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Journal of Bacteriology, February 1999, p. 998-1004, Vol. 181, No. 3
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The SPI-3 Pathogenicity Island of
Salmonella enterica
Anne-Béatrice
Blanc-Potard,
Felix
Solomon,
Jayson
Kayser, and
Eduardo A.
Groisman*
Department of Molecular Microbiology, Howard
Hughes Medical Institute, Washington University School of Medicine,
St. Louis, Missouri 63110
Received 9 October 1998/Accepted 12 November 1998
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ABSTRACT |
Pathogenicity islands are chromosomal clusters of pathogen-specific
virulence genes often found at tRNA loci. We have determined the
molecular genetic structure of SPI-3, a 17-kb pathogenicity island
located at the selC tRNA locus of Salmonella
enterica serovar Typhimurium. The G+C content of SPI-3 (47.5%)
differs from that of the Salmonella genome (52%),
consistent with the notion that these sequences have been horizontally
acquired. SPI-3 harbors 10 open reading frames organized in six
transcriptional units, which include the previously described
mgtCB operon encoding the macrophage survival protein MgtC
and the Mg2+ transporter MgtB. Among the newly identified
open reading frames, one exhibits sequence similarity to the ToxR
regulatory protein of Vibrio cholerae and one is similar to
the AIDA-I adhesin of enteropathogenic Escherichia coli.
The distribution of SPI-3 sequences varies among the salmonellae: the
right end of the island, which harbors the virulence gene
mgtC, is present in all eight subspecies of
Salmonella; however, a four-gene cluster at the center of
SPI-3 is found in only some of the subspecies and is bracketed by
remnants of insertion sequences, suggesting a multistep process in the evolution of SPI-3 sequences.
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INTRODUCTION |
The gram-negative bacterium
Salmonella enterica is responsible for a variety of
diseases, which include gastroenteritis and typhoid fever, depending on
the nature of the infected host and on the serovar of the infecting
bacteria. Salmonella has a complex life cycle in infected
animals, and a large number of genes have been implicated in
Salmonella virulence. Several of these virulence determinants are clustered within pathogenicity islands, i.e., large
segments of horizontally acquired sequences present in pathogenic species but absent from closely related nonpathogenic species (17,
24). Pathogenicity islands constitute major elements in the
evolution of bacterial pathogens, because their incorporation can, in a
single step, transform a normally benign organism into a pathogen.
In addition to several small pathogenicity islets, five large
pathogenicity islands have been identified in Salmonella
(21, 51, 52). SPI-1, at 63 min on the S. enterica
serovar Typhimurium chromosome, is a 40-kb island that governs the
ability to invade epithelial cells (10, 38) and is required
for Salmonella-induced macrophage apoptosis (9).
The SPI-2 island is also 40 kb in length, maps downstream of a
tRNAVal locus at 31 min (25), and harbors genes
required for intramacrophage survival and systemic infection (40,
44). The SPI-3 island is located at 82 min, immediately behind
selC, a tRNA locus that is the insertion site for distinct
pathogenicity islands in enteropathogenic and uropathogenic strains of
E. coli (3, 5, 33). Recently, a 27-kb
Salmonella-specific DNA fragment at 92 min was designated the fourth Salmonella pathogenicity island because it
includes a macrophage survival locus (34). A fifth
pathogenicity island, containing genes mediating Salmonella
enteropathogenesis, is located downstream of a tRNASer
locus at 20 min in the chromosome (52).
The SPI-3 island harbors mgtC, a
Salmonella-specific gene that is required for
intramacrophage survival, virulence in mice, and growth in
low-Mg2+ media (3). The mgtC gene is
transcriptionally controlled by the PhoP-PhoQ regulatory system,
which governs the adaptation to low-Mg2+ environments
(15, 46) and is the major regulator of virulence functions
in Salmonella (14, 18). The mgtC gene
is cotranscribed with mgtB (45), a
Mg2+ transporter gene dispensable for virulence in BALB/c
mice (3). SPI-3 is 17 kb long and may contain additional
genes that contribute to virulence or to other
Salmonella-specific attributes.
