The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri

Molecular Microbiology (2000) 38(4), 760±771

The virulence plasmid pWR100 and the repertoire ofproteins secreted by the type III secretion apparatus ofShigella flexneri

Carmen Buchrieser,1 Philippe Glaser,1 Christophe

Rusniok,1 Hafed Nedjari,1 HeÂleÁne d'Hauteville,2 Frank

Kunst,1 Philippe Sansonetti2 and Claude Parsot2*1Laboratoire de GeÂnomique des Microorganismes

PathogeÁnes, and 2UniteÂ de PathogeÂnie Microbienne

MoleÂculaire, INSERM U389, Institut Pasteur, 28 rue du

docteur Roux, 75724 Paris Cedex 15, France.

Summary

Bacteria of Shigella spp. are the causative agents of

shigellosis. The virulence traits of these pathogens

include their ability to enter into epithelial cells and

induce apoptosis in macrophages. Expression of

these functions requires the Mxi±Spa type III secretion

apparatus and the secreted IpaA±D proteins, all of

which are encoded by a virulence plasmid. In wild-type

strains, the activity of the secretion apparatus is

tightly regulated and induced upon contact of bacteria

with epithelial cells. To investigate the repertoire of

proteins secreted by Shigella flexneri in conditions of

active secretion, we determined the N-terminal

sequence of 14 proteins that are secreted by a mutant

in which secretion was deregulated. Sequencing of the

virulence plasmid pWR100 of the S. flexneri strain

M90T (serotype 5) has allowed us to identify the genes

encoding these secreted proteins and suggests that

approximately 25 proteins are secreted by the type III

secretion apparatus. Analysis of the G1C content and

the relative positions of genes and open reading

frames carried by the plasmid, together with informa-

tion concerning the localization and function of

encoded proteins, suggests that pWR100 contains

blocks of genes of various origins, some of which

were initially carried by four different plasmids.

Introduction

Bacteria of Shigella spp. are Gram-negative, non-spor-

ulating, non-encapsulated, facultative aerobic, non-motile,

straight rods that belong to the family Enterobacteriaceae

and to the tribe Escherichiaeae. They are divided into four

groups (designated species), Shigella boydii, Shigella

dysenteriae, Shigella flexneri and Shigella sonnei, all of

which cause shigellosis, a human disease characterized

by the association of fever, abdominal cramps and

diarrhoea containing blood and mucus. Infections are

localized in the colon and restricted to the outermost layer

of the intestinal wall. Invasion of the tissue by bacteria

induces a strong inflammatory reaction and leads to

destruction of the epithelium.

Essential attributes of Shigella spp. required for

virulence are their ability to enter into and disseminate

within epithelial cells and to induce apoptosis in infected

macrophages (Takeuchi et al., 1965; Clerc et al., 1986;

Zychlinsky et al., 1992). The role of a 200 kb virulence

plasmid in cell invasion was demonstrated 20 years ago;

transposon insertions and deletions within the plasmid

abolished the ability of bacteria to enter into epithelial

cells, and mobilization of the plasmid from S. flexneri to

Escherichia coli conferred an invasive phenotype to the

transconjugants (Sansonetti et al., 1982; 1983a; Sasa-

kawa et al., 1986). Genes necessary for entry are

clustered within a 31 kb region of the plasmid, designated

the entry region (Maurelli et al., 1985; Sasakawa et al.,

1988). This region contains (i) the mxi and spa genes

encoding components of a type III secretion apparatus

(Andrews and Maurelli, 1992; Venkatesan et al., 1992;

Allaoui et al., 1993a; Sasakawa et al., 1993); (ii) the ipaA,

B, C and D and ipgD genes encoding proteins secreted by

this machinery (Baudry et al., 1988; Sasakawa et al.,

1989; Venkatesan and Buysse, 1990; Menard et al.,

1993; Allaoui et al., 1993b; Niebuhr et al., 2000); (iii) the

ipgC gene encoding a cytoplasmic chaperone required for

stability of IpaB and IpaC (Menard et al., 1994a) and the

ipgE gene encoding a cytoplasmic chaperone required for

stability of IpgD (Niebuhr et al., 2000); (iv) the virB gene

encoding a protein required for transcription of the mxi,

spa and ipa genes (Adler et al., 1989); and (v) other

genes, the functions of which have not been elucidated.

The ability of bacteria to move within the cytoplasm of

infected cells relies on the expression and proper

localization of the outer membrane protein IcsA (VirG),

the gene for which lies outside the entry region (Makino

et al., 1986; Bernardini et al., 1989; Lett et al., 1989).

