the pyv plasmid of yersinia encodes a lipoprotein,

8/9/2019 The pYV plasmid of Yersinia encodes a lipoprotein,

1/10

icrobiology (1990) 4(9), 158 5-1593

YV plasm id of

Yersinia

encodes a l ipop rotein,

B. China, T. Michiels and G. R. ComelJs*

Unite de f>/licrobioiogie. Universite Catholique de

Louvain. U.C.L 5 4-90. 54 Avenu e Hippocrate,

B-1200

Brussels, Belgium.

Summary

A series of lipoproteins w as detec ted n the membrane

fraction of

Yersinia enterocolitica

W227, a typical

strain from serotype 0:9. At least two of them, YlpA

and YIpB, are encoded by the pYV plasmid. The

sequence of

ylpA

reveals the presence of a typical

lipoprotein signal peptide. The mature YtpA protein

would be 223 residues long with a calculated molecu-

lar weight of 23798 for the proteic moiety of the

molec ule. YlpA shares 88 identical residues with the

TraT protein encoded by plasmid pED208, 80

identity with TraT proteins encoded by plasmids R100

and F, and 77 identity with the TraT protein encoded

by the virulence plasmid of

Salmonella typhimurium.

Th e ylpA gene hybridized with the pYV plasmid of

Yersinia pseudotubercutosis

suggesting that this

gene is conserved among

Yersinia

spp. The produc-

tion of YlpA is controlled by

virF

and only occurs at

37 C in the absence of Ca^' ions. This co-regu lation

with the

yo p

genes suggests that

ylpA

is a virulence

determinant. However, mutations n ylp clearly a ffect

neither the resistance to human serum nor the

virulence for intravenously inoculated mice.

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 temperature-

dependent properties. These include a Ca^

*

requirement

for growth at 37°C, the secretion of large amounts of

plasmid-encoded proteins called Yops and the pro-

duction of an outer membrane protein called PI (Bolin e t

ai

1982; for review see Straley, 1988; Cornelis et

ai

1989a).

Received 3 January, 1990; revised 17

ApnI.

1990. 'For correspondence.

Tel.

(2) 7645488; Fax (2)

7645481;

Earn/Bitnet CORNELIS at

BUCLLN11.

The secretion of Yops occurs in growth-restriction

con ditions , i.e. at 37°C in the absence of Ca^* ions. Both

phenomena are controlled by a ±2 0k b region of the PYV

plasmid called the 'calcium region'. This region contains

several transcription units designated virA. virB. wrCand

virF in Yersinia enterocolitica and IcrA. B. C and F in

Versin/a pesf/s (Goguen et al., 1984; Cornelis

etai,

1986;

Yother et al., 1986). The virF/lcrF locus of the calcium

region encodes a DNA-bind ing protein respon sible for the

transcriptional activation of yo p genes at 37^C (Yother et

ai , 1986; Cornelis

etai.

1987; 1989b). Vop proteins

are

thought to be involved in the resistance of yersiniae

towards host immune defences but little is known about

their individual role. Yop2 b from Yersinia pseudotubercu-

/os/s(thecounterpartofYop51 from

Y.

e nterocolitica) has

been reported to be involved in phagocytosis inhibition

(Rosqvist et ai, 1988) while Yo pM from V. pestis (the

counterpart of Yop48 from

Y.

enterocolitioa) was found to

share homology vi/ith the a-chain of the human platelet

membrane glycoprotein Ib, which suggests that this

protein might interfere with blood coagulation (Leung and

Straley, 1989).

In contrast to Yops, the membrane protein P l, encoded

by gene yopA. is produ ced at 37''C irrespective of the

Ca^

concentration and independently of the presence of VirF

(Bolin ef ai , 1982; Bolin and Wo lf-Watz, 1984; Skurnik,

1985;

Cornelis

etai.

1986), This protein has been reported

to be involved in autoag glutination(Sk umik efa/. . 1984), in

the intestinal colonization of mice (Kapperud et ai, 1987),

in the bindin g of collagen fibres (Emody ef a i. 1989) and in

resistance to the bactericidal activity of human serum

(serum resistance) (Balligand e t ai, 1985). A clone d yopA

gene can restore the serum resistance of a yopA mutant ot

y. e nterocolitica but not that of a pYV strain, suggesting

that serum resistance in

Y.

enterocolitica is multi-factorial

(Balligand ef al., 1985). In contrast, Martinez showed

recently (1989) that Escherichia co//strain MM294 prod uc-

ing PI from a cloned y. enterocolitica gene became

serunn-resistant.

