Laboratorio de Microbiología, Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda no. 340, Santiago, Chile1
Author for correspondence: Guido C. Mora. Tel: +56 2 6862849. Fax: +56 2 2225515. e-mail: gmora{at}genes.bio.puc.cl
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ABSTRACT |
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Keywords: Salmonella, outer-membrane protein, smvA, genetic variability, host specificity
Abbreviations: OMP, outer-membrane protein
a Present address: Programa de Microbiología, Instituto de Ciencias Biomédicas, Universidad de Chile, Independencia no. 127, Santiago, Chile.
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INTRODUCTION |
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Because the sequences of conserved genes between S. typhi and S. typhimurium are nearly identical, they are said to be homeologous (Zahrt & Maloy, 1997 ; Zahrt et al., 1994
). However, it is unlikely that the minor fraction (12%) of nucleotide base pair differences between genes conserved in these two serovars contributes significantly to their remarkably different host specificities. Rather, the striking differences in S. typhi and S. typhimurium host specificity is most likely due to the gross rearrangements one observes when comparing their structures at a more macroscopic level. The substitutions and insertions between S. typhi and S. typhimurium genomes range in size from islands comprised of more than 100 genes to smaller substitutions corresponding to single ORFs (McClelland & Wilson, 1998
). In addition, about 20% of the S. typhi genome is not present in S. typhimurium and vice versa (Lan & Reeves, 1996
). Therefore, it is possible that as many as 900 S. typhi genes could be serovar-specific, a subset of which is likely to be involved in determining host range (Bäumler et al., 1998
).
In this work, we describe one of the smaller interserovar differences in genome structure between S. typhi and S. typhimurium. The region of the S. typhimurium genome including smvA, ompD and yddG genes is different in S. typhi. These genes encode proteins that confer resistance to methyl viologen, to an abundant outer-membrane porin and to a putative transmembrane permease, respectively. The primary structure of the region containing these functions is genetically variable among Salmonella serovars.
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METHODS |
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Preparation of outer-membrane proteins (OMPs).
Outer-membrane fractions were prepared as described by Lobos & Mora (1991) , based on a modification of the method of Schnaitman (1971)
. Bacteria were grown overnight at 37 °C and harvested by centrifugation at 13000 g for 15 min. Cell pellets were washed and resuspended in 1 ml 10 mM Tris/HCl pH 8·0 buffer. Bacteria were disrupted by sonication (Vibra Cell, Sonics & Materials) for 100 s and centrifuged at 8000 g for 5 min. Supernatants were collected and centrifuged at 13000 g for 30 min. Supernatants were then discarded and pellets were resuspended in 500 µl buffer containing 10 mM Tris/HCl pH 8·0, 10 mM MgCl2, 2% Triton X-100. After incubation at 37 °C for 30 min, mixtures were centrifuged at 13000 g for 30 min. The insoluble outer-membrane fraction was resuspended in buffer consisting of 100 mM Tris/HCl pH 8·0, 2% SDS. SDS-PAGE was performed in 12·5% polyacrylamide slabs as described by Lobos & Mora (1991)
.
PCR amplifications.
All amplifications were conducted using a Perkin Elmer thermal cycler (GeneAmp PCR System 2400) and Taq polymerase (Gibco-BRL). The final volume in the tubes for amplification was 50 µl and consisted of 1xTaq PCR buffer, 1·5 mM MgCl2, each dNTP at a concentration of 200 µM, 50 pmol of each primer, 0·1 mg DNA and 1·25 U Taq polymerase. Standard conditions for amplification were 30 cycles at 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min, followed by a final extension step at 72 °C for 5 min. Primers OMPD1 (5'-GAC AAA GAC AAA ACC CGT T-3') and OMPD2 (5'-CGT CCA GCA GGT TGA TTT T-3') were used to amplify a 740 bp internal fragment of the ompD gene (Singh et al., 1996 ). Primers SMVA1 (5'-CTT AAC CGC CCG CTA TGA T-3') and SMVA2 (5'-GCT GAA CCA CAT CCC TAC C-3') were designed from the reported smvA sequence (GenBank accession no. D26057) and used to amplify a 940 bp fragment internal to smvA.
Southern hybridizations.