Expression of both icsA and virB is controlled by VirF, a

transcriptional activator of the AraC family that is encoded

Q 2000 Blackwell Science Ltd

Accepted 6 September, 2000. *For correspondence. E-mail [email protected]; Tel. (133) 1 45 68 83 00; Fax (133) 1 45 68 89 53.

by the plasmid (Tobe et al., 1993). The virulence plasmid

also carries the sepA gene, which encodes a secreted

serine protease of the IgA1 protease family (Benjelloun-

Touimi et al., 1995). Two chromosomal pathogenicity

islands encoding an aerobactin iron acquisition side-

rophore system and a secreted protease related to SepA,

respectively, have also been described recently (Moss

et al., 1999; Vokes et al., 1999; Al-Hasani et al., 2000). In

addition, numerous chromosomal genes that are not

specific to Shigella spp. are involved in the regulation of

expression, production or proper localization of the

virulence plasmid-encoded proteins (Maurelli and Sanso-

netti, 1988; Okada et al., 1991; Sandlin et al., 1996).

The type III secretion pathway is involved in transloca-

tion of effector molecules from the bacterial cytoplasm to

or beyond the cytoplasmic membrane of eukaryotic cells

(reviewed by Hueck, 1998). The S. flexneri type III

secretion apparatus is weakly active during growth of

bacteria in laboratory media, and its activity is induced

upon contact of bacteria with epithelial cells (Menard et al.,

1994b; Watarai et al., 1995a). There is evidence that IpaB

and IpaC insert within the cytoplasmic membrane of

target cells and might form a channel allowing transloca-

tion of other proteins, such as IpaA and IpgD (Tran Van

Nhieu et al., 1997; Blocker et al., 1999; Niebuhr et al.,

2000). Parts of IpaB and IpaC might also be directly

involved in altering cellular signalling processes to result

in internalization of bacteria and triggering of apoptosis

(Zychlinsky et al., 1994; Tran Van Nhieu et al., 1999). The

Mxi±Spa secretion apparatus can also be activated by

such external inducers as the dye Congo red, extracellular

matrix components or bile salts (Pope et al., 1995;

Watarai et al., 1995a; Bahrani et al., 1997). Constitutive

secretion, i.e. secretion in the absence of inducers, is

observed after inactivation of the ipaB or ipaD genes

(Menard et al., 1994b), and analysis of the culture

medium of a DipaBCDA mutant indicated that < 15

proteins are secreted by this strain (Parsot et al., 1995).

To analyse the repertoire of proteins secreted by S.

flexneri in conditions of active secretion, conditions that

might be relevant to those encountered by bacteria during

infection of the host, we have determined the N-terminal

sequence of 14 proteins that are secreted by the

DipaBCDA mutant. Sequencing of the virulence plasmid

pWR100 allowed us to identify all the genes encoding

these secreted proteins. Sequence analysis suggests that

< 25 proteins might be secreted by the Mxi±Spa

secretion apparatus and indicates that pWR100 contains

blocks of genes of different origins.

Results and discussion

N-terminal sequence of proteins secreted by the type III

secretion apparatus

To identify proteins secreted by the Mxi±Spa secretion

apparatus, we determined the N-terminal sequence of

proteins secreted by the DipaBCDA strain SF635, a

derivative of the wild-type S. flexneri strain M90T

(serotype 5) that secretes constitutively (Parsot et al.,

1995). Secreted proteins were separated by SDS±

PAGE, and proteins ranging in size from 10 to 65 kDa

were used for N-terminal sequence determination. Some

species contained two proteins, the N-terminal sequence

of which could nevertheless be identified unambiguously

on the basis of the relative intensities of the signals

obtained at each sequencing cycle. Thus, the N-terminal

sequence of 14 proteins was determined (Table 1). Two

of these sequences corresponded to the N-terminal

sequences of VirA and IpaH9.8 (Uchiya et al., 1995;

Demers et al., 1998). In addition, these data indicated

that four proteins encoded by the entry region, MxiC,

MxiL, Spa32 and IpgB1, were secreted (see below). The

genes encoding the other secreted proteins were then

identified by determining the complete sequence of the

virulence plasmid pWR100 from the wild-type strain

M90T.

Sequencing of pWR100

To determine the sequence of pWR100, we used both a

library of 10 cosmids carrying overlapping inserts of

40 kb (Maurelli et al., 1985) and recombinant plasmids

carrying most of the BamHI and SalI fragments

subcloned from pWR100. The final sequence assembly

of the virulence plasmid was confirmed by comparing the

restriction patterns predicted from the sequence with

those obtained by digestion of pWR100 with different

enzymes. No major differences were detected between

the sequence determined here and previously for genes

identified on pWR100, pMYSH6000 (the virulence

plasmid from a S. flexneri strain of serotype 2a;

Table 1. N-terminal sequence of proteins secreted by the DipaBCDAmutant SF635.