In this study, we determined the molecular genetic structure of the
SPI-3 island, examined the functions of the genes it carries, and
investigated the distribution of SPI-3 sequences among salmonellae. We
establish that at least 10 genes are encoded within SPI-3, some of
which show similarity to known virulence factors from other
bacterial species, and that the evolution of SPI-3 sequences occurred
through a multistep process.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, bacterial genetic techniques, and
growth conditions.
Strains used in this study are listed in Table
1. The strains used in this study are
derived from 14028s, except for TT10288 and AA3007, which are derived
from LT2. Bacteria were grown at 37°C in Luria-Bertani broth (LB)
(35). Ampicillin and kanamycin were used at 50 µg/ml, and
chloramphenicol was used at 10 µg/ml. Phage P22-mediated transduction
was carried out as described previously (12).
Construction of the
marT1::
cat strain
EG10207 was performed as follows. A 2.3-kb
HindIII-
BspMII fragment from plasmid pEG9106
(
3) carrying
marT was subcloned into pUC19
between the
HindIII
and
XmaI sites to form
plasmid pEG9109. A
cat-containing 0.8-kb
BamHI
fragment from plasmid pKRP10 (
43) was introduced into
the
unique
BglII site in
marT in plasmid pEG9109. The
resulting
plasmid (pEG9110) was used to transfer the
marT1::
cat mutation
into the
Salmonella chromosome as described previously
(
23).
The structure of the
marT gene in the
mutant strain was verified
by Southern hybridization with both
marT- and
cat-specific probes
(data not
shown).
MudJ is a derivative of bacteriophage Mu that harbors a gene conferring
resistance to kanamycin and a segment of the
lac operon
devoid of its promoter sequences (
7). To isolate MudJ
insertions
in SPI-3, a P22 lysate grown in TT10288 was used to infect
strain
EG10207. A lysate grown on a pool of 25,000 kanamycin-resistant
transductants was used to infect 14028s, with selection for both
kanamycin and chloramphenicol resistance. To establish the orientation
and approximate position of each MudJ insertion, PCR was performed
with
primers complementary to the ends of MudJ (i.e.,
attL or
attR) and to known SPI-3
sequences.
Molecular biological techniques.
The nucleotide sequence of
the 12-kb segment between the selC and mgtB genes
was determined on both strands by using plasmid pEG9106 DNA as the
template, starting with primers complementary to selC
(selC-F) (11) and to the 3' end of
mgtB (3'mgtB-F,
5'-ATCGTCGTGGTTTAACCGCCGTCC-3') and walking with newly
synthesized primers. DNA sequence analysis and protein sequence
alignments were performed by using the GeneWorks (IntelliGenetics) and
Genetics Computer Group (GCG) (University of Wisconsin) software packages.
PCRs were carried out on purified chromosomal DNA with
Taq
polymerase (Gibco BRL) according to the manufacturer's protocol.
For
amplification of long DNA fragments, we used the TaqPlus Long
PCR
system (Stratagene). To examine whether SPI-3 sequences are
linked to
selC in different
Salmonella subspecies, we used
primers
selC-F (
11),
selC-1-25
(5'-GGAAGATCGTCGTCTCCGGTGAGGC-3'), and
slsA-R (5'-TTGTACAAAATCGGCATTATCCCAGGC-3').
To determine whether
mgtC and
orf307 are
linked in different
Salmonella subspecies,
we used primers
mgtC-R (5'-GCCCGCCCCCAGAAAGCCAATCCC-3') and E07-R
(
11).