In this work, we report that the pYV plasmid directs the

produ ction of at least two lipo proteins. One of

them,

YlpA,

is related to TraT, a protein kn own to be involved in surface

exclusion during conjugation (Achtman et ai. 1977) but

also in serum resistance (Moll ef ai , 1980) and in the

inhibition of phagocytosis by macrophages (Aguero ef ai ,

1984).


2/10

1586 S, China,

T.

Michiels and

G.

R. Cornelis

Y l p A

Y I p B

3 0 -

2 0 -

1 4 -

L P S

L PP

Fig, 1.1 den 11 fi cat on of lipoproteins encod ed by Yersinia W22703 or

W22708 (serotype 0:9) pH)-palm ittc acid-labe lled memb ranes extracted

trom Y. enterocolitica W227 strains were analysed by SDS-PAGE and

fluorography. Lane 1, W22703(pWe22 7) (pBC5) incubated at 3 7 C In

BHIo., lane 2. W22708(pYL4) incubated at 37 C in BH b. ; lane 3.

W22703(pYVe227) incubated at 37 C in BHIr,.; lane 4, W22703

incubaled at 37-C in BHIo.; lane 5, W22703(pYVe227) incubated at 25' C

in BHIo., lane 6, W22703(pYVe227) incubated at 37 X in BHica; lane 7,

W22703(pYVe2?7) incubated at 37''C in BHIo.. Two proteins, YlpA and

YIpB, produced by the wild-type strain incubated at 3 r C in BHIo< (lanes

3 and 7) were not detected when the strain was grown at 25 C (lane 5) or

in the presence of Ca^ ions (lane 6). The broad band labelled LPS

probably corresponds to the lipopolysaccharide since it did not

disappear upon 1 h incubation at 37 C with up lo 1 mg rnl ' pronase or

proteinase K (data not shown). Lpp identities an abundant, low

molecular-weight lipoprotein inferred to correspond to the ma|Or E. coli

fipoprotein. 30. 20 and 14 are molecular m ass markers (kD).

Results

identification of ptasmid-encoded tipoproteins

The lipoproteins of

Y.

e nterocolitica W227 03 were labelled

in vivo with [^H]-palmitic acid and analysed by SDS -PAG E

and fluorography (Fig. 1). This typical O:9 strain produ ced

several lipoproteins, two of which appeared in higher

amo unts on the gels. These were a 19 kD protein and a low

molecular-weight (


3/10

YlpA. a pYV-encoded lipoprotein of Yersinia 1587

B a s a

I I

He Me

E4 (9.4 kb i

pQCS

DBC4

g . 2. Genetic map of pYVe227 and cloned fragments.

p ot pYVe227 integrating previous data (see Mulder e(a (.. 1989): yop are the genes of the released proteins; yo pfl enc odes protein P I: vi r

repBA-oriR is the replication region and incD is the stabilization region (Vanooteghem and Cornelis, 1990). Arrows

fTnflf^,

mini-Mu diac or Jr\2S07) inserted into pYV. M . operon fusion

{cat o r lacZ) at 37 X , • I, lack of transcription, •^. weak transcription.

ISR or pBC19R den vat ves carrying subfragments of fragment EcoRI 4 cf pYV439 80 and used to sequence th e/Ip A gene; restriction sites are B,

E, EcoRI; He, HincW a nd H, H/ndlll. Only the Hi nd i and the Hin dlll sites used for cloning are shown.

No copy of ylpA was detected on the chromoso me

W22 703 by Southern blot hybridization

a not shown).

ylpA

1), which suggests that it is regulated by virF like th e

p genes (Cornelis ef a/. , 1989b). In order to confirm this

onitored the expression of ylpA in a pair

virF ^

an d

virF~

strains. The wr f mutant was constructed

virF

{C.