Size (kDa) N-terminal sequence Protein

62 MLPINNFS IpaH9.862 MFSVNNTH IpaH7.862 MKPINNH IpaH4.558 MNISETLSA OspC146 MQTSNITHE VirA40 MLDVK MxiC34 ALDNINL Spa3231 MNILDGVRPY OspB28 MPIKKP OspF25 MQILNKIL IpgB125 MSINNYGLH OspD124 MKITSTIIQ OspG10 MLTQTIFP OspE110 MINQINAS MxiL

The virulence plasmid of S. flexneri 761

Q 2000 Blackwell Science Ltd, Molecular Microbiology, 38, 760±771

762 C. Buchrieser et al.


Sasakawa et al., 1986) or the virulence plasmids from

other S. flexneri strains (Sasakawa et al., 1992; Parsot

and Sansonetti, 1999).

pWR100 is composed of 213 494 bp (Fig. 1).

Sequence analysis identified 93 DNA fragments that

have over 90% identity with known insertion sequence

(IS) elements and constitute < 58 kb of pWR100

(Table 2). Several DNA fragments were tentatively

identified as parts of seven putative new IS elements,

designated ISSfl1±ISSfl7 (Mahillon and Chandler,

1998). These new ISs correspond to 29 fragments

that account for 19 kb of sequence. Accordingly, one-

third of the plasmid appears to correspond to IS

elements. Outside these ISs, we identified < 100 open

reading frames (ORFs) that correspond to potential

genes and represent 100 kb of sequence. These ORFs

were labelled according to the co-ordinates (in kb) of

their position on the sequence, and we used the current

nomenclature to designate most of the ORFs that

correspond to genes identified previously (Sasakawa

et al., 1992). Sequence analysis indicated that pWR100

carries several multigene families encoding related

proteins. For the sake of clarity, genes belonging to

the same family were designated using the same

generic name, e.g. ospD, followed by a different

number, e.g. ospD1, ospD2 and ospD3. Accordingly,

the previously characterized senA gene that was

proposed to encode an enterotoxin (Nataro et al.,

1995) was designated ospD3.

The entry region

Genetic analysis has shown that a 31 kb fragment of the

virulence plasmid is necessary and sufficient for entry of

bacteria into epithelial cells (Maurelli et al., 1985;

Sasakawa et al., 1988; 1993). This fragment contains

34 genes that are clustered in two regions transcribed in

opposite directions. The first region contains 10 genes,

from icsB to virB, and the second region contains 24

genes, from ipgD to spa40 (Fig. 1). Many of these genes

have already been studied (see References in the

Introduction and Hueck, 1998 for a review) and will not

be described here.

N-terminal sequencing of proteins secreted by SF635

indicated that, in addition to IpaA±D and IpgD, several

other proteins encoded by the entry region are secreted:

Spa32, MxiC, MxiL and IpgB1. Both Spa32 and MxiC

exhibit sequence similarities to components of the type III

secretion apparatus of Yersinia and Salmonella spp., and

Spa32 has been shown to be involved in the activity of the

Mxi±Spa secretion apparatus (Watarai et al., 1995b). No

protein homologous to MxiL was detected in protein

sequence databases; however, the position of the mxiL

gene within the mxi operon suggests that MxiL is involved

in the secretion apparatus. Inactivation of ipgB1 had no

effect on secretion of the IpaA±D proteins (R. MeÂnard, P.

Sansonetti and C. Parsot, unpublished results), which

suggests that IpgB1 is not a component of the secretion

apparatus. IpgB1 exhibits 20% sequence identity with (i)

TrcA (Tobe et al., 1999a) and LEE19 (Elliott et al., 1998),

which are encoded by the chromosomal LIM and LEE loci,

respectively, of enteropathogenic E. coli (EPEC) strains;

(ii) TrcP, which is encoded by the adherence factor

plasmid of EPEC (Tobe et al., 1999b); and (iii) IpgB2,

which is also encoded by pWR100. The most closely

related proteins are IpgB2 and TrcA, which exhibit 37%

sequence identity. TrcA has been proposed to be a

cytoplasmic chaperone required for the production of

BfpA and intimin (Tobe et al., 1999b). Genes encoding

proteins related to BfpA and intimin are not present on

pWR100, and the observation that IpgB1 is secreted by

the type III secretion apparatus of S. flexneri suggests that

related proteins are also secreted.

Genes located between virB and spa40 all have a low

G1C content (average 34.2%). On the left side, the

Fig. 1. Genetic map of pWR100. The position and direction of transcription of the various genes and ORFs are indicated by arrows. ORFs forwhich no function could be proposed are labelled according to their co-ordinates (in kb) on the sequence. Genes truncated or inactivated byframeshifts are indicated in parenthesis. The colours refer to the G1C content of each ORF: red, , 40%; blue, between 40% and 50%; green,. 50%. The position of ISs is indicated by yellow bars. The sequence of pWR100 has been submitted to the DDJ/EMBL/GenBank databasesunder accession number AL391753.

Table 2. IS elements detected on pWR100.

