Department of Microbiology, University of Illinois, Urbana, IL 61801, USA1
Author for correspondence: Robert A. Edwards. Tel: +1 901 448 8101. Fax: +1 901 448 8462. e-mail: redwards{at}utmem.edu
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ABSTRACT |
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Keywords: pili, salmonellosis, regulation, genomics, pathogenicity islands
The GenBank accession number for the sequence reported in this paper is AF239978.
a Present address: Department of Molecular Sciences, University of Tennessee Health Sciences Center, MSB101 858 Madison Ave, Memphis, TN 38163, USA.
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INTRODUCTION |
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Most fimbriae (with the notable exception of the type-IV pili) have a similar mechanism of translocation and biogenesis. The fimbrial shaft is composed of a major subunit that is translocated across the cytoplasmic membrane via the general secretory (sec-dependent) system. In the periplasm the subunits are prevented from premature aggregation by binding to a chaperone. Resolution of the crystal structure of two fimbrial chaperone/subunit complexes elegantly revealed the interactions involved in regulating the periplasmic embrace between these two partners, simultaneously preventing premature subunitsubunit interactions but allowing subunit translocation across the outer membrane (Choudhury et al., 1999 ; Sauer et al., 1999
). A specific usher protein mediates export across the outer membrane, with a distinct chaperoneusher pair encoded by each fimbrial operon. Although chaperones and ushers from different fimbrial systems are very homologous, the usher and chaperone work together and are normally specific for the fimbrial subunits a chaperone and usher pair will not usually mediate translocation of fimbrial subunits from another operon. Many fimbrial systems also encode one or more minor components (so called because they are much less abundant than the major subunit) that are exported as part of the fimbrial structure (Edwards et al., 1996
; Edwards & Puente, 1998
). These subunits often form the adhesin that mediates the interaction between the fimbriae and the solid surface (Khan & Schifferli, 1994
).
A variety of fimbriae have previously been characterized in Salmonella enterica, including the plasmid-encoded (PEF), long polar (LPF), thin aggregative (AGF) and type I (FIM) fimbriae (Bäumler et al., 1997 ; Townsend et al., 2001
). Many of the fimbriae from Salmonella enterica subsp. enterica serovar Enteritidis are similar to fimbriae from Salmonella Typhimurium, although a number of these fimbriae were initially given alternate names (for example the Salmonella Enteritidis AGF homologue was initially called SEF17 and the FIM homologue was initially called SEF21). In addition to the fimbriae that are shared between serovars, Salmonella Enteritidis contains fimbriae such as the Salmonella Enteritidis SEF14 fimbriae that are not found in Salmonella Typhimurium. The SEF14 fimbriae require four proteins for biogenesis: the major subunit (SefA), chaperone (SefB) and usher (SefC) have previously been characterized (Clouthier et al., 1993
; Thorns et al., 1990
; Turcotte & Woodward, 1993
). The minor subunit (SefD) was also previously identified but not thought to be part of the SEF14 fimbriae (Clouthier et al., 1994
).
We recently reported the role of the SEF14 fimbriae in virulence and showed that SEF14 fimbriae are essential for binding to macrophages (Edwards et al., 2000 ). In this report we describe a multi-faceted approach to dissecting the sef operon, utilizing genomics and genetics to define and inactivate genes. We identify the principal regulatory element that controls sef gene expression and characterize environmental regulation of sef gene expression.
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METHODS |
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Bacterial strains, plasmids, phage and growth conditions.
All bacterial strains, plasmids and phage used in this study are given in Table 1. All strains were grown in LuriaBertani (LB) medium, supplemented with the following antibiotics where necessary (final concentrations, µg ml-1): kanamycin, 50; chloramphenicol, 30; tetracycline, 10; ampicillin, 90. All transductions were performed using P22 HT int. Transductants were purified on Evans Blue Uranine (EBU) plates and checked for lysogens by cross-streaking against phage P22 H5 as described previously (Maloy et al., 1996
).
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To introduce the mutations onto the chromosome, the suicide plasmid was electroporated either into TYT3362 (a sef+ derivative of Salmonella Typhimurium LT2 whose construction is described below) or wild-type LK5 with selection for the MudJ-encoded KanR marker. Enrichment for second recombination events that eliminated the suicide vector was performed by growth on sucrose (Edwards et al., 1998 ) and confirmed by checking for loss of the plasmid-encoded TetR marker and by PCR to confirm inheritance of the mutant allele.
