om im r y z s a tuvwx p q 1 3 assembly secretion atp folding 2
TRANSCRIPT
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OM
IM
RY ZSA
TUVWX
P
Q
1
3
Assembly
Secretion
ATP
Folding
2
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TypIV piliation (Pil)
PilD
A
OM
IM
Q
CBATP
R
Y Z
TUVWX
S
ATP
XcpA
Q
Protein secretion (Xcp)
P
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Bacteria overproducing XcpT are covered with extracellular pilus-like appendages.
Transmission Electron Microscopy, Negative Staining
Individual fibrils
« Bundled »
The pseudopilus hallmarks
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XcpT is assembled in a multifibrillar structure calledtype II pseudopilus
TEM analysis, Immunogold labelling anti-XcpT
Type II pseudopilus
Type IV pilus
Flagella
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Image FilteringOriginal Image
Fourier Transform
Filtered image
Enlargement
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The Secreton-Dependent Pathway (Type II)
C
P
Xcp
IM
OM
XcpQ
XcpR
ATPADP + Pi
Secretin
Traffic ATPase
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SÉCRÉTINE « TRAFFIC ATPASE »
SÉCRÉTION TYPE II YES YES
SÉCRÉTION TYPE III YES NO
SÉCÉRTION TYPE IV NO YES
DNA UPTAKE
GRAM+
NO YES
DNA UPTAKE
GRAM-
YES YES
TYPE IV PILI YES YES
PHAGE
FILAMENTEUX
YES NO
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Molecular Mechanisms involving:
• Secretion
– Type II
– Type III
• Type IV Piliation
• Filamentous phage extrusion
• DNA uptake/Natural competence
• Assembly of S-layer proteins
• Secretion
– Type II
– Type IV
• Type IV Piliation
• Bacterial Conjugation
• DNA uptake/Natural competence
SECRETINS TRAFFIC ATPASES
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BfpB SecretinE. coli EPEC
Scmidt et al., J Bacteriol 2001 183:4848-4859
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LES SÉCRÉTINES TRANSPORTENT DE LARGES COMPLEXES
YscC PilQ XcpQ pIV
5 5-6 9-10 8
20 18 19-20 14
Type IIISecretion
Type IVpili
Type IISecretion
Filamentousphages
Inner diameter (nm)
Outer diameter (nm)
Process
Bitter & Tommassen, 1999, Trends in Microbiol., 7: 4-7
(12-14 subunits)
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PAO1 GENOME SEQUENCE
Stover et al., 2000, Nature, 406: 959-964
Traffic ATPase
Secretin
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hxcR
hplR
xcpR
pilT/pilU
hvbA
pilB
hxrA
0
1
2
3
4
5
66,3
Mb
Type IV piliation
Type IV piliation
Type II secretion
Traffic ATPases
W U P V T X Y Z Q R S
0
1
2
3
4
5
66,3
hxcQ
xqhC
xqhB
pscC Type III secretion
xqhA Type II secretion
xcpQ Type II secretion
pilQ Type IV piliation
Secretins
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P. aeruginosa hxc gene cluster
WT xcp hxc94 kDa
67
43
30
20
(Supernatant fraction, Phosphate starvation)
W U P V T X Y Z Q R S
QPRSTUVWXYZ
hxc
xcp
ApR
Mass Spectrometry
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0
50
100
150
200
250
300
100 200 300 400 500
growth time, min
PAO1 pMP220
PAO1 pMPTZ
PAO1 pMPVZ
phxcV-lacZ phxcT-lacZ
LapA (368) MFKRSLIAASLSVAALVSAQAMA-VTGGGASLPAELYK
lapB
LapB (370) MLKPMFGVVALTLLTSHTTLG-ATVTGGGASMPAKLYK
lapAhxc gene cluster
Tan & Worobec, 1993
Low molecular weight Alkaline Phosphatase
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• Identification of a second xcp-like gene cluster involved in type II secretion
• Hxc system is specifically involved in secretion of low molecular weight alkaline phosphatases
• Expression of the hxc gene cluster appeared to be coregulated with lap genes expression
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Which alternative systems can assemble the type II pseudopilus ?