IS(name)

Length(bp)

GC(%)

Fragments(number)

Total size(bp)

CompleteIS

IS629 1310 54.0 16 10 972 5IS600 1264 49.1 16 9962 2IS1294 1689 55.0 6 9904 2IS2 1331 53.0 9 4567 2IS100 1953 51.4 6 4372 0IS4 1426 53.5 5 3059 1IS1F 768 51.9 4 2983 3IS91 1829 52.9 4 2521 0IS3 1258 53.6 8 2510 0IS630 1153 52.2 6 2417 1IS1N 765 51.0 6 1786 0IS911 1250 51.6 2 1352 0IS150 1443 44.1 2 1097 0IS21 2131 52.3 3 705 0ISSfl1 1301 44.0 2 1610 1ISSfl2 . 718 49.9 2 718 0ISSfl3 2729 52.2 2 5458 2ISSfl4 1451 57.4 3 2949 2ISSfl5 . 2339 55.1 12 3914 0ISSfl6 . 684 49.8 4 1814 0ISSfl7 924 50.1 4 2753 1



fragment located between virB and the closest IS element

(IS100) is 3 kb long, has a G1C content of 38.7% and

contains one ORF, ipaJ (Buysse et al., 1997). On the right

side, the fragment located between spa40 and IS600 is

0.9 kb long, has a G1C content of 33.2% and contains

two ORFs, orf131a and orf131b. This suggests that these

genes and their flanking regions have the same origin as

the ipa, ipg, mxi and spa genes. The IS elements located

on both sides of the entry region are truncated, and each

lacks the extremity directed towards the entry region,

which indicates that rearrangements, including deletion of

one extremity of each IS and its flanking sequences, had

taken place after acquisition of the entry region.

The osp genes

The genes encoding all the proteins secreted by SF635

(Table 1) were identified on pWR100. These include virA

(Uchiya et al., 1995), ipaH4.5, ipaH7.8, ipaH9.8 (Hartman

et al., 1990; Venkatesan et al., 1991; Demers et al., 1998;

see below) and six new genes that were designated osp

(outer Shigella proteins): ospB, ospC1, ospD1, ospE1,

ospF and ospG. In addition, genes encoding proteins with

sequence similarities to Osp proteins were also detected

on pWR100. These sequence similarities suggested that

the products of these genes might also be secreted, and

these genes were designated ospC2, ospC3, ospC4,

ospD2, ospD3 and ospE2. The N-terminal sequences of

OspE1 and OspE2 are identical, and the 10 kDa protein

secreted by SF635 could correspond to OspE1, OspE2 or

a mixture of both. The sizes of OspD2, OspD3, OspC2

and OspC3 are similar to those of IpaH proteins (about

60 kDa), which might explain why these proteins were not

resolved from IpaH proteins by SDS±PAGE. Alterna-

tively, the conditions of expression of these proteins might

be different from those used to grow SF635. The

nucleotide sequences of ospE1 and ospE2 and those of

ipaH1.4 and ipaH2.5, which are located downstream from

ospE1 and ospE2, respectively, are almost identical,

indicating that these regions result from a duplication

event. This duplication was followed by the insertion of IS

elements upstream from ospE1, within the ospE2±

ipaH2.5 intergenic region, and downstream from

ipaH2.5. The sequences of ospC2, ospC3 and ospC4

exhibit 96% identity to each other. However, two deletions

within ospC4, at positions 98 and 560, result in frame-

shifts that inactivate this gene. The sequence of ospC1

exhibits only 74% identity with those of the other ospC

genes, suggesting a more ancient duplication event.

Members of the ospD family are more distantly related

compared with those of the ospE and ospC families.

OspD2 (569 residues) and OspD3 (SenA; 565 residues)

exhibit 38% sequence identity over their entire length, and

the C-terminal region of both proteins contains six repeats

of 44 residues (Fig. 2). A similar repeat is present three

times in the C-terminal region of OspD1 (225 residues).

No proteins sharing sequence similarity with Osp

proteins were detected in protein sequence databases,

except for OspF, which exhibits 63% sequence identity

with SpvC, a protein encoded by the virulence plasmid of

Salmonella typhimurium. The spvC gene is part of a locus

comprising five genes that are required for S. typhimurium

to cause systemic infections of the reticuloendothelial

organs during experimental infections of animals (Gulig

et al., 1993). The observation that OspF is secreted by S.

flexneri suggests that SpvC might also be secreted.