Lambert

at.,

in preparation). As show n in Fig. 5, both

pB disappe ared in the virF~ strain.

ylpA and yIpB are members of the yo p

In order to check whether ylpA is transcribed from its

33, a yop20 insertion mutant of pYVe227 (Mulder et

, 1989). As show n in Fig. 5, YlpA was still produc ed by

ylpA do not

Searching for homologies

Four genes were foun d to share significant hom ology w ith

gene yIpA. These were the fra fge ne s of plasmid pED208

from Satmonella fyp/j/(Finlay and Paranehych, 1986), of

plasm ids RIOO (Ogata et ai. 1982) and F (Jalajakumari ef

ai , 1987) and of the virulence-as sociated plasm id of S,

typhimurium (Sukupolvi ef ai . 1990). The homology to

ylpA was 76 % for fr a ff ro m pED208 (fra7 pED208) and 6 8%

fo r

traTf,

traTp^oo a n d traTs. typhimunum- The s imi lar i ty was

even more pronounced when the amino acid sequences of

mature proteins were compared (Fig. 6). The five TraT

proteins aligned without gaps. Only 26 out of 223 amino

acids differed between YlpA and TraTpEopoa (88%

identity). The number of divergent residues was higher for

the other TraT proteins: 44/223 for TraTpioo

(80%

identity),

45/223 for TraTp (80% identity) and 51/223 for

TraJs.,ypt,,mtjr.ijm (77% iden t i t y ) .

Role of YlpA in pathogenicity

We mo nitored the virulence for the m ouse of Y. enterocoli-

tica W22703(pYVe227) and of the ylpA mutant W22708-

(pYL4). The mice were inoculated intravenously (i.v.), as

was done previously, to assess the virulence of yo p2 0 and

yop48 mutants (Mulder ef ai . 1989). As shown in Fig, 7,

the bacterial coun ts in the spleen and the liver did not differ


4/10

1588 e. China. TMichiels and

G.

R. Cornelis

HetGluAspAspMetLysLys

CTGGATCCCATTTTGGTCAACCGTGGCTATGGGAGGTGCCATGCCCTGTTTTAAATGGAAGATGATAreAAGAAA

1960 Hindi

.

2000

AsnMetLysLeul leAlal lfThrAUValLeuS«>rS TValLeuValLeuSerGlyCysr.l>AIaMetScrThr

AACATCAAGTTAATAGCAATAACTGCCGTACTGTCCTCAGTGTTAGTCCTCTCCGGCTGTGGTGCGATGAGCACA

Z

AlalleLysLysArgAsnLeuGluValLysThrGlnMetSerGluThrlleTrpLeuGluCroSerSerGlnlys

GCAATCAAAAAACGTAATCTGGAAGTGAAAACGCAGATGAGTGAAACGATTTGGTTAGAGCCGTCTTCACAGAAA

ThrValTyrLeuGlnllrLysAsnThrSerAspLysAsnMetLeuGlyLeuAlaProLyslleThrLysAlaVal

ACCGTTTATCTACAGATAAAAAATACCTCAGATAAAAATATGCTTGGCTTAGCCCCCAAAATCACAAAAGCTGTG

2200

. .

GlnAapLysGlyTyrThrValThrScrSerProGluAspAlaHisTyrTrpl icIilnAlaAsnValLpuLjsAla

CAGGATAAGGGGTATAtCGTGACATCGTCCCCAGAAGATGCACATTACTGGATCCAGGCTAATGTCCTGAAAGCC

2300 aMl

AspLyaMetAspLeuArgGluAlaGluGlyPheLeuSerGlnGlyTyrGlnGlyAiaAULeuGlyAlaAiaLeu

GATAAAATGGATTTGCGTGAAGtTGAAGGATTTCTGAGTCAGGGGTATCAGGGGGCTGCGCTGGGGGCCGCATTA

2400

GlyAlnGIyl leThrGlyTyrAsnScrAKiiScrAlaGlyAiaSprl.ciitilyVulrilyLcuAlnAlaGiyl.PuVal

GGGGCTGGTATTACAGGCTACAACTCTAACTCAGCGGGAGCrTCGTTAGGAGTTGGATTGGfGGrTGGTCTTGTT

GlyHetValAUAspAlaMetValGluAspIleAsnTyrThrMetValThrAspValGlnl leSerGluLysThr

GGGATGGTCGCGGATGCGATGGTCGAGGACATCAATTATACTATGCTGACGGATGTCCAGATTTCCGAGAAAACG

2500 . . . . .