Southern hybridization analysis was carried out with chromosomal DNA as
described previously (
11). To investigate the distribution
of the
orf269 gene, a PCR-generated probe corresponding to
the
Escherichia coli K-12
orf269 open reading
frame (ORF) was used
for hybridization to DNAs from
E. coli K-12,
E. coli D,
Shigella flexneri,
S. enterica serovar Typhimurium,
Citrobacter freundii,
Enterobacter
aerogenes,
Enterobacter cloacae,
Klebsiella
pneumoniae,
Serratia odifera,
Yersinia
enterocolitica,
Yersinia pestis,
Haemophilus influenzae,
Mycobacterium avium, and
Pseudomonas
aeruginosa. To
investigate the distribution of SPI-3 sequences
among salmonellae,
probes were hybridized to DNAs from strains of the
Salmonella Reference Collection C (
6) and from
E. coli K-12 strain MC1061.
Probe 1 (410 bp) was
generated from a PCR DNA fragment by using
primers
selC-415
(5'-AGATGATGTGGCTGGCG-3') and
selC-R
(
11),
probe 2 (780 bp) was generated by using primers
described previously
(
3), and probes 3, 4, 6, 7, 8, and 9 were generated by using
primers complementary to the 5' and 3' ends of
the
sugR,
rhuM,
marT,
slsA,
mgtB, and
mgtC genes, respectively. Probe 5 was
generated
from a 4-kb
EcoRI-
HindIII
restriction fragment (
3).
Virulence and 
-galactosidase assays.
Macrophage survival
assays with the macrophage-like cell line J774 and invasion assays with
canine kidney epithelial (MDCK) cells were conducted as described
previously (28). Virulence assays were performed with 7- to
8-week-old female BALB/c mice (10 mice per mutant) inoculated orally
with 100 µl of bacteria diluted in phosphate-buffered saline.
-Galactosidase assays were carried out in triplicate with bacteria
grown exponentially in LB as described previously (35).
Nucleotide sequence accession number.
The sequence reported
in this paper has been deposited in the GenBank database (accession no.
AF106566).
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RESULTS AND DISCUSSION |
Molecular analysis of SPI-3 genes and encoded proteins.
We
have previously identified a pathogenicity island downstream of the
selC gene in the S. enterica serovar Typhimurium
chromosome (3). This island includes the
mgtCB operon, which codes for the virulence protein MgtC and
the Mg2+ transporter MgtB. To further examine the role of
SPI-3 in Salmonella, we determined the molecular
genetic structure of the DNA region between the selC and
mgtB genes (Fig. 1).
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FIG. 1.
Physical and genetic maps of the SPI-3 pathogenicity
island. (Top) G+C content of the SPI-3 island. The graph was created by
using the program Cricket Graph with data generated by the program
Windows (GCG) (window, 100 bp; sliding increment, 100 bp). The line at
52% indicates the overall G+C content estimated for the S. enterica serovar Typhimurium chromosome. (Bottom) Positions and
orientations of ORFs encoding products larger than 120 amino acids and
containing potential Shine-Dalgarno sequences. DNA sequences reported
in Table 2 are indicated by numbers (IS-like sequences are represented
by gray squares). The map positions of MudJ insertions in the SPI-3
region are indicated by triangles. RSA refers to a family of protected
sequences present in the genomes of members of the family
Enterobacteriaceae.
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The SPI-3 island is 17 kb long, and in addition to the
mgtCB
operon, it harbors eight ORFs, all of which contain potential
Shine-Dalgarno sequences (Fig.
1). (Additional small ORFs
[encoding
<120 amino acids] lacking a clear translation start site
and similarity
to proteins in the sequence databases are not
reported here).
The 10 genes carried within SPI-3 appear to be
organized in six
transcriptional units. Characteristics of the
SPI-3 ORFs, as well
as features of DNA stretches that have
identity with sequences
in the databases, are described in Table
2. Except for the
rmbA gene,
which is part of region with a very low G+C content, the
codon usages
of SPI-3 ORFs do not appear to be significantly different
from those of
highly expressed
E. coli and
Salmonella
genes.
The first gene of the island,
sugR, encodes a protein that
exhibits closest similarity to the PgaA antigen of the
periodontopathogen
Porphyromonas gingivalis (
43a)
and to a putative ATP binding
protein encoded in the genome of a
clinical isolate of
E. coli (
29). The
SugR protein contains an imperfect nucleotide-binding
Walker A
motif (APNGAGKT) that is missing the first conserved
G of the consensus
Walker sequence (GXXGXGKS/T) (
48).
The MisL (for membrane insertion and secretion) protein exhibits
similarity to the immunoglobulin A1 protease family of autotransported
proteins, which have been found only in pathogenic bacteria (
26,
32). These proteins consist of an N-terminal effector domain
and
a C-terminal conserved domain that forms a pore in the outer
membrane
through which the N-terminal domain is translocated.
The
similarity between MisL and the AIDA-I protein from
enteropathogenic
E. coli and the VirG protein from
S. flexneri is limited to the
C-terminal region (Fig.
2A), suggesting a similar autotransporter
function rather than specific functional similarities with these
two
proteins, which have been implicated in diffuse adherence
to HeLa cells
(
2) and cell-to-cell spreading (
16),
respectively.
Neither the 955-amino-acid MisL protein nor the
1,286-amino-acid
AIDA-I protein contains cysteine residues, a feature
that precludes
the formation of disulfide bonds and is believed to be
crucial
for membrane translocation (
26). MisL also contains
a predicted
N-terminal signal sequence required for the translocation
of the
protein across the inner membrane.
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FIG. 2.
(A) Alignment of the C-terminal domains of the S. enterica MisL protein, the plasmid-encoded AIDA-1 protein from
enteropathogenic E. coli, and the VirG protein of
S. flexneri. An 18-amino-acid duplicated region rich in Pro,
Asp, and Val within MisL is indicated by a horizontal line. Amino acids
that are identical between the MisL and AIDA-1 proteins or between the
AIDA-1 and VirG proteins are linked by vertical lines. Amino acids that
are identical between the MisL and VirG proteins are indicated by a
short underline in the VirG residue. (B) Alignment of the N-terminal
domains of the MarT protein, ORF269 (O269) of E. coli,
and the ToxR regulator from V. cholerae. Highly conserved
residues among OmpR homologs are indicated by dots (30).
Amino acids that have been shown to be important for ToxR function
(41) are marked by asterisks. Amino acids that are identical
between the MarT and ORF269 proteins or between the ORF269 and ToxR
proteins are linked by vertical lines. Amino acids that are identical
between the MarT and ToxR proteins are indicated by a short underline
in the ToxR residue. Alignments were performed by using the PILEUP
program (GCG).
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The MarT (for membrane-associated regulator) protein has homology with
a protein from
E. coli K-12 (ORF269) and exhibits
similarity
in its N-terminal domain to the ToxR protein from
Vibrio cholerae (Fig.
2B). ToxR is a transmembrane
regulatory protein that is
required for the synthesis of cholera toxin
in
V. cholerae (
36).
It consists of an N-terminal
cytoplasmic domain, which is homologous
to the OmpR family of
transcription factors and probably involved
in DNA binding, and a
C-terminal domain which is thought to be
involved in sensing
environmental signals (
30,
37). Like ToxR,
MarT contains a
potential transmembrane domain in its central
region and exhibits
similarity with the putative DNA binding domain
of the CadC
transcriptional activator of
E. coli K-12
(
49),
another member of the OmpR family, suggesting
that the
marT gene
encodes a regulatory
protein.
Finally, the
rhuM,
rmbA,
fidL,
slsA, and
cigR gene products do not exhibit
sequence similarity to proteins with known functions
in the sequence
databases. FidL and the glycine- and asparagine-rich
CigR contain
putative signal sequences and might be exported
proteins.
Expression of SPI-3-encoded genes.
To examine the expression
of the genes encoded within SPI-3, MudJ transposon insertions
were isolated in this region of the Salmonella genome,
and the -galactosidase activities produced by the
resulting strains were determined (see Materials and Methods) (Fig. 1). When the strains were grown in LB broth, -galactosidase activity was produced by lac gene fusions to the
sugR, rhuM, and marT genes but not by
the misL-lac fusion (Table
3), suggesting that the
misL gene may respond to signals not present in laboratory media. A lac fusion to the intergenic region between
sugR and rhuM, located approximately 200 to 250 bp upstream of rhuM, produced a level of -galactosidase
activity comparable to that of the rhuM-lac fusion,
suggesting that sugR and rhuM may constitute an
operon even though the distance between sugR and
rhuM is 580 bp.
Expression of horizontally acquired genes is often controlled by
regulatory proteins encoded by linked genes within the acquired
sequences. For example, several genes in the
Salmonella
SPI-1
island are regulated by the HilA and InvF proteins, which are
also encoded within SPI-1 (
1,
27). Likewise, a two-component
system encoded in the
Salmonella SPI-2 island governs
expression
of several SPI-2 genes (
46-47). Despite its
similarity to the regulatory
protein ToxR, MarT does not appear to
control expression of the
sugR,
rhuM,
misL, and
mgtC genes, because similar levels of
-galactosidase
were displayed by isogenic
marT+ and
marT mutant strains when
bacteria were grown in LB broth
(Table
3). MarT does not appear to
regulate its own expression
either, because the wild-type
marT gene on a multicopy plasmid
did not modify the
-galactosidase activity of a
marT2::MudJ strain
(data not shown). The MarT protein has different amino acids than
the
ToxR protein at three of four positions shown to be important
for ToxR
regulatory function (Fig.
2B) (
41), which raises the
possibility of MarT being involved in a function other than
transcriptional
regulation. However, it is also possible that the MarT
protein
governs transcription of other genes within SPI-3 or under
different
growth
conditions.
To coordinate their expression with that of the rest of the genome,
foreign sequences often recruit host regulators in addition
to
those encoded within the acquired sequences. One striking example
is
provided by the regulatory protein PhoP, which is present in
both
pathogenic and nonpathogenic bacterial species (
19) and
controls expression of several horizontally acquired sequences
involved
in
Salmonella virulence, including the SPI-3-carried
mgtC gene (
18). However, expression of
sugR,
rhuM,
misL, and
marT
is not dependent on the PhoP regulatory protein, since similar
levels
of
-galactosidase were displayed by isogenic
phoP+ and
phoP mutant strains (Table
3).
Virulence properties of SPI-3 mutants.
Because the
misL and marT genes encode proteins with
similarity to known virulence factors, we investigated whether these genes were required for Salmonella virulence. Strains with a
misL1::MudJ mutation (EG10755) or with a
marT1::cat mutation (EG10207) exhibited wild-type levels of survival within macrophages and invasion of epithelial cells (data not shown). Moreover, their ability to cause a
lethal infection in mice was as efficient as that of the wild-type
parent when tested orally on BALB/c mice at doses of 2 × 106 and 1.5 × 107 CFU (the 50% lethal
dose of the wild-type strain is 6 × 105 CFU)
(22). These results indicate that the misL and
marT genes are not essential for virulence under the
conditions investigated. However, these genes could be involved in
other aspects of pathogenesis, such as chronic infection and host
specificity, or they could play a role in processes specific to
Salmonella that are unrelated to virulence.
Similarity of SPI-3 proteins to E. coli proteins
encoded by horizontally acquired sequences.
Consistent with the
notion the SPI-3 island was acquired by horizontal gene transfer, its
overall G+C content is 47.5%, which is much lower than that of the
Salmonella chromosome (52%) (39). Moreover,
SPI-3 is located next to the selC tRNA gene, and tRNA genes are preferential sites of insertion of foreign sequences, including phages, plasmids, and pathogenicity islands (8, 20, 24). Furthermore, several SPI-3 gene products exhibit similarity with proteins encoded by horizontally acquired DNA sequences in other
bacterial species. For example, the sugR gene product
exhibits similarity with a protein from a clinical isolate of
E. coli encoded by a gene that was probably acquired by
lateral gene transfer, because it is part of a region with a low G+C
content, located downstream of the thrW tRNA locus
(29), and is absent from the E. coli K-12
genome (4).