The G1C content of osp genes ranges from 31.2%

(ospC1) to 37.9% (ospF), which suggests that these

genes have the same origin as the entry region (34.2%

G1C). Indeed, the G1C content of the genes appears as

a discriminatory criterion to classify genes carried by

pWR100. Of the 109 genes and ORFs detected on

pWR100, 56 have a G1C content lower than that of

spa47 (39.3%), which is the gene of the entry region that

Fig. 2. The repeated motifs of OspD proteins.The sequences of the C-terminal domain ofOspD1 (225 residues), OspD2 (569 residues)and OspD3 (565 residues) have been alignedto show the 44-residue repeated motif.Residues that are identical in at least sixrepeats are underlined and indicated in theconsensus sequence. Dots indicate gaps thathave been introduced to maximize thealignment.



has the highest G1C content. In addition to genes of the

entry region, these include all the osp genes and only a

few other genes: virF (30.4%), orf137 (31.0%), orf13

(31.6%), orf81 (34.1%), orf212 (34.5%) and icsP (sopA)

(37.6%). Both virF and orf81 encode proteins with

similarities to transcriptional activators of the AraC family.

VirF is required for the expression of virB and therefore is

functionally related to the entry region. No convincing

similarities were detected between the sequences of

Orf13, Orf137 or Orf212 and proteins present in data-

bases. The icsP gene encodes an outer membrane

protease that is involved in cleavage of IcsA and does

not appear to be related to genes of the entry region (Egile

et al., 1997; Shere et al., 1997). Whether the products of

the ORFs other than icsP that have a low G1C content

have any functional relationship with the Mxi±Spa

secretion machinery or Osp proteins will require further

investigation.

The ipaH genes

Previous analysis has shown that pWR100 carries five

ipaH genes, which were designated ipaH1.4, ipaH2.5,

ipaH4.5, ipaH7.8 and ipaH9.8 according to the size of the

HindIII fragment of the virulence plasmid that carries each

gene. The present analysis indicates that ipaH1.4 and

ipaH2.5 are not incomplete, as was proposed (Venkate-

san et al., 1991), and corrects the reading frame of the

beginning of ipaH7.8 (Hartman et al., 1990). This

correction is consistent with the N-terminal sequence

determined for a protein of the size of IpaH7.8 that is

secreted by SF635. Except for the presence of an IS629

element at the 3 0 end of ipaH2.5, the entire sequences of

ipaH1.4 and ipaH2.5 are almost identical and, for the sake

of clarity, the ipaH2.5 gene will not be discussed further.

Members of the ipaH family are characterized by (i) a 5 0

variable region of 600±760 bp that encodes six to eight

repeats of a 20-residue motif; and (ii) a 3 0 constant region

of 839 bp that is identical in all genes (Venkatesan et al.,

1991). In fact, the region of identity between ipaH9.8,

ipaH7.8 and ipaH4.5 is extended by 98 bp at the 5 0 end of

the constant region, and the region of identity between

ipaH7.8 and ipaH4.5 is extended by 29 bp further

upstream (Fig. 3). Downstream from the 3 0 end of the

constant region, defined by the site of insertion of an

IS629 element in ipaH4.5 (and ipaH2.5), the sequences of

ipaH7.8, ipaH9.8 and ipaH1.4 are identical over 55 bp,

except for a small deletion within ipaH7.8. In contrast,

there is little sequence similarity among the 5 0 parts of

ipaH genes, even though the deduced amino acid

sequences are related. The difference in sequence

conservation of the 5 0 and 3 0 parts of ipaH genes cannot

be explained in a classical scheme of divergent evolution

after duplication of an ancestral copy, even by hypothe-

sizing that functional constraints on the C-terminal domain

of IpaH proteins might be more important than those on

the N-terminal domain. Moreover, the G1C contents of

the variable and constant regions of ipaH genes are

clearly different; the G1C content of the variable regions

ranges from 35.2% for ipaH7.8 to 39.3% for ipaH4.5 and

that of the constant region is 53.9%. These observations

indicate that the two parts of ipaH genes have different

origins and suggest that the constant region of ipaH

genes might result from independent conversion events

on pre-existing copies of ipaH `genes' by an unknown

mechanism.

The icsA and virK regions

The icsA (virG) gene encodes the outer membrane protein

that is directly responsible for the motility of bacteria within

the cytoplasm of infected cells (Makino et al., 1986;

Bernardini et al., 1989; Lett et al., 1989; Goldberg and

Theriot, 1995; Egile et al., 1999), and the virK gene

encodes a protein that is required for proper production or

localization of IcsA (VirG) (Nakata et al., 1992).

Fig. 3. Schematic representation of the constant region of ipaH genes. The sequences of various fragments of each ipaH gene are shown,separated by dashes. Numbers on the first line refer to the co-ordinates of the nucleotides (indicated by stars) within the coding sequence ofipaH7.8. Regions that are identical between several ipaH genes are boxed, and their length is indicated on the last line. For the sake of clarity,the sequence of ipaH2.5, which is identical to that of ipaH1.4, is not shown.