To construct both the constitutively expressed sefR construct (pRE234) and the fusion between sefR and the arabinose-inducible promoter (pRE235), a sefR PCR product was cloned into either pUC19 or pBAD18, respectively, using primers 5'-GGTCAGAATTCCTCAGCCCATAA-3' and 5'-CGGAGTCTGCAGTGAAGCGTAAAAA-3'. This 1078 bp product encodes the 3' end of the sefD gene, all of the sefR gene and 38 bp upstream of sefR. The expression of sefR in the pUC19 clone is controlled by the lac promoter in this plasmid, and the pBAD18 construct contains the arabinose-inducible promoter and araC, the gene encoding the transcriptional activator for expression from that promoter (Guzman et al., 1995 ).
Identification of linked markers and genetic manipulations.
To identify markers linked to the sef island, plasmid pBSL142 (a delivery vehicle for Tn5dGen; Alexeyev et al., 1995 ) was electroporated into Salmonella Enteritidis sefA1::Kan that had been heated to inactivate the restriction modification system (Edwards et al., 1999
). Because pBSL142 is a suicide plasmid, any GenR colonies should result from transposition events. Colonies were pooled and used as donors in a second transduction with wild-type Salmonella Enteritidis as the recipient, selecting for both GenR and KanR simultaneously. The resulting colonies contain both sefA1::Kan and a linked Tn5dGen. The GenR marker was subsequently separated from the sefA1::Kan allele by transduction. Several GenR alleles were transduced from Salmonella Enteritidis to Salmonella Typhimurium LT2. The transductants were screened for the presence of the sef locus by a PCR assay for each of the sef genes.
To transduce the sefD::MudJ fusion into a strain harbouring sefA1::Kan, the fusion was linked to a GenR allele that lies outside of the sef island and has no effect on the regulation of expression of the sefD::MudJ fusion. The markers were moved into Salmonella Enteritidis LK19 by transduction followed by selection for GenR and screening for both sefA1::Kan and sefD::MudJ by PCR. The presence of sefB and lacZ were confirmed by PCR both before and after expression studies to confirm that the sefA1::Kan and MudJ elements did not recombine. If the sefA1::Kan and sefD::MudJ elements recombined both sefB and lacZ would be deleted. Although this may happen at a very low frequency, such recombination events were never observed.
One of the Tn5dGen mutations located very close to the sef operon, but not within the operon itself co-transduces with sefA::Kan approximately 80% of the time. This insertion does not affect virulence and lies outside the sef insertion in the genome. This transposon insertion was used to backcross the sefA mutation into an otherwise wild-type background by isolation of a strain with the transposon linked to the in-frame deletion, followed by P22 transductions of the transposon into LK5 and screening for co-inheritance of the
sefA allele.
ß-Galactosidase assays.
Assays were performed as described elsewhere and the activities are reported in Slauch units (Maloy, 1990 ; Slauch & Silhavy, 1991
).
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RESULTS |
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Three group D Salmonella serovars have been shown to contain the sef operon inserted at leuX: Salmonella Enteritidis, Salmonella Typhi and Salmonella Paratyphi. Phage genomes separate leuX and the IS1230 element/sef island in the latter two serovars, but not in Salmonella Enteritidis (Fig. 1). Sequencing data suggests that the Salmonella Typhi sef operon contains multiple frameshift mutations. These have been confirmed by independent sequencing, suggesting that the sef genes are not expressed in Salmonella Typhi (Townsend et al., 2001
).
Unlike Salmonella Typhi and Salmonella Typhimurium, there is no phage genome inserted between the IS1230 element and leuX in Salmonella Enteritidis. However, the entire leuX gene has been substituted by a putative transposon containing a different leuX gene. The transposon contains 16 bp direct inverted repeats that delimit the foreign DNA inserted into the Salmonella Enteritidis genome and a transposase gene immediately adjacent to one inverted repeat (Fig. 1).
Salmonella Typhimurium, which is not a member of the group D Salmonella, does not contain any of the sef island, IS1230 element, transposon or phage genome at leuX. Based upon analysis of the Salmonella Typhimurium genome sequence, we have identified a previously uncharacterized 25337 bp pathogenicity island inserted at this site (data not shown). This island contains very limited homology to sequences in the databases; however, one ORF is similar to the Shigella flexneri virulence gene, shiB, and another was identified as being specifically induced inside macrophages (Pfeifer et al., 1999 ). This island has not been characterized further; however, it indicates that as in Escherichia coli there have been multiple independent acquisitions of pathogenicity islands at leuX in the Salmonella chromosome: the sef island was probably inherited after group D Salmonella diverged from other groups and Salmonella Typhimurium inherited a separate island.
