1. 3
Secretion OM Q 2 P
Folding Z IM A S Y R
ATP 1
Assembly
TUVWX

4. S C R B TypIV piliation (Pil) Protein secretion (Xcp) Q Q PilD Z P
TUVWX S
ATP
XcpA Q
Protein secretion (Xcp) P OM Q
PilD IM C B
ATP A

5. The pseudopilus hallmarks
Bacteria overproducing XcpT are covered with extracellular pilus-like appendages.
Transmission Electron Microscopy, Negative Staining
Individual
fibrils
« Bundled »
The pseudopilus hallmarks

6. XcpT is assembled in a multifibrillar structure called
type II pseudopilus
TEM analysis, Immunogold labelling anti-XcpT
Flagella
Type II pseudopilus
Type IV pilus

7. Image Filtering Original Image Filtered image Enlargement Fourier
Transform
Filtered image
Enlargement

8. The Secreton-Dependent Pathway (Type II)
Secretin OM
XcpQ P
Xcp IM
XcpR C
ATP
ADP + Pi
Traffic ATPase

10. Molecular Mechanisms involving:
SECRETINS
TRAFFIC ATPASES
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

11. BfpB Secretin E. coli EPEC
Scmidt et al., J Bacteriol :

12. LES SÉCRÉTINES TRANSPORTENT DE LARGES COMPLEXES
(12-14 subunits)
YscC
PilQ
XcpQ
pIV
Inner diameter (nm) 5
5-6
9-10 8
Outer diameter (nm) 20 18
19-20 14
Process
Type III
Secretion
Type IV
pili
Type II
Secretion
Filamentous
phages
Bitter & Tommassen, 1999, Trends in Microbiol., 7: 4-7

13. PAO1 GENOME SEQUENCE Stover et al., 2000, Nature, 406: 959-964
Traffic ATPase
Secretin

14. Traffic ATPases Secretins Mb W U P V T X Y Z Q R S hxcR hxcQ 1 1 xqhB
hxcR
hxcQ 1 1
xqhB
pscC
Type III secretion 2 2
xqhA
Type II secretion 3
hplR 3
xcpR
Type II secretion
xcpQ
Type II secretion 4 4
pilT/pilU
Type IV piliation
hvbA
xqhC 5 5
pilB
Type IV piliation
pilQ
Type IV piliation
hxrA 6 6
6,3
6,3

15. P. aeruginosa hxc gene cluster
ApR W U P V T X Y Z Q R S
hxc
xcp WT
xcp
hxc
94 kDa 67 43 30 20
(Supernatant fraction, Phosphate starvation)
Mass Spectrometry

16. Low molecular weight Alkaline Phosphatase
LapA (368) MFKRSLIAASLSVAALVSAQAMA-VTGGGASLPAELYK
Tan & Worobec, 1993
lapB
LapB (370) MLKPMFGVVALTLLTSHTTLG-ATVTGGGASMPAKLYK
phxcV-lacZ
phxcT-lacZ
lapA
hxc gene cluster

17. 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

18. Which alternative systems can assemble the type II pseudopilus ?Q
Pil system
(type IV pili)
pil A B C
Prepilin peptidase
xcpA/pilD
Pilin
Xcp system
(type II secreton)
xcp Z Y X W
V U T S R P Q
ATP fixation motif
Pseudopilins
Secretin
Hxc system
(type II secreton)
hxc
W U P V T X Y Z Q R S

19. xcpRS/hxcR/pilQ - - - No
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

20. Traffic ATPases Secretins Mb hxcR hxcQ 1 1 xqhB pscC 2 2 xqhA 3 hplR 3
hxcR
hxcQ 1 1
xqhB
pscC
Type III secretion 2 2
xqhA
Type II secretion 3
hplR 3
xcpR
Type II secretion
xcpQ
Type II secretion 4 4
pilT/pilU
Type IV piliation
hvbA
xqhC 5 5
pilB
Type IV piliation
pilQ
Type IV piliation
hxrA 6 6
6,3
6,3

21. A flp / tad gene cluster in the P. aeruginosa genome
Secretin
Traffic ATPase
Actinobacillus actynomycetemcomitans
Kachlany S.C. et al., (2000), J. Bact.,
Secretin
Traffic ATPase

22. A. actinomycetemcomitans adherence system: ( electron microscopy)
the « Flp pili »
Kachlany S.C. et al., (2001), Molecular microbiology, 40,
- 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
A. actinomycetem-
comitans
(negativ contrast)
Bundled pili
( electron microscopy)

23. Phylogeny of genes for secretion NTPases: Development of a diagnostic
key for gene classification
Planet et al.,
Proc. Natl. Acad. Sci. USA, 2001, 98,

24. The Secreton-Dependent Pathway (Type II)
ETA SP
Sec OM
XcpQ P
Xcp
Tat IM
XcpR C
ATP
ADP + Pi

25. The P. aeruginosa Tat system
E. coli
P. aeruginosa
Amino acid identity (%)
in corresponding proteins 39 30 44 57
tatA
tatC
hisE
tatD
tatE
tatB
PA5071
KmR
Is the system functional ?

26. Viability of E. coli and P. aeruginosa strains upon pfColA production
The colicin A assay OM IM
OmpF
BtuB
PAL
Tol
pfColA
Colicin A ( A B
Tat RR
Sec SP
 Viability of E. coli and P. aeruginosa strains upon pfColA production
E. coli
 P. aeruginosa WT
WT (cai)a
tatA-E
tatC
tatC (pTATC)
pMMB67EH +
pRR-pfColA67 -

27. The P. aeruginosa Tat substrates ?
PAK-TC
PAK 94 67 43 30 20
PAK
PAK-TC
PAKDxcpRZ
Mass spectrometry
MISKSRRSFIRLAAGTVGATVATSMLPSSIQAALAI
PlcN
35 aa
Phospholipases C
MTENWKFRRRTFLKHGAQAATLAGLSGLFPETLRRALAV
PlcH
38 aa

28. Phospholipase C activity* in cell fractions
4.21
0.57
0.68
Cytoplasmic membrane
0.04
<0.01
0.01
PAK-TC (tatC)
0.16
4.74
0.03
PAK-∆xcpRZ
0.10
0.50
2.80
PAK
Wild-type
Cytoplasm
Periplasm
Supernatant
Strain
* p-nitrophenyl-phosphatidylcholine as substrate

29. The Secreton-Dependent Pathway (Type II)
ETA SP
Sec
Plc’s RR
Tat OM
XcpQ P
Xcp IM
XcpR C
ATP
ADP + Pi