Plasmid pNK2883 (Kleckner et al., 1991 ) was cleaved with BamHI, and the 3 kb fragment with the tetRA genes was purified, labelled using the Bioprime DNA labelling system (Gibco-BRL) and used as the probe for Tn10. Hybridization probes for ompD and smvA were generated by PCR using a mixture of dNTPs containing biotin-14-dCTP and purified by using the Concert gel extraction system (Life Technologies). Genomic DNA of Salmonella serotypes was prepared as described by Sambrook et al. (1989)
, restricted with PstI (Gibco-BRL) and the fragments were separated on a 0·8% agarose gel. The DNA was then transferred onto a nylon membrane and cross-linked by UV irradiation. Hybridization was performed in solutions without formamide at 65 °C. Two 15 min washes were performed at 65 °C in a buffer consisting of 0·5 M Na2HPO4 pH 7·2, 2% SDS, 1 mM EDTA. Hybridization was detected by using the nonradioactive Photogene nucleic acid detection system (Life Technologies) and XAR-5 Kodak films.
Tests for resistance to methyl viologen (paraquat).
Each of the Salmonella strains tested was isolated on MacConkey-lactose agar, and single colonies were streaked onto LB agar plates containing 0·5 mM methyl viologen, an agent that generates superoxide and oxygen radicals (Farr & Kogoma, 1991 ). Plates were incubated overnight at 37 °C and scored for bacterial growth. To determine the minimal dose of methyl viologen required to kill Salmonella strains, a lawn of the strain being tested was seeded on LB agar plates and 10 µl of serial dilutions of methyl viologen stock were added. Plates were incubated overnight at 37 °C and scored for bacterial growth.
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RESULTS |
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The finding that S. typhi strains do not produce OmpD can be explained by one of two alternative hypotheses. S. typhi may have an ompD gene that is not expressed, or there may be no homologous ompD gene in S. typhi. Because the current sequence data produced by the S. typhi Sequencing Group at the Sanger Center (ftp://ftp.sanger.ac.uk/pub/pathogens/st/ST.dbs) do not include an OmpD coding sequence, sequence data alone do not allow the distinction between these two hypotheses. Therefore, a combination of genetic and biochemical experiments was designed to determine whether the ompD gene is absent from the S. typhi genome.
PCR and DNA hybridization analyses indicate that the ompD gene is absent from the S. typhi genome
To determine whether the ompD gene is present in S. typhi, we first attempted to amplify a 740 bp fragment internal to ompD using PCR, with ompD-specific oligonucleotide primers (Singh et al., 1996 ). With these primers, no product was obtained when DNA isolated from S. typhi strain Ty2, or DNA isolated from each of 26 independent S. typhi clinical isolates, was used as template (see Fig. 2b
for example). In contrast, a product of the predicted size was amplified using the same primers and DNA templates prepared from all the other Salmonella serovars tested (Fig. 2c
).
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In a first cross, we introduced an ompD::Tn10 allele from donor S. typhimurium strain MST2944 into recipient S. typhi strain Ty2 by generalized transduction with phage P22 and selection for recombinants that had acquired resistance to tetracycline. PCR amplification of the ompD gene from S. typhimurium strain LT2 ompD::Tn10 and S. typhi strain Ty2 ompD::Tn10 DNA templates yielded the expected 740 bp product, revealing that the amplified fragment of ompD does not include the site of the ompD::Tn10 insertion (Fig. 2a). The presence of the ompD::Tn10 allele in the chromosome of these interspecific hybrids was confirmed by Southern hybridization, using a probe specific to the tetRA genes of Tn10. As shown in Fig. 4(a
), the Tn10 probe hybridizes with a band of approximately 14·3 kb in PstI-restricted DNA samples obtained from S. typhi strain Ty2 ompD::Tn10 and S. typhimurium strains MST2944 ompD::Tn10 and LT2 ompD::Tn10. A comparison of the OMP profiles by SDS-PAGE clearly showed that S. typhimurium strain LT2 ompD::Tn10 and S. typhi strain Ty2 ompD::Tn10 do not express the OmpD porin (Fig. 5
).
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S. typhi is highly sensitive to methyl viologen
The observation that S. typhi strain Ty2, as well as the clinical strains, are missing the ompD gene is suggestive of a deletion in the S. typhi genome with respect to the S. typhimurium genome that may extend beyond the ompD gene. Analysis of the S. typhimurium chromosome reveals that ompD is adjacent to smvA (Hongo et al., 1994 ). It has been suggested that SmvA is an inner-membrane protein involved in the export of ammonium quaternary substrates, such as methyl viologen, by exporting them to the extracellular space (Hongo et al., 1994
). Methyl viologen generates superoxide and oxygen radicals (Farr & Kogoma, 1991
) and has been used in vitro to mimic the oxidative environment found within macrophages (Buchmeier et al., 1997
).