SecretinATP
fixation motif
Pseudopilins
Xcp system(type II secreton)
W PSTXYZ QR V Uxcp
Q Pil system
(type IV pili)A B C
Pilin
pil
Prepilin peptidase
xcpA/pilD
W U P V T X Y Z Q R S
Hxc system(type II secreton) hxc
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Requirement for the Xcp, Hxc or Pil system in the assembly of the type II pseudopilus
STRAINS MOLECULAR MACHINERIES PSEUDOPILUS ASSEMBLY
Xcp Hxc Pil
WT + + + Yes
xcpZ-Q - + + Yes
xcpA - - - No
pilQ/hxcR + - - Yes
xcpRS/pilQ - + - Yes
xcpRS/hxcR - - + Yes
xcpRS/hxcR/pilQ - - - No
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hxcR
hplR
xcpR
pilT/pilU
hvbA
pilB
hxrA
0
1
2
3
4
5
66,3
Mb
Type IV piliation
Type IV piliation
Type II secretion
Traffic ATPases
0
1
2
3
4
5
66,3
hxcQ
xqhC
xqhB
pscC Type III secretion
xqhA Type II secretion
xcpQ Type II secretion
pilQ Type IV piliation
Secretins
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A flp / tad gene cluster in the P. aeruginosa genome
Kachlany S.C. et al., (2000), J. Bact., 6169-6176Actinobacillus actynomycetemcomitans
Traffic ATPaseSecretin
Secretin Traffic ATPase
flp
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A. actinomycetemcomitans adherence system:the « Flp pili »
A. actinomycetem-comitans
(negativ contrast)
Kachlany S.C. et al., (2001), Molecular microbiology, 40, 542-554
Bundled pili( electron microscopy)
- Gram-negative cocobacillus, agent of the juvenil periodontal disease
- Produces long fibrils of bundled pili responsible for non-specific adherence and autoaggregation
- Forms a tenacious biofilm on teeth
- Structural subunit of bundled pili is encoding by flp-1 gene. tad genes cluster encodes the assembly machinery of Flp pili
- Flp-1 is a member of the type IVB pilus sub-familly
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Phylogeny of genes for secretion NTPases: Development of a diagnostickey for gene classification
Planet et al., Proc. Natl. Acad. Sci. USA, 2001, 98, 2503-2508.
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C
P
Xcp
IM
OM
ETA
SP
Sec Tat
XcpQ
The Secreton-Dependent Pathway (Type II)
XcpR
ATPADP + Pi
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The P. aeruginosa Tat system
E. coli
P. aeruginosa
Amino acid identity (%)in corresponding proteins
39 30 4457
tatA
tatA
tatC
tatC
hisE
tatD tatE
tatD
tatB
PA5071 tatB
Is the system functional ?
KmR
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The colicin A assay
OM
IM
OmpFBtuB
PALTol
pfColAColicin A (A B
Viability of E. coli and P. aeruginosa strains upon pfColA production
E. coli P. aeruginosa
WT WT (cai)a tatA-E WT tatC tatC (pTATC)
pMMB67EH + + + + + +
pRR-pfColA67
- + + - + -
Sec
SP
Tat
RR
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The P. aeruginosa Tat substrates ?PAK-TC PAK
94
67
43
30
20
PAK PAK
PAK-TC PAKxcpRZ
MTENWKFRRRTFLKHGAQAATLAGLSGLFPETLRRALAVPlcH38 aa
MISKSRRSFIRLAAGTVGATVATSMLPSSIQAALAIPlcN35 aa
Phospholipases C
Mass spectrometry
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Phospholipase C activity* in cell fractions
4.21
0.57
0.68
Cytoplasmic membrane
0.04<0.010.01PAK-TC (tatC)
0.164.740.03PAK-∆xcpRZ
0.100.502.80PAK
Wild-type
CytoplasmPeriplasmSupernatant Strain
* p-nitrophenyl-phosphatidylcholine as substrate
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C
P
Xcp
IM
OM
ETA
SP
Sec
Plc’sRR
Tat
XcpQ
The Secreton-Dependent Pathway (Type II)
XcpR
ATPADP + Pi