The central region of SPI-3 includes four genes, i.e.,
rmbA,
misL,
fidL, and
marT, three of which
code for proteins with sequence
similarity to
E. coli
K-12 ORF products encoded at 64 min in the
chromosome (
rmbA,
fidL, and
marT are similar to
orf230,
orf164,
and
orf269, respectively) (
4).
However, the genetic organizations
of these genes are different in
E. coli and
Salmonella (Fig.
3).
The
orf230,
orf269, and
orf164 genes appear to have been
acquired
horizontally into
E. coli K-12, because they
have an atypical
codon usage and are part of a 13-kb region with a very
low G+C
content (37.1%) that is located downstream of the
glyU tRNA gene
(
4). Consistent with the notion
that this region is not ancestral
to enteric bacteria but rather that
it was incorporated into
E. coli K-12 by horizontal
gene transfer, Southern hybridization
experiments revealed that
orf269-hybridizing sequences are absent
from 12 bacterial
species, including
S. flexneri, which is considered
to be
part of the
E. coli species (see Materials and Methods)
(data not shown).
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FIG. 3.
Organization of the rmbA, misL,
fidL, and marT genes in S. enterica serovar Typhimurium SPI-3 in comparison to the
orf230 (o230), orf269
(o269), and orf164 (o164) genes of E. coli K-12. The deduced amino acid sequences of rmbA,
fidL, and marT are about 40% identical to the
deduced proteins encoded by o230, o164, and
o269, respectively (Table 2), suggesting that these proteins
are orthologues rather than homologues.
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Evolution of SPI-3 sequences.
The G+C content is not uniform
along SPI-3 (Fig. 1): genes with the lowest G+C content
(rmbA, fidL, and marT, with 37.7, 45.2, and 47.3%, respectively) are located in the central region
of SPI-3. However, this region, which is homologous to an E. coli K-12 gene cluster (see above), is interrupted by the
misL gene, which has a G+C content of 53%, suggesting that
the incorporation of misL was a genetic event separate from
that mediating the acquisition of rmbA, fidL, and
marT. The central region of SPI-3 is surrounded by DNA
segments with a very low G+C content that contain remnants of insertion
sequences (Fig. 1): between the rhuM and rmbA
genes, a 200-bp segment is homologous to the left inverted repeat and the 40 first residues of the transposase gene of the IS1351
element of Salmonella enteritidis (6a), and the
region that separates the marT and slsA genes
harbors a 100-bp sequence similar to a repetitive element from
E. cloacae that is related to IS10
(31) and to the left inverted repeat of the IS911
element of Shigella dysenteriae (42). Taken
together, these data suggest that SPI-3 has a composite structure and
that the central region might have an independent origin.
To further examine the evolution of the SPI-3 island, we investigated
the
Salmonella Reference Collection C, which includes
strains that encompass the eight subspecies of the genus
Salmonella (
6), for the presence of SPI-3
sequences. Southern hybridization
experiments established that
sequences hybridizing to the 5.3
kb at the right end of the island
(including the
slsA gene and
the
mgtCB operon)
are present in all eight subspecies of
S. enterica (Fig.
4). In contrast, the 5.5-kb central
region did not hybridize
to DNAs from strains of groups IIIa, IV, and
VII and from one
representative of group IIIb. Surprisingly, sequences
hybridizing
to this region were detected in
Salmonella
bongori (group V),
the more divergent form of
Salmonella. These results are consistent
with the hypothesis
that the central region of SPI-3 was incorporated
as a separate genetic
event, as suggested by the G+C composition
and presence of surrounding
insertion sequences (ISs). Thus, this
portion of SPI-3 might have been
acquired independently in
S. bongori, or,
alternatively, it may have been introduced into the
Salmonella lineage or in the donor chromosome from which
SPI-3
originated and then have been deleted in a subset of
Salmonella subspecies. The left end of SPI-3 (including
sugR and
rhuM) appears
to be less conserved than
the central region and might have been
the subject of deletions. As
expected, a probe complementary to
a region outside SPI-3, which
included most of the
selC gene and
350 bp of upstream
sequences, hybridized to DNAs from all
Salmonella subspecies
and from
E. coli K-12.
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FIG. 4.
Phylogenetic distribution of SPI-3 sequences among
salmonellae based on Southern blot experiments carried out as described
in Materials and Methods. Positions of the remnants of ISs are
indicated. +, presence of a positive hybridization signal with the
designated strain. Evolutionary relationships of Salmonella Reference
Collection C strains are based on variation in the nucleotide sequences
of five housekeeping genes (6). The roman numerals indicate
the eight Salmonella subspecific groups.
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The
selC tRNA locus is the site of insertion of SPI-3 in
Salmonella and of the PAI-1 and LEE islands in pathogenic
strains
of
E. coli (
3,
5,
33). While this
suggests a common mechanism
for the acquisition of these foreign
sequences, the SPI-3 island
does not encode an integrase-like protein
or harbor long repeated
sequences. This is in contrast to PAI-1, which
contains a cryptic
integrase gene in its left end and is flanked
by short direct
repeats (
5,
24), and to LEE, which
harbors remnants of a
transposase gene in its right end
(
13). The sequences of the
Salmonella
SPI-1, SPI-2, and SPI-4 islands and their boundaries
have, thus far,
not revealed long sequence repeats, phage attachment
sites, or
remnants of integrase genes, which could be responsible
for the
stability of these regions in the
Salmonella genome.
The LEE pathogenicity island was originally identified at the
selC locus of enteropathogenic strains of
E. coli (
33), but
recent work indicates that LEE can be
found at locations other
than
selC (
50). We
investigated whether SPI-3 is located at
the
selC locus in
the different
S. enterica subspecies by carrying
out
PCRs with primers complementary to the
selC and
slsA genes
(these genes are 12 kb apart in
S. enterica serovar Typhimurium,
and
selC- and
slsA-hybridizing sequences have been detected in
all
Salmonella subspecies). The
slsA gene is linked
to the
selC gene in most subspecies, because a 12-kb
fragment was obtained
when DNAs from
Salmonella subspecies
I, VI, and II were used as
templates, and fragments smaller than 12 kb
were amplified from
at least one representative of each of the other
subspecies (two
different primers within
selC were used to
exclude nonspecific
amplifications). At the right end of the island,
the
mgtC gene
appears to be located next to
orf307 in all eight subspecies of
S. enterica, because PCR experiments with primers complementary
to
mgtC and
orf307 resulted in the amplification of
the same 0.76-kb
fragment.
Conclusion.
The molecular analysis of SPI-3 sequences and
their phylogenetic distribution among the different subspecies
that comprise S. enterica indicate that SPI-3 has
a mosaic structure, most likely the result of a multistep evolutionary
process, and encodes proteins that are not obviously functionally
related. This is in contrast to the Salmonella SPI-1 and
SPI-2 pathogenicity islands, which were likely acquired through single
horizontal gene transfer events and encode functionally related
proteins, which include type III export systems and secreted effector
proteins (38, 40, 44). The different distribution of SPI-3
sequences may reflect the functional role of encoded genes: sequences
present in only a subset of Salmonella subspecies could be
involved in host specificity, tissue tropism, and disease
manifestation, as recently proposed for a group of
Salmonella-specific sequences of atypical base composition
recovered by the in vivo expression technology procedure (11).
| |
ACKNOWLEDGMENTS |
We thank Matthew Chung-Ying Lo for help in strain construction
and Howard Ochman for comments on an earlier version of the manuscript.
This work was supported by NIH grant GM54900 to E.A.G., who is an
Associate Investigator of the Howard Hughes Medical Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Howard Hughes
Medical Institute, Washington University School of Medicine, Department of Molecular Microbiology, 660 South Euclid Ave., Campus Box 8230, St.
Louis, MO 63110. Phone: (314) 362-3692. Fax: (314) 362-1232. E-mail:
groisman{at}borcim.wustl.edu.
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Top
Abstract
Introduction