Therefore, S. typhi strains, as well as other Salmonella serovars were tested for their sensitivity or resistance to methyl viologen following the same procedure used to identify the S. typhimurium smvA gene (Hongo et al., 1994 ). Among 35 Salmonella serovars tested, only S. typhi strain Ty2 was unable to grow on Luria agar plates supplemented with 0·5 mM methyl viologen. In addition, the 26 clinical isolates of S. typhi used in this study were also unable to grow in the presence of the same amount of methyl viologen. To determine the level of resistance to methyl viologen, some strains were tested for their ability to grow in the presence of a range of concentrations of this compound. The highest concentration that allowed the growth of S. typhi strain Ty2 and S. typhimurium strain LT2 were 0·4 mM and 50 mM, respectively. These results suggest either that S. typhi is missing the smvA gene, or that the smvA gene is not functional in this serovar.
To distinguish between these two alternatives, primers from the reported smvA sequence (GenBank accession no. D26057) were designed to amplify a portion of this gene. Amplification of a S. typhi strain Ty2 DNA template did not yield a product internal to smvA. In contrast, amplification of chromosomal DNA isolated from S. typhimurium strains LT2 ompD+ and LT2 ompD::Tn10, as well as S. typhi strains Ty2 ompD::Tn10 and Ty2 ompD+, resulted in the expected 940 bp product (Fig. 6a). Remarkably, DNA obtained from four randomly chosen clinical isolates of S. typhi, which were sensitive to methyl viologen, yielded an amplification product of the expected size (Fig. 6b
). Thus, although the SmvA function is absent from all tested S. typhi strains, the smvA gene appears to be present in S. typhi clinical strains.
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DISCUSSION |
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To construct a recombinant strain with a single copy of ompD in the proper genetic position, we introduced an ompD::Tn10 allele from S. typhimurium MST2944 donor strain into recipient S. typhi strain Ty2 by generalized transduction with S. typhimurium phage P22. This transduction was originally performed by Liu & Sanderson (1995a , b
) as one of a systematic set of transductions used to assign genes on the S. typhi strain Ty2 genome to positions corresponding to those on the S. typhimurium genetic map. Using this approach, Liu & Sanderson (1995a
, b
) concluded that the ompD gene is located in the centre of a 500 kb inversion which includes the replication terminus of the S. typhi strain Ty2 chromosome. The ompD::Tn10 allele from S. typhimurium can be transduced into the S. typhi genome even though the recipient does not have the corresponding homeologous gene. This is because P22 packages about 44 kb of DNA, and P22-mediated generalized transduction permits homologous recombination events to occur in genes flanking a region of DNA that is not present in the recipient genome.
Our Southern blot analyses show that all S. typhi strains tested retain an smvA coding sequence, but they are nevertheless sensitive to methyl viologen, suggesting that the gene is not expressed in these strains. Alternatively, they may be lacking or be defective in additional genes required for methyl viologen resistance. In Escherichia coli, a related Gram-negative bacillus, at least three genes dispersed on the bacterial genome are required for methyl viologen resistance, and mutations in each of these genes confer sensitivity to methyl viologen (Morymio, 1988 ; Morymio et al., 1992
). Transductants of S. typhi strain Ty2 carrying the ompD+ allele and the cotransduced smvA gene express the OmpD porin in the outer membrane, and acquire resistance to methyl viologen (unpublished observation), suggesting that the smvA gene in clinical isolates of S. typhi may not be functional. Thus, the ompDsmvA region is genetically variable among different Salmonella serovars.
The ompD and smvA genes have been mapped at centisomes 33·7 and 38·6, respectively, in the S. typhimurium chromosome (Sanderson et al., 1995 ). Because both genes are cotransducible with phage P22, they cannot be that far apart. Instead, our results confirm those of Singh et al. (1996)
that place three S. typhimurium genes, ompD, smvA and narU, at centisome 33·7 (GenBank accession no. D26057). Analysis of the S. typhi genome sequence data released by the Sanger Center (http://www.sanger.ac.uk/Projects/S_typhi/blast_server.shtml and ftp://ftp.sanger.ac.uk/pub/pathogens/st/ST.dbs) reveals that S. typhi has two nar operons, 85% and 81% identical at the nucleotide level to the E. coli narGHJI and narZYWV operons, respectively. Both nar operons are also present in S. typhimurium (Barrett & Riggs, 1982
; Spector et al., 1999
), but only one of them (narGHIJ) has been mapped, lying at centisome 38·6 (Casse et al., 1973
; Sanderson et al., 1995
). In E. coli, the narU gene is located immediately upstream of the narZYWV operon (Gennis & Stewart, 1996
). Given the remarkable conservation of gene order between E. coli strain K12 and S. typhimurium strain LT2 (Krawiec & Riley, 1990
), we can assign the narZYWV operon to a map position at centisome 33·7 on the S. typhimurium genome, adjacent to the ompDsmvAnarU region. Recently, the Genome Sequence Center from the Washington University School of Medicine (http://genome.wustl.edu/gsc/Projects/bacterial/salmonella.shtml) sequenced an ORF upstream of the S. typhimurium ompD gene. This ORF, designated yddG, encodes a putative transmembrane permease. Notably, yddG is not present in the S. typhi genome sequence data released by the Sanger Center, suggesting that the genetic variability in the ompDsmvA region of the Salmonella genome also includes the yddG locus.
Because S. typhi is the only Salmonella serovar missing the ompD gene, the determination of its presence in other Salmonella serovars has a clinical significance. This kind of assay allows us to distinguish S. typhi from S. paratyphi serovars that cause systemic human infections presenting nearly identical symptoms. Thus, a PCR assay could be developed by using primers hybridizing upstream and downstream of the presumed deletion in the S. typhi strains, where the length of the amplified segment should discriminate between the different Salmonella serovars responsible for clusters of typhoid-like fevers and thus allow a more accurate assessment of their epidemiology.
What is the role of the ompD gene in Salmonella infection? The ompD gene does not appear to play a major role in the virulence of Salmonella serovars, because the effects of mutations in the ompD gene on the virulence of S. typhimurium are subtle. Dorman et al. (1989) found that a strain with a Tn10 insertion in ompD has a slightly greater LD50 than its otherwise isogenic wild-type parent. However, Meyer et al. (1998)
found that there was no statistically significant difference between the LD50 of S. typhimurium wild-type and ompD mutant strains.
Although it seems likely that the ompD gene plays only a minor role in virulence, it may be important for determining host specificity. Many studies have characterized the genetic determinants of Salmonella virulence; however, very little is known about the genetic determinants of host specificity. Initial attempts to define the genetic basis of host specificity revealed that its determinants in Salmonella are likely to be multifactorial. In generalized transduction experiments with phage P22 grown in a S. typhimurium donor and with S. typhi recipients, no virulent recombinants that could kill mice were obtained (Zahrt, 1998 ). These negative results indicate that multiple genetic differences between these serovars must contribute to host specificity.
We hypothesize that the ompDsmvA region contributes to host specificity, because the presence of ompD is strongly correlated with the ability of Salmonella serovars to grow in alternative, non-human hosts. OmpD is present in all Salmonella serovars, such as S. typhimurium and S. enteritidis, that grow in multiple mammalian hosts. In contrast, OmpD is absent from S. typhi, which can grow only in a human host. Although the S. paratyphi A, B and C serovars express OmpD, and were once thought to cause systemic infections only in humans, several reports have shown that S. paratyphi serovars B and C are not restricted to a human host and can infect cattle and poultry (George et al., 1972 ; Thomas, 1978
; Ojeniyi, 1984
). The almost perfect correlation between the absence of ompD and a host range restricted to humans raises the important question of whether S. paratyphi A also has an alternative vertebrate host.
In recent years, it has become clear that the outer-membrane porins are involved in the interactions of Gram-negative species, including Helicobacter pylori (Tufano et al., 1994 ), Neisseria gonorrhoeae (Bauer et al., 1999
), Shigella flexneri (Bernardini et al., 1993
), S. typhi (Blanco et al., 1997
) and possibly Pasteurella haemolytica (Davies et al., 1997
), with their specific hosts. The OmpD porin must play a critical role in the survival of S. typhimurium in certain environments, because OmpD facilitates the entry of some critical nutrients (Nikaido, 1996
). Furthermore, OmpD may also play a critical role in the interactions between S. typhimurium and host cells, and is likely to be involved in the adhesion of S. typhimurium to murine macrophages (Negm & Pistole, 1998
).
The characterization of this important genomic difference between S. typhi and other Salmonella serovars that includes ompD is significant for the following three reasons. First, the report of similar small differences in genome structure among different serovars will enable the development of additional PCR assays for a more robust assessment of the epidemiology of Salmonella outbreaks. Second, by demonstrating that generalized transduction can be used to construct interspecific hybrids between different Salmonella serovars, it is possible to take a genetic approach to assessment of the contributions of small genomic differences between two serovars to differences in host specificity. Third, the correlation between the presence of the ompD gene and the ability of Salmonella serovars to infect vertebrate, non-human hosts is the first reported correlation between an individual gene and the host range of Salmonella serovars.
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ACKNOWLEDGEMENTS |
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Received 27 February 2001;
accepted 26 March 2001.