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Previous Article | Next Article 
Journal of Bacteriology, February 1999, p. 998-1004, Vol. 181, No. 3
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
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.
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.
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).
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The SPI-3 Pathogenicity Island of
Salmonella enterica
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results and discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results and discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results and discussion
References
TABLE 1.
Salmonella strains used in this study
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 |
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Top
Abstract
Introduction
Materials and methods
Results and discussion
References
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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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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.
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-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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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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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).
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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* 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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