The icsA and virK genes are not linked on the plasmid;

however, they are both present within clusters of genes that

have a G1C content of approximately 42%. The first cluster

includes four genes, icsA (41.0% G1C), orf149 (41.7%

G1C), ushA (40.5% G1C) and phoN1 (42.4% G1C),

which are separated by ISs (Fig. 1). Part of the sequence

deduced from orf149 exhibits 82% identity with an internal

fragment of PapC (between residues 268 and 349), the

outer membrane usher protein of Pap pili from E. coli. The

ushA gene encodes a protein that exhibits 76% identity with

the UshA protein of E. coli and S. typhimurium, a

periplasmic UDP-sugar hydrolase (Burns and Beacham,

1986; Edwards et al., 1993). The phoN1 (phoNsf) gene

encodes a periplasmic acid phosphatase (Uchiya et al.,

1996) that exhibits 50% sequence identity with the product

of phoN2 (apy), another periplasmic phosphatase encoded

by the virulence plasmid (Bhargava et al., 1995). The

second cluster of genes includes orf185, orf186, virK and

msbB2. The orf185±orf186, orf186±virK and virK±msbB2

intergenic regions are 2, 4 and 63 bp long, respectively,

suggesting that these genes and ORFs might be part of the

same operon. The products of orf185, orf186 and virK

exhibit 90% sequence identity with the products of three

adjacent ORFs carried by pAA2, a plasmid harboured by

enteroaggregative E. coli (Czeczulin et al., 1999). Likewise,

the products of orf185, orf186 and msbB2 exhibit 65%

sequence identity with the products of ORFs carried by

pO157, the virulence plasmid of an EPEC strain (Burland

et al., 1998; Makino et al., 1998). Genes encoding proteins

related to Orf185, Orf186, VirK and MsbB2 are also present

on the chromosome of various bacteria, including, E. coli, S.

typhimurium and Neisseria meningitidis, indicating that,

although widespread among virulence plasmids, these

genes are not specific to plasmids. The MsbB2 protein

exhibits 70% sequence identity with the chromosomally

encoded MsbB protein of E. coli, an acyltransferase that is

involved in lipid A modification (Clementz et al., 1997).

The similar G1C content of icsA, orf149, ushA and phoN1

and their genetic linkage suggest that these genes might

have the same origin, and the presence of a remnant of the

papC gene (orf149) suggests that, in the past, this region

might have carried a pap operon. The function of VirK is not

yet known; however, a functional relationship between VirK

and IcsA is suggested by the phenotype of the virK mutant

(Nakata et al., 1992). In that respect, it is noteworthy that the

icsA and virK regions have a similar G1C content that is

different from other parts of the plasmid.

Replication, partition and transfer functions

Hybridization studies indicated that pMYSH6000 is a

RepFIIA (IncFII) replicon (Makino et al., 1988; Silva et al.,

1988). This system consists of (i) oriR, which is the origin

of replication; (ii) RepA, which is required for replication at

oriR; (iii) CopB, which represses transcription at the repA

promoter; (iv) TapA, which is required for expression of

RepA; and (v) CopA, which is an antisense RNA

functioning as a copy number control element (Blomberg

et al., 1994; Malmgren et al., 1997). Each of these

elements was detected on pWR100. RepA, CopB and

TapA of pWR100 exhibit 95±100% sequence identity with

the corresponding proteins from plasmids R100, a 90 kb

Fig. 4. The par and stb regions of pWR100. The position and orientation of the par and stb genes of pWR100, R100 and P1 are shown byarrows, with the number of residues of encoded proteins. The nucleotide sequences of the 3 0 part of parS sites (indicated by boxes) areshown, with repeated motifs indicated in bold characters. Upstream from the stbA genes, the cis-acting sites are indicated by boxes (not toscale), and their A1C contents are indicated within boxes. The sequences and numbers of occurrences of repeats detected in the cis-actingsites are shown on the left.



conjugative IncFII resistance plasmid from a clinical

isolate of S. flexneri (Nakaya et al., 1960), and pO157.

As for other IncFII replicons, a DnaA box and a putative

origin of replication were detected downstream from repA

on pWR100. The putative CopA antisense RNA of

pWR100 exhibits over 90% sequence identity with those

of plasmids R100 and pO157, which suggests that the

copy number of pWR100 might be similar to that of R100,

i.e. one or two copies per chromosome.

Partitioning systems are essential for inheritance of low-

copy-number plasmids in daughter cells. Sequence

analysis revealed the presence of two partitioning

systems on pWR100 (Fig. 4). The first system, desig-

nated par (bp 29 020±31 196), is similar to the parABS

system of bacteriophages P1 and P7 (Ludtke and Austin,

1987; Chattoraj and Schneider, 1997) and plasmid pMT1

of Yersinia pestis (Lindler et al., 1998), whereas the

second system, designated stb (bp 158 164±159 515), is

similar to the stbAB system of plasmids R100 (NR1) (Miki

et al., 1980; Tabuchi et al., 1988) and pB171, the

adherence factor plasmid of EPEC (Tobe et al., 1999b).