AspThrProLeuGlnThrAspAsnValAIaAlaLeuLyaGlnGlyThrSerGlyTyrLysValGlnThrSerThr

GACACCCCCCTACAGACTGACAATGTGGCGGCGCTGAAGCAAGGCACCTCTGGCTATAAAGTTCAGACCAGCACA

2600

G l n Th rG l y A s n L y s H i s G l n Ty rG l n Th rA rg V a l V a l S e rS e rA l a A s n l . y s V a l A s n L e u L y s P h e G l u G l u

CAGACGGGtAACAAACATCAATACCAGACTCGCGTGGTTTCTTCGGCTAACAAGQTtAACCTGAAATTTGAAGAA

.Hindi . 2700

AlaGlnProValLeuGluAspGlnLeuAlaLyBSerlleAlaAsnlleLeu

GCCCAGCCGCTTCTGGAAGACCAGCTAGCGAAGTCTATCGCCAATATCCTGTAAGTCATAAGCATCCTGGTATGA

. 277fi

Fig. 3, Sequence of the ylpA gene from pYVe

80 and of (fs gene prod uct. The 875 bp

sequenced fragment corresponds to c o-ordin

±7.1 to

6.2kb

of the pYV plasmid (see Fig. 2

This sequence was numbered from nucleofid

1952 to nucleotide 2776 to allow an easy

connection w ith the immediately p receding

sequence containing gene /o p 20 and presen

elsewhere (T Michiels et al.. submitted for

publication). ylpA starts about 500bp

downstream of the end of yop20 an d is

transcribed in the same onentation. The large

open reading frame (249 codons) starts at

nucleotide 2006 and ends at nucleotide 2752

the sequence. The actual ylpA gene presuma

starts at the second ATG (underlined twice).

Cleavage by signal peptidase II would occur

the Leu-Ser-Gly v Cys sequence (underlined

typical of TraT proteins. Bam HI and Hincll

restriction sites are indicated. These sequenc

data will appear in the EMBL'GenBan k/DDBJ

Nucleotide Sequence Data Libranes under th

accession number X52753 (ylpA).

significantly between both strains.

It is

noteworthy that

only one mouse out of 13 inoculated with the ylpA strain

exhibited abcesses on the liver 96 hours after inocu lation,

whereas these abcesses occurred

in

13/13 m ice inocu -

lated with the ylpA * strain.

Discussion

Plasmid pYV from Y. enterocolitica 0:9 encodes at least

two lipoproteins designa ted 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 nucleo tide seque nce 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

virulence-

associated plasmid of S. typhimurium (Finlay and Paran-

ehych,

1986; Ogata

ef

ai

1982; Jalajakukumari

ef

ai.

1987;

Sui


5/10

YlpA, a pYV-en coded lipoprotein o f yersinia 1589

y/p/i

4.9 kb

14.9 kb

3.6 kb

B2( 13.6 kb)

B

^

^ Q

i >

a. a. a.

14 9

13 6

3.6

4. Mapping of the I n3 insertion in pYL4 and detection of ylpA in

Y pseudotubercutosis.

no. 2 |82) of pYVe2 27, (ii) transpo son T n3, and {IIMV) the two

Hinc\\-BamH\ 300bp

ylpA gene. The probe

give nse to two S amHI fragments of pVL4. hybridizing with

the probe hybndized with two fragments ot 3,6 and

ylpA

This is the case for TraT proteins from plas mids R l 00 (also

known as NR1) (Reynard and Beck, 1976; Taylor and

Hughes, 1978), R6-5 (Moll

ef ai

1980), and from the

virulence plasmid

of S.