The sequence of the Salmonella Enteritidis sef region from more than 1 kb upstream of sefA to a few base pairs downstream of sefD was available from GenBank as several different sequences (accession nos L11008, L11009, L11010, U07129 and X98516); however, sequence information in the region downstream of sefD was not available. Using primers designed to be compatible with Salmonella Enteritidis sefD and Salmonella Typhi mcrD regions, the DNA downstream of sefD was PCR-amplified and sequenced. The sequence adjacent to sefD in Salmonella Enteritidis was similar to the corresponding region from the Salmonella Typhi genome and contains both sefR, encoding the transcriptional activator, and dlp, encoding the thiol:disulphide oxidoreductase. The sequence was compiled together with the previously published sequences and the entire contig was deposited in the GenBank database with the accession number AF239978. The sequence of adjacent regions in the Salmonella Enteritidis genome have been released as part of the genome sequencing project and can be retrieved from http://www.salmonella.org.
Transduction of the sef island to Salmonella Typhimurium LT2
A genetic approach was taken to characterize the islands and genes that were identified from the genomic analysis. Salmonella Enteritidis contains a potent restriction system that limits genetic manipulations in this strain. The restriction system was circumvented by moving the entire sef island from Salmonella Enteritidis to Salmonella Typhimurium. Several Tn5dGen transposon insertions linked to the sef operon were identified. Following transduction from Salmonella Enteritidis to Salmonella Typhimurium two classes of insertion were found. The first were 100% linked to the sefA operon in transductions between Salmonella Enteritidis and Salmonella Typhimurium, although there was variable linkage in transductions between Salmonella Enteritidis and Salmonella Enteritidis. This class comprised insertions located in the sef island (Fig. 2a). The Salmonella EnteritidisSalmonella Enteritidis transductions reflect the expected genetic linkage based upon physical distance between the two markers on the chromosome, but because Salmonella Typhimurium does not contain the sef island, transduction of the Tn5dGen from Salmonella Enteritidis to Salmonella Typhimurium always resulted in inheritance of the sef island. The second class of insertions had a greatly reduced linkage with the sef operon in transductions between Salmonella Enteritidis and Salmonella Typhimurium compared to transductions between Salmonella Enteritidis and Salmonella Enteritidis. This class comprised insertions near the sef island but in chromosomal DNA that is common between Salmonella Enteritidis and Salmonella Typhimurium. Transduction of these markers did not demand inheritance of the sef genes (Fig. 2b
).
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Growth-phase regulation of sef gene expression
To investigate the regulation of sef gene expression, ß-galactosidase activities were compared at different time points during growth. Expression of both sefR and sefD was optimal during late exponential growth and declined during stationary phase (Fig. 5). Growth-phase-dependent regulation of gene expression appears to occur at the sefR promoter as expression of both sefR and sefD were regulated by growth phase and we have shown that sefR regulates sefD expression. We had previously noted that sef gene expression was higher in a Salmonella Typhimurium LT2 background than in a wild-type Salmonella Enteritidis background. Salmonella Typhimurium has several genetic defects accumulated over years of maintenance in the laboratory. One such defect is a mutation that reduces rpoS expression; thus we reasoned that RpoS may be responsible for both the difference in expression between Salmonella Enteritidis and Salmonella Typhimurium and the growth-phase-dependent regulation of sef gene expression. To investigate whether the difference in expression levels observed between Salmonella Enteritidis and Salmonella Typhimurium could be explained by rpoS effects, an rpoS::Pen mutation was transduced from Salmonella Typhimurium to Salmonella Enteritidis. Because there was no detectable difference in growth rate, ß-galactosidase activity was measured after 16 h of growth, when sef expression is normally repressed (Fig. 5
). Inactivation of rpoS relieved this repression and the sef genes were expressed at a high level at this time. In contrast, when rpoS was restored on a plasmid the repression returned (Fig. 6
). Together these results suggest that expression of the sef operon is repressed, either directly or indirectly, by the stationary phase sigma factor
s in Salmonella Enteritidis.
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DISCUSSION |
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Analysis of the available genome sequences from various Salmonella serovars revealed a novel large pathogenicity island in all four serovars. From hybridization and PCR studies it was known that the sef operon was located on an island which is absent from many other Salmonella but the proximity of phage genomes to this operon had not been appreciated. It remains to be determined whether the phage genomes adjacent to the sef operon are responsible for the transmission of this island between strains. The Salmonella Typhimurium genome sequence revealed a large insertion in which only a single ORF had been previously identified. Our analysis suggests that there may be other, as yet uncharacterized, virulence genes in this region.