ParA and ParB of pWR100 exhibit 75% and 58% sequence

identity to ParA and ParB, respectively, of P1, and the

region located downstream from parB on pWR100

contains sequence motifs that are similar to those of the

parS site of P1 (Hayes and Austin, 1994). On the other

hand, StbA and StbB of pWR100 exhibit 39% and 29%

sequence identity with StbA and StbB, respectively, of

R100, and a putative cis-acting site exhibiting a strong

A1C/T1G bias and several repeats (Tabuchi et al., 1988)

is present upstream from stbA on pWR100 (Fig. 4). These

similarities suggest that both the par and the stb systems of

pWR100 are functional. Another protein encoded by

pWR100, VirB, exhibits sequence similarities with mem-

bers of the ParB family (Adler et al., 1989). However, no

gene encoding a protein homologous to ParA is present in

the vicinity of virB or elsewhere on the virulence plasmid

(except for the parA gene described above), which

suggests that VirB is not involved in plasmid partitioning.

VirB is required for transcription of genes of the entry

region by a mechanism that has not yet been elucidated.

Plasmids often carry a system that is involved in post-

segregational killing of bacteria that have not received a

copy of the plasmid. pWR100 encodes two proteins that

exhibit 85% and 95% sequence identity to CcdA and

CcdB, respectively, of plasmid F, which are responsible

for killing of segregant cells (Jaffe et al., 1985). In addition,

pWR100 carries the mvpT and mvpA genes that are

located on the complementary strand of the trbH gene

and encode a toxin and an antidote, respectively, which

can promote stable inheritance of recombinant plasmids

carrying the origin of replication of pMYSH6000 (Rad-

nedge et al., 1997; Sayeed et al., 2000).

Analysis of pWR100 revealed the presence of a 8.4 kb

region that exhibits 96% sequence identity with parts of the

transfer region of plasmid F. This region is limited by two

copies of IS600 and corresponds to the 3 0 part of traD, a

complete trbH gene, a traI gene interrupted by an internal

deletion leading to a frameshift and the traX and finO

genes. Other tra genes were not detected on pWR100,

which is consistent with previous results suggesting that

pWR100 is non-conjugative (Sansonetti et al., 1982).

The sequence of six adjacent ORFs (orf159b, 160, 161a,

161b, 162 and 163) covering a 3.6 kb region exhibits 94%

identity with that of ORFs carried by pCollIB-P9 and pO157.

The presence of these genes on different plasmids and the

absence of related genes on bacterial chromosomes

suggests that theymightbe involved inplasmidmaintenance

or transfer. The G1C content of this region (56.9%) is similar

to that of the tra region discussed above (57.1%).

Concluding remarks

Sequence analysis of pWR100 indicated that one-third of

the plasmid is composed of IS elements. These are likely

to be responsible for the heterogeneity detected in

restriction patterns of the virulence plasmid harboured

by strains of different species or of different serotypes

within the same species (Sansonetti et al., 1983b).

Differences in G1C content among genes carried by

pWR100 provide strong evidence that the plasmid is

composed of a mosaic of blocks of genes from different

origins. Genes of the entry region and osp genes have a

similar G1C content (average 34%), which is different

from those of the transfer and replication regions (55%

G1C), the icsA and virK regions (41% G1C) or sepA

(49% G1C). This suggests that the entry region and osp

genes have the same origin and were probably acquired

simultaneously, even though they are not linked on the

plasmid. Insertion of IS elements and DNA rearrange-

ments involving deletions (indicated by the presence of

truncated ISs adjacent to the entry region), duplications

(indicated by the presence of several almost identical

copies of ospC genes) and inversions (suggested by the

opposite orientations of the identical ipaH1.4 and ipaH2.5

genes) have modified the organization of the ancestral

blocks and have probably led to the loss of some genetic

information initially carried by these blocks (as suggested

by the absence of the impABC genes on pWR100;

Runyen-Janecky et al., 1999). The presence of two

seemingly functional partition systems (parAB and

stbAB) and of the remnant of a third one (virB) suggests

that parts of pWR100 come from three plasmids. More-

over, the G1C content of the replication system (repA,

58%) is different from those of the above-mentioned

partition systems, virB (34%), stbAB (39%) and parAB

(43%), which suggests that the virulence plasmid contains

elements that were initially carried by four plasmids.



Determination of the N-terminal sequence of proteins

present in the culture medium of strain SF135 allowed us

to identify new proteins secreted by the type III secretion

machinery of S. flexneri. These and previous results

indicate that 19 proteins are secreted by the Mxi±Spa

secretion apparatus: Spa32, MxiC and MxiL, which might

be involved in the assembly or regulation of the secretion

apparatus, and IpaA±D, IpgB1, IpgD, OspB±G, VirA and

three IpaH proteins. Analysis of pWR100 also revealed

the presence of eight genes encoding proteins with

sequence similarities to secreted proteins, which sug-

gests that the repertoire of proteins secreted by the Mxi±

Spa secretion apparatus consists of < 25 proteins.