typhimurium LT2 (Rhen

and

Sukupolvi,

1988: Sukupolvi

et ai

1990). However, the

deletion of the region enco ding TraT in plP135G, the

virulence plasmid of S. typhimurium C52, did not have a

significant effect on the virulence for orally infected mice

as estimated by bacterial growth in the spleen (Michiels ef

ai , 1987),

Plasmid pYV-encoded components are known to be

involved in the resistance of Yersinia to human serum at

37X (Heesemann

et ai

1983; Pai and DeStephano,

1982). Protein PI has been shown to participate in that

activity since mutants lacking PI were rapidly killed upon

incubation in 5% human serum , irrespective of the grow th

temperature (Balligand et al., 1985). Furthermore, E. coli

MM294 expressing protein

PI

was reported

to

resist

human serum (Martinez, 1989). However, the production

of protein

PI

from

a

cloned gene restored the serum

resistance of a pYV* yers/n/a strain mutated in P I b ut did

not confer serum resistance to a pYV strain (Balligand ef

ai , 1985). This suggests that

at

least one additional

plasmid-encoded factor is involved in serum resistance.

The YlpA protein is a likely candidate in view of its high

homology with TraT, However, Balligand et

al.

(1985) did

not detect

any

difference betwee n

Y.

enterocolitica

W22708 strains carrying either the wild-type plasmid

pYVe227 or the ylpA mutant plasmid pYL4 with respect to

their ability to resist human serum. This sugge sts that YlpA

is not essential for resistance to human serum , at least in

the experimental conditions used by these authors.

Production

of

YlpA appeared to be regulated by

virF.

-^ igkD

-•YIpB

19kD

Fig.

5. Expression of YlpA and YIpB in virF and

yop2 0 mutants. Lipcprcteins were analysed by

SOS-PAGE and fluorography after incorporation

ot pH|-palmitic ac id during incubation at 37 X in

BHIo,. Unes 1-3, 16% SDS-PAGE analysis of

total cell proteins from Y. enterocolitica:

W22708(pYL4}. the ylpA muta nt {lane 1);

W22703(pBC6), the virF mutan t (lane 2):

W22703(pYVe227), the wild-type strain (lane

31.

Both YlpA and YIpB disappear in the virF

mutant, while the strain containing pYL4 only

lacks YlpA Lanes 4-7 . i d % SDS-PAGE analysis

of pHlpalmitic acid-labelled outer-membrane

proteins extracted from W22703, the plasm d ess

derivative (lane 4); W 22703(pYVe227), the

wild-

type strain (lane 5}; W22708(pYL4), the ylpA

mutant (lane 6); and U'22703(pBM33), the yop20

mutant (lane 7). YlpA was not detected in the

ylpA mutant pYL4 (lane 6), but was detected in

the yop20 mutant, pBM33 (lane 7), indicating that

yop20 and ylpA are net part ot a single operon.

Note that a chromosome -encoded protein

migrates nearly to the same position as YlpA on

this 14% acrylamide gel, while this protein was

separated from YlpA on the 16% acrylamide gel.


6/10

1590

S.

China,

T.

Michiels

and

G .

R . Cornells

pVV*.439-B0

pK[)2U8

^

Riao

.S .

(

1 phimuri UBl

- 2 0 - 1 0

MKKNMKLIAITAVLSSVLVLSG

- - » - • • •

M M l i i -

H K - -

MK - -

• - M - M

•K l .HMV -LV -

• K L M M \ - L V -

KLMMVTLV-

• - T - A - - -

• - T - A

• - T - A

pYVp439-80

ptD20fl

RIOO

1 10 20 30 10 50

rGAHSTAIKKRNLb:\KT9MSETIWLEPSS(JhTVVLalKNTSDKNMLGLAPKITKAV8DKG

A-tN--F D-S--wr.--AI)--KA--

W-^-HD-\-fB--*>--\ I)-SD-WSL-A-DI-A--

70 KO

30 100 110 120

pVVe4 39-H0 VTVTSSi EDAHVl. IWANVLKADKMllLKKAFCFLSQGYWCAAl.CAAl GAGITGVNMNSAGA

pEDZOB 1 DS T-tt F--IA S

F -«-VT--DK-> Sg W-NR--E:---\ -G

RiOO -«-\T--DK-V S« li NH t V

S.

t.vptiimurium

VT--DK-V Sg-y>-NH--E--LS

A- --

pED208

F

RIOO

S.

ip/ji

pED20B

RIOO

5 t

yph

mtir

1.10 no 150 160 170 180

SLGVGLAAGLVGHVADAMVEDI WTHVTIlVal St:KTDTPLWTDM\ ,VALK«GTSGYKVgTS

T A -- [ TASV-- - - -

T

A V I

A-H-KATVT

R A-1

T

A \ [

A-R-KATVT

R A-1

T

A 1

R-KAT\T

R A-I

l y d ZOO 210 22a

TeTGNKliaY8TRV\ SSANKVNl,KFFEA«F Vl.EDyLAKSl ANI L

9-K

1 K \

E---a-K

-J K - - - -

-K---a-K S K

-K y -h --N K K-

IDENTITY

197/223

178/223

79/223

172/223

Fig.

6 .

Similarities between TraT proleins.

T

amtno acid sequence of Yip A trom PYVe43

js aligned with those

of

TraT from p6 D20e

and Pafanchych, 1986], F (Jalaiakuman

et

a

J986),R100 (Ogata e(aA, 1982).

and S,

typhimurium (Sukupoivi

el

ai.. 1990). The a

adds

are

numbe red from ifie c ysteine that

conslitLrtes the first residue of the mature p

Dashes symbolize residues identical

to

thos

YipA. Divergent residues are indicated.

like the production of the Yops. This co-regulation sug-

gests that, like Yop s, YlpA could

be

involved

in

pathogen-

icity. 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 dem onstrate

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 com ponen t involved in com pleme nt neutral-

ization because normal mouse serum has been shown to

have a poor bactericidal effect against E. coli (Vaara ef

ai.

1984), Thus

our

result sugg esting that YlpA

is not a

clear

virulence factor must

be

interpreted c autiously.

Alternatively, the presence of YlpA on the pYV plasm id

could simply be an evolutionary vestige of this p lasmid.

Indeed, the

replication fun ction

of

pYV

is

related

to

that

of

plasmid RIOO (Vannooteghem

and

Cornells, 1990) while

the partition

and

stabilization fu nction of

pYV is

homolo-

gous

to

that of

F

(Bakour

ef ai.

1983; Biot

and

Cornells,

1988), both plasmids containing

a

fra fg en e. However,

one argument contradicts this hypothesis: ylpA is not part

of a transfer op eron in pYV (Bakour ef at.. 1983) while in

both RIOO and

F

traTis included in

the

ra

operon.

Experimental procedures

Bacteriat strains and plasmids

Y.

enterocolitica W22703 (nalidixic acid-resistant) and

Y.

en

colitica W22708 (slreptomycin-resistant)

are

derivatives o

same Res Mod mutant

of Y.

enterocolitcaVJ227 (serotyo

(Cornells and Colson, 1975). Y. enterocotitica 439-80

wild-type

0:9

strain.

The pYV

plasmids of

all

these strain

indistinguishable

by

SamHI, EcoRI or Ss fll res triction ana

(Laroch eefa/., 1984).

E. coliJM^ 01 (Yanisch-Perron

etal..

1985)and LK111 (rec

from

M.

Zabeau)

are

F . /acZ delta

M15

strains. Strain

S

containing a copy

of

RP4 integrated into

its

chromosom e (S

etal., 1983). was used

to

mobilize onT-containing plasmids

Phasmids pT Zi8R

and

pTZ19R

are

from Pharmacia, pT

(similar to pTJS81) is a

pUC

derivative containing

the

tra

origin (oriT) from piasmid

RK2

(Schmidhauser

and

Hel

1985).

Construction

of

mobilizable derivatives ofpTZtSR

an

pTZ19R

In order

to

facilitate

the

introduction

of

recombinant plasm

yersiniae, we constructed mobilizable denvatives of the ver

cloning vectors pTZ18R

and

PTZ19R. A ±760bp Hinc\\-S


7/10

YlpA,

a pYV-encoded tipoprotein of \ersm\a 1591

o

7

6

5

3

A

r

i ^ ^ ^

^ ^

O pYV*

D pVL4

1 I I I

0 6 12 18 24 30 36 42 48 54 60 66 7? 78 84 90 96

0 6 12 18 24 30 364 2485 460 66 72 78 84 90 96

Hours

7. Growth of strains K enterocolitica Vi/22703(pYVe227) and

yipA derivative, in the spleen (A) and the liver (B) of

v. with 5 x 10' bad eria (arrow).

a 4-day

Each point was the mean value of a group of four mice;

XUe

. pYVe227 ' bacteria; L], pYL 4' bacteria.

plasmid pTJS 82, containing the transfer origin of

{oriT) was cloned into pTZ19R linearized by partial

I

dige stion. The recom binants ca rrying the o/'/7 fragment were

E. coli S17.1 as

E.

co / /SI

7.1:

i) all thelranstorm ants, selected on am piciflin, were collected m a

Y .

W22703; (iii) the transfer of mobilizable derivatives

contain

fragm ent inserted in the Dral site at co-ordinate 1372 of

aced the 346 bp PvuU fragment containing the multicloning

e of pBO l 9R with that of pTZ I 8R. The constructed mobilizable

on analysis, pBC I 8R lost the D ral site at co-ordinate 1353

ty to be pa ckaged as single-stranded

7, to complemen t the /acZ delta M l5 mutation or to be

E. coli S17.1.

pH]-patmitic acid tabelling of tipoproteins

Yersinia strains were inoculated to an optical density ot 0.1 ODsoo

in 5-1 0m l of brain heart infusion broth

fBHI,

Difco) supplem ented

with 0,4% (w/v) glucose as well as either 5mM OaCtz (BHIca) or

200mM Na oxalate and 200mM MgClp (BHIoJ. After 2h

incu-

bation at room temperature with shaking, 250-500[iCi of [9.10-

^H]-palmitic acid (30Ci mmol ', from Dupont-NEN) was added to

the culture and bacteria were further incubated at either 37 C or

25°C for 4h . Membrane proteins were prepared acco rding to

Achtman ef al. (1978). Proteins were analysed by SDS-PAGE, on

gels containing 14% or 16% of 29/1 (w/w) acrylamide/bisacryl-

amide (Serva). The gel was treated with En^Hance (Dupont-NEN)

for fluorography as recommended by the manufacturer. Auto-

radiography was performed over 2-10 days at -7 0°C using FUJI

NIF X-ray films.

Infection of mice

Specific pathogen-free BAL8/c female mice (bred at the Uni-

versity of Louvain) 6 weeks old were given (intraperitoneally)

0.5ml of saline (NaCI 0.15M) containing 20 mg m l ' desferriox-

amine (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,

wash ed onc e and then susp ende d in saline. Grovt̂ 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 we re counted after inc ubation for 4 8h at 28°C. Minimal

detectable limits were 50 bacteria per organ. Results were

expressed as the log,o of bacterial counts.

Proteinase treatment of membrane fractions

In order to discriminate between lipoproteins and other lipid-con-

taining components, membrane tractions were incubated with

increasing quantities (10fi.g ml ' to 1 mg ml ')o f either pronase

(Serva) or proteinase K (Serva). After 30 min incuba tion at 3 7 X ,

membranes were either pelleted by a 60 min centrifugation at

15000

r.p.m.

or were precipitated by the ad dition of

4

vol. acetone

and subsequently harvested by centrifugation.

Southern blot analysis

DNA was dige sted, electrophoresed on a 1 % agarose gel,

denatured and transferred into a nylon membrane (Hybond-N,

Amersham) by standard methods (Maniatis er al.. 1982). The

probe was electroeluted from a polyacryiamide gel and labelled

with p^Pj-dATP (D upont-NEN, lOOOCi mm ol ') by nick trans-

lation (M aniatis et a(., 1982).

Nucieotide sequence and sequence analysis

The nucleotide sequence was determined by the method of

Sanger ef al . (1977), using T7 DNA polymerase for eiongation

(Sequenase, from USB). Single-stranded DNA was obtained from


8/10

1592 a China, T. Michiets and

G.

R. Cornetis

strains LK111 or JM101 overinfected with the M13K07 helper

phage.

DNA and protein sequences were analysed on a Micro Vax

Computer (Digital Equipment Corp.) with the program packag e of

Claverie (1984) and the FASTN, FASTP (Lipman and Pearson.

1985), ALIGN, and NAQ software from the Protein Identification

Resource program package.

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 fo r discussion. This

work was supported by the Belgian Ministry for Sciences (Action

Concertee 86/91686) and by the Belgian Fund for Medical

Research (FRSM — co nven tion 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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the pyv plasmid of yersinia encodes a lipoprotein,

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