
• Hybridation on pan-genomic E.coli
  micro-arrays (GODMAP)

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Hybridation on E. coli micro-array
ompC
ompA
gDNA
gDNA
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RNA-RNA band-shift
ompA mRNA
RseX-ompA mRNA
RseX
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Putative pairing of RseX with 5 ompA and ompC
mRNA UTRs
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Outer membrane protein gel
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ompA/ompC mRNA northern blot
ompA/ompC mRNA northern blot
pRSEXaTc
pRSEX-aTc
pControl
ompA
ompC
ssrA
rseX
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RseX degradation is likely RNaseE dependent
rne(ts) pPLtetRseX
30 -aTc
44 -aTc
30 aTc
44 aTc
C
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DompA DompC is a suppressor of DrseP
Parental strain PLtetO-1rseP DrseP
aTc
-aTc
Parental strain
DompA
DompC
DompF
DompA DompC
DompA DompF
DompC DompF
DompS1
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DompA DompC is a suppressor of DrseP
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Absence of porins and sE response
lacZ
sE
250
200
150
Miller Units
100
50
0
WT
DompF
DompA
DompS
DompC
DompCF
DompAC
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Hfq represses the sE response
lacZ
sE
250
200
150
Miller Units
100
50
0
WT
hfq
Dhfq
DompF
DompA
DompS
DompC
DompCF
DompAC
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OmpF
OmpA
OmpC