Several secreted proteins are encoded by multigene

families (ipaH, ipgB, ospC, ospD and ospE), and it is not

known whether or not all members of a family are

expressed in the same environmental conditions. No

genes encoding potential chaperones were detected in

the vicinity of osp and ipaH genes, which suggests that

the requirement for specific cytoplasmic chaperones

concerns only a subset of proteins that are secreted by

the type III secretion pathway (Wattiau et al., 1996).

Including components of the secretion apparatus, secreted

proteins, chaperones and regulators, the type III secretion

system of S. flexneri comprises about 50 proteins. It seems

unlikely that all this genetic information is involved solely in

entry of bacteria into epithelial cells. According to the

current model of the type III secretion pathway (Hueck,

1998), Osp and IpaH proteins are candidates for being

translocated into target cells. The type of cells in which

these proteins might be translocated and the cellular

processes they might affect remain to be investigated.

Experimental procedures

Bacterial strains

pWR100 is the large virulence plasmid harboured by M90T, aS. flexneri strain of serotype 5 (Sansonetti et al., 1983b). E.coli strain DH5a was used for cloning SalI and BamHIfragments of pWR100 into pK19, and XL2-Blue was used forthe construction of shotgun libraries in pcDNA2.1.

Shotgun cloning and DNA sequencing and analysis

To determine the sequence of pWR100, we first used 10cosmids constructed previously (Maurelli et al., 1985) thatcontained overlapping inserts of 40 kb, resulting from apartial digestion of pWR100 with Sau3AI. DNA (5 mg) fromeach cosmid was sheared by nebulization, end-repairedusing T4 DNA polymerase and ligated to BstXI adaptors. Theligation mixture was fractionated by agarose gel electrophor-esis, and fragments from 1 to 3 kb were ligated into BstXI-digested pcDNA2.1. Recombinant plasmids were used astemplates for cycle sequencing reactions consisting of 35cycles (968C for 30 s; 508C for 15 s; 608C for 4 min) in a

thermocycler. Samples were precipitated and loaded onto a96-lane, 4% polyacrylamide gel, and electrophoresis wasperformed on a mModel ABI Prism 377 automatic DNAsequencer (Perkin-Elmer) for 10 h. Assembly of thesequences of individual cosmids indicated that the virFgene, known to be carried by pWR100, was not present inany of the cosmids. To identify the fragment that was missingin the cosmid library, the DNA of pWR100 was digested withBamHI or SalI, and the restriction fragments were cloned intothe vector pK19. Ligation mixtures were used to transformDH5a, and transformants were selected on plates containingkanamycin and Xgal. Plasmid DNA was prepared from 96transformants that did not express b-galactosidase activityand used to determine the sequence of both ends of theinserts. Plasmids carrying different inserts were character-ized further with respect to the size and the restriction map ofthe inserts. This approach allowed us to identify four BamHIand three SalI restriction fragments that were not present inthe first assembly. The sequence of these overlappingfragments was determined, and the final sequence assemblyof the virulence plasmid was confirmed by comparing therestriction patterns predicted from the sequence with thoseobtained by digestion of pWR100 with different enzymes.Retrospectively, it appeared that the virulence plasmid thathad been used to construct the cosmid library probably had adeletion of a 35 kb fragment encompassing the virF gene.Apart from this region, no differences were detected betweenfragments carried by the cosmids and the virulence plasmid.

The sequence was assembled and edited using the PHRED

(Ewing and Green, 1998; Ewing et al., 1998), PHRAP (P.Green, unpublished) and CONSED (Gordon et al., 1998)software. Sequence analysis and annotation was managedby ARTEMIS (EMBL sequence viewer and annotation program,Sanger Centre, UK) and the GCG software package.Determination of similarities with known proteins involvedinterrogation of BLAST2N, BLAST2X, BLAST2P (Altschul et al.,1997) and FASTA (Pearson and Lipman, 1988). The sequencedata have been submitted to the DDJ/EMBL/GenBankdatabases under accession number AL391753.

Acknowledgements

We are pleased to acknowledge Zineb Benjelloun-Touimi andBrigitte Demers for mapping of some cosmids, EduardRocha, Ivan Mozer and Lionel Frangeul for discussions andhelp with sequence analysis, Jacques d'Alayer for determi-nation of amino acid sequences, Mick Chandler and PatriciaSiguier for analysis of insertion sequences, and Maria Mavris,Dana Philpott and John Smith for critical reading of themanuscript. This work was supported in part by a grant fromthe GIP-HMR (Hoechst-Marion-Roussel).

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The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri