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Introduction
Pathogenic strains of Yersinia harbour closely related
70kb plasnnids involved in virulence designated pYV
{Gemski ef ai. 1980; Zink et ai, 1980; Biot and Cornelis,
1988). The pYV plasmids specify several temperaturedependent properties. These include a Ca^ * requirement
for growth at 37C, the secretion of large amounts of
plasmid-encoded proteins called Yops and the production of an outer membrane protein called PI (Bolin et
ai, 1982; for review see Straley, 1988; Cornelis et ai,
1989a).
1586
YlpA
30-
YIpB
14-
LPS
LPP
Results
identification of ptasmid-encoded tipoproteins
The lipoproteins of Y. enterocolitica W22703 were labelled
in vivo with [^H]-palmitic acid and analysed by SDS-PAGE
and fluorography (Fig. 1). This typical O:9 strain produced
several lipoproteins, two of which appeared in higher
amounts on the gels. These were a 19 kD protein and a low
molecular-weight (<1G000) protein probably corresponding to the major E. coli lipoprotein. Two lipoproteins were
only produced by the plasmid-bearing strain, suggesting
that they are plasmid-encoded. These proteins, with
apparent molecular weights of 29000 and 27000, were
called YlpA and YIpB, respectively (for Yersinia lipoprotein). They were only detected when the strain was
incubated at 37''C in the absence of Ca^' ions.
Plasmid pYL4 is a Tn3 insertion mutant of the pYVe227
plasmid obtained earlier in ourlaboratory (Balligand efa/.,
as
a
I
He
E4
I
Me
1587
(9.4 kbi
pQCS
DBC4
Regulation of ylpA
YlpA was only produced at 37''C in absence of Ca^* (see
Fig. 1), which suggests that it is regulated by virF like the
yop genes (Cornelis efa/., 1989b). In order to confirm this
hypothesis, we monitored the expression of ylpA in a pair
of virF'^ and virF~ strains. The wrf mutant was constructed
by homologous recombination between pYVe227 and
pGCS904, a 'suicide plasmid' (Miller and Mekalanos,
1988) containing an internal fragment of virF {C. Lambert
de Rouvroit ef at., in preparation). As shown in Fig. 5, both
lipoproteins YlpA and YIpB disappeared in the virF~ strain.
Thus genes ylpA and yIpB are members of the yop
regulon.
In order to check whether ylpA is transcribed from its
own WrF-regulated promoter or from the promoter of the
yop20 neighbouring gene, we tested its expression in
pBM33, a yop20 insertion mutant of pYVe227 (Mulder et
ai, 1989). As shown in Fig. 5, YlpA was still produced by
W22703(pBM33), indicating that yop20 and ylpA do not
form an operon.
1588
HetGluAspAspMetLysLys
CTGGATCCCATTTTGGTCAACCGTGGCTATGGGAGGTGCCATGCCCTGTTTTAAATGGAAGATGATAreAAGAAA
1960
Hindi
,
.
2000
AsnMetLysLeul leAlal lfThrAUValLeuS>rS(TValLeuValLeuSerGlyCysr.l>AIaMetScrThr
AACATCAAGTTAATAGCAATAACTGCCGTACTGTCCTCAGTGTTAGTCCTCTCCGGCTGTGGTGCGATGAGCACA
ZIOO
AlalleLysLysArgAsnLeuGluValLysThrGlnMetSerGluThrlleTrpLeuGluCroSerSerGlnlys
GCAATCAAAAAACGTAATCTGGAAGTGAAAACGCAGATGAGTGAAACGATTTGGTTAGAGCCGTCTTCACAGAAA
ThrValTyrLeuGlnllrLysAsnThrSerAspLysAsnMetLeuGlyLeuAlaProLyslleThrLysAlaVal
ACCGTTTATCTACAGATAAAAAATACCTCAGATAAAAATATGCTTGGCTTAGCCCCCAAAATCACAAAAGCTGTG
2200
,
,
.
.
,
GlnAapLysGlyTyrThrValThrScrSerProGluAspAlaHisTyrTrpl icIilnAlaAsnValLpuLjsAla
CAGGATAAGGGGTATAtCGTGACATCGTCCCCAGAAGATGCACATTACTGGATCCAGGCTAATGTCCTGAAAGCC
2300 BaMl
AspLyaMetAspLeuArgGluAlaGluGlyPheLeuSerGlnGlyTyrGlnGlyAiaAULeuGlyAlaAiaLeu
GATAAAATGGATTTGCGTGAAGtTGAAGGATTTCTGAGTCAGGGGTATCAGGGGGCTGCGCTGGGGGCCGCATTA
2400
GlyAlnGIyl leThrGlyTyrAsnScrAKiiScrAlaGlyAiaSprl.ciitilyVulrilyLcuAlnAlaGiyl.PuVal
GGGGCTGGTATTACAGGCTACAACTCTAACTCAGCGGGAGCrTCGTTAGGAGTTGGATTGGfGGrTGGTCTTGTT
GlyHetValAUAspAlaMetValGluAspIleAsnTyrThrMetValThrAspValGlnl leSerGluLysThr
GGGATGGTCGCGGATGCGATGGTCGAGGACATCAATTATACTATGCTGACGGATGTCCAGATTTCCGAGAAAACG
2500
.
.
.
.
.
AspThrProLeuGlnThrAspAsnValAIaAlaLeuLyaGlnGlyThrSerGlyTyrLysValGlnThrSerThr
GACACCCCCCTACAGACTGACAATGTGGCGGCGCTGAAGCAAGGCACCTCTGGCTATAAAGTTCAGACCAGCACA
2600
GlnThrGlyAsnLysHisGlnTyrGlnThrArgValValSerSerAlaAsnl.ysValAsnLeuLysPheGluGlu
CAGACGGGtAACAAACATCAATACCAGACTCGCGTGGTTTCTTCGGCTAACAAGQTtAACCTGAAATTTGAAGAA
.Hindi
.
2700
AlaGlnProValLeuGluAspGlnLeuAlaLyBSerlleAlaAsnlleLeu
GCCCAGCCGCTTCTGGAAGACCAGCTAGCGAAGTCTATCGCCAATATCCTGTAAGTCATAAGCATCCTGGTATGA
. 277fi
Discussion
Plasmid pYV from Y. enterocolitica 0:9 encodes at least
two lipoproteins designated YlpA (29kD) and YIpB (27 kD).
As in the case of most pYV-encoded functions, these
proteins were found to be produced at 37''C only in the
absence ot Ca^* ions.
According to the nucleotide sequence analysis, the
YlpA lipoprotein of Y. enterocolitica is closely related to
the TraT proteins encoded by the conjugative plasmids
pED208, RIOO and F as well as by the virulenceassociated plasmid of S. typhimurium (Finlay and Paranehych, 1986; Ogata ef ai, 1982; Jalajakukumari ef ai.
Fig. 3, Sequence of the ylpA gene from pYVe43980 and of (fs gene product. The 875 bp
sequenced fragment corresponds to co-ordinates
7.1 to 6.2kb of the pYV plasmid (see Fig. 2).
This sequence was numbered from nucleofide
1952 to nucleotide 2776 to allow an easy
connection with the immediately preceding
sequence containing gene /op 20 and presented
elsewhere (T Michiels et al.. submitted for
publication). ylpA starts about 500bp
downstream of the end of yop20 and is
transcribed in the same onentation. The largest
open reading frame (249 codons) starts at
nucleotide 2006 and ends at nucleotide 2752 of
the sequence. The actual ylpA gene presumably
starts at the second ATG (underlined twice).
Cleavage by signal peptidase II would occur in
the Leu-Ser-Gly v Cys sequence (underlined),
typical of TraT proteins. BamHI and Hincll
restriction sites are indicated. These sequence
data will appear in the EMBL'GenBank/DDBJ
Nucleotide Sequence Data Libranes under the
accession number X52753 (ylpA).
4.9 kb
14.9 kb
3.6 kb
B
^
CD
a.
^
-i
Q)
>
a.
a.
14.9
13.6
3.6
-^ igkD
-YIpB
19kD
1590
-20
pVV*.439-B0
pK[)2U8
Riao
.S . ( 1 phimuri UBl
10
20
-M-M
MMlii- Kl.HMV-LV- - T - A - - HK-- KLMM\-LV- -T-A
MK-- KLMMVTLV- -T-A
- - -
pYVp439-80
ptD20fl
30
10
50
rGAHSTAIKKRNLb:\KT9MSETIWLEPSS(JhTVVLalKNTSDKNMLGLAPKITKAV8DKG
A-tN--F
RIOO
D-S--wr.--AI)--KA--
W-^-HD-\-fB--*>--\
70
pVVe4 39-H0
pEDZOB
F
RiOO
-10
MKKNMKLIAITAVLSSVLVLSG
KO
30
I)-SD-WSL-A-DI-A--
100
110
120
S. t.vptiimurium
VT--DK-V
Sg-y>-NH--E--LS
1.10
no
150
160
A-
170
--
180
S. (ip/ji
pED20B
lyd
ZOO
210
22a
TeTGNKliaY8TRV\ SSANKVNl,KFFEAF'Vl.EDyLAKSl ANI L
9-K
1
K
\
pED208
F
RIOO
IDENTITY
RIOO
5 . t yph I mtir I
'E---a-K
-K---a-K
-K
y-h
-J
S
--N
K
K
K
---
K-
like the production of the Yops. This co-regulation suggests that, like Yops, YlpA could be involved in pathogenicity. However, our observations with i.v.-inoculated mica
did not support this hypothesis. The i.v,-inoculated mouse
model was selected because tt allowed us to demonstrate
the role of yop20 and yop48 in virulence (Mulder ef al..
1989). This model may not be the best one to piri-point the
influence of a component involved in complement neutralization because normal mouse serum has been shown to
have a poor bactericidal effect against E. coli (Vaara ef ai.
1984), Thus our result suggesting that YlpA is not a clear
virulence factor must be interpreted cautiously.
Alternatively, the presence of YlpA on the pYV plasmid
could simply be an evolutionary vestige of this plasmid.
Indeed, the replication function of pYV is related to that of
plasmid RIOO (Vannooteghem and Cornells, 1990) while
the partition and stabilization function of pYV is homologous to that of F (Bakour ef ai. 1983; Biot and Cornells,
1988), both plasmids containing a frafgene. However,
one argument contradicts this hypothesis: ylpA is not part
of a transfer operon in pYV (Bakour ef at.. 1983) while in
both RIOO and F traTis included in thefraoperon.
197/223
178/223
I 79/223
172/223
Experimental procedures
Bacteriat strains and plasmids
Y. enterocolitica W22703 (nalidixic acid-resistant) and Y. enterocolitica W22708 (slreptomycin-resistant) are derivatives of the
same Res Mod ' mutant of Y. enterocolitcaVJ227 (serotyoe 0:9)
(Cornells and Colson, 1975). Y. enterocotitica 439-80 is a
wild-type 0:9 strain. The pYV plasmids of all these strains are
indistinguishable by SamHI, EcoRI or Ssfll restriction analysis
(Larocheefa/., 1984).
E. coliJM^01 (Yanisch-Perron etal.. 1985)and LK111 (received
from M. Zabeau) are F ' . /acZ delta M15 strains. Strain S17.1.
containing a copy of RP4 integrated into its chromosome (Simon
etal., 1983). was used to mobilize onT-containing plasmids.
Phasmids pTZi8R and pTZ19R are from Pharmacia, pTJS82
(similar to pTJS81) is a pUC derivative containing the transfer
origin (oriT) from piasmid RK2 (Schmidhauser and Helinski,
1985).
1 A
6
5
^
i"
1591
^ ^ ^
O pYV*
D pVL4
3
1
0 6 12 18 24 30 36 42 48 54 60 66 7? 78 84 90 96
Infection of mice
0 6 12 18 24 30 364248546066 72 78849096
Hours
Fig. 7. Growth of strains K enterocolitica Vi/22703(pYVe227) and
W22708(pYL4). the yipA derivative, in the spleen (A) and the liver (B) of
the mouse. Mice were inoculaled i v. with 5 x 10' baderia (arrow).
Bacterial growth was followed in the liver and in the spleen over a 4-day
period. Each point was the mean value of a group of four mice; XUe
standard deviation was less Ihan 0.7 log.o bactena (not shown). All mice
inoculated with both the pYVe227- and pYL4-containing strains died at
day 5. This expenment was repeated twice with similar results. Symbols:
O . pYVe227 ' bacteria; L], pYL4' bacteria.
Specific pathogen-free BAL8/c female mice (bred at the University of Louvain) 6 weeks old were given (intraperitoneally)
0.5ml of saline (NaCI 0.15M) containing 20mg ml ' desferrioxamine (Desteral, Ciba-Geigy). Twenty-four hours later, mice were
inoculated intravenously (i.v.) with 0.5ml of a V. enterocolotica
suspension in saline. Bacterial challenges were prepared from
overnight cultures at room temperature in tryptic soy broth,
washed once and then suspended in saline. Grovt^h of bacteria in
the spleen and liver of animals was followed in relation to time
after the i.v. injection. Groups of four mice were sacrificed by ether
anaesthesia and the organs were removed aseptically and
hon^iogenized separately in saline: 0.1-nnl volumes of serial
10-fold dilutions in saline were spread on McConkey agar and
colonies were counted after incubation for 48h at 28C. Minimal
detectable limits were 50 bacteria per organ. Results were
expressed as the log,o of bacterial counts.
In order to discriminate between lipoproteins and other lipid-containing components, membrane tractions were incubated with
increasing quantities (10fi.g ml ' to 1 mg ml ')of either pronase
(Serva) or proteinase K (Serva). After 30 min incubation at 37X,
membranes were either pelleted by a 60 min centrifugation at
15000 r.p.m. or were precipitated by the addition of 4 vol. acetone
and subsequently harvested by centrifugation.
1592
Acknowledgements
We acknowledge L. Houdmont for skilful help in testing the
virulence in mice. We thank H. Wolf-Watz for the gift of V.
pseudotuberculosis VPIII. and H. Makela for discussion. This
work was supported by the Belgian Ministry for Sciences (Action
Concertee 86/91686) and by the Belgian Fund for Medical
Research (FRSM convention 3.4514.83). B.O. is a fellow of the
Belgian Institute for Scientific Research applied to Industry and
Agriculture (IRSIA). T.M, is Senior Research Assistant of the
Belgian National Fund for Scientific Research (FNRS).
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