A region of homology between the virulence plasmid and the chromosome was identified downstream of the sef operon. Salmonella Typhi does not contain the virulence plasmid but contains the chromosomal homologue of this region and Salmonella Typhimurium contains the region on the virulence plasmid but does not contain the chromosomal homologue. The homology between plasmid and chromosome is potentially only found in a few Salmonella serovars, including Salmonella Enteritidis, which contain both virulence plasmid and chromosomal homologues. Such regions of homology may allow the plasmid to integrate into the chromosome by homologous recombination. As the virulence plasmid is self-transmissible this would result in the formation of an Hfr capable of transferring virulence factors and other genetic elements between serovars (Ahmer et al., 1999 ). The formation of an Hfr in this way could have profound impact on the emergence of infectious disease and the spread of antibiotic resistance genes.
After this manuscript was first submitted, another group also described the chromosomal region surrounding the sef island (Collighan & Woodward, 2001 ). There appears to have been a rearrangement around the sef island when their description of the island is compared to Fig. 1
. In the strain of Salmonella Enteritidis used in their study, leuX is located downstream of sefD and the sef island is flanked by two insertion elements. They also identify an AraC-like transcriptional activator that they designate SefE. However, when this manuscript was prepared this sequence was not available in the DNA or protein databases and therefore could not be compared to the sequence described here. A third group has also reported the sequence of an AraC-like regulator that is involved in the regulation of the sef operon. This has been deposited in GenBank with the accession number 7330248 (J. A. Botten, I. Kotlarski & R. Morona, unpublished). The two available SefR sequences only differ at two of their 271 residues.
A previous study indicated that sefABC were probably encoded in a single operon (Clouthier et al., 1993 ). This study also noted that accessory proteins in addition to SefA, SefB and SefC must be required for export of SEF14 fimbriae as no fimbriae were detected on the surface of a smaller clone of the island (Clouthier et al., 1993
). The clone that was used in this study was a HindIII subclone from a cosmid. There is a HindIII site in sefD which would produce a clone of the size observed (5·3 kb), suggesting that clones lacked both sefD and sefR, and providing a likely explanation why SEF proteins were not expressed. Another report described a unique 18 kDa fimbria, ascribed to sefD. In this report, antibodies were prepared against the fimbrial protein and used to probe for the distribution of the fimbriae amongst enteric bacteria. Genomic DNA was also probed with a sefD-specific PCR product. Both these techniques suggested that sefD, unlike sefABC, had a widespread distribution among enteric bacteria (Clouthier et al., 1994
). Several lines of evidence refute this distribution. (i) There is no homology between sefD or its predicted product and the DNA and protein sequences in the E. coli genomic databases even though it was reported that the 18 kDa fimbria can be found in E. coli. (ii) More exhaustive studies using either PCR or Southern hybridization were unable to identify a sefD homologue outside the group D Salmonella (data not shown). (iii) Our studies have shown that a single transcript initiated upstream of sefA and extending through sefD encodes these fimbrial proteins. Moreover, the close coupling of the UGA stop codon for sefC and the AUG initiation codon for sefD indicates that SefC and SefD proteins are co-expressed as expected for components of the same fimbriae. There are no known cases where subunits that form different fimbriae are co-expressed from the same operon, so this close coupling suggests that SefD is part of the SEF14 fimbriae.
Comparison of growth-phase-dependent regulation of gene expression, and the expression of the sef genes in wild-type Salmonella Enteritidis and Salmonella Typhimurium LT2, a natural rpoS mutant, indicated that the sef genes are repressed by s, the stationary phase sigma factor. These results do not identify whether rpoS affects sef gene expression directly or indirectly and therefore the precise role of rpoS in regulation of sef gene expression, as well as the role of other genetic elements outside of the sef operon remains to be determined.
A previous report showed that the sef genes require a rare arginine tRNA (tRNA-UCU) for expression (Clouthier et al., 1998 ). It is not known why this tRNA is required. However, genomic analysis of the sef operon showed that the mean G+C content of this region is 35·4 mol%. In comparison, the entire Salmonella Enteritidis genome has a mean G+C content of 52 mol%. The arginine codon recognized by the rare tRNA is AGA. Of the arginine codons in the sef island, 58% are AGA and only 2% are CGC, the preferred Salmonella arginine codon. The limited distribution of the sef island suggested that it is a recent acquisition on the chromosome and this is supported by its unusual codon usage profile. The regulation of the sef operon by tRNA-UCU may simply reflect this different codon usage which presumably betrays some ancestral host with an A+T-rich genome, rather than a specific regulatory mechanism for sensing arginine concentrations.
Although many other fimbrial systems from Salmonella serovars have been identified and characterized, type I fimbriae and the aggregative fimbriae encoded by agfBAC were the only examples for which a transcriptional regulator has been identified (Romling et al., 2000 ; Tinker & Clegg, 2000
, 2001
; Tinker et al., 2001
). It remains to be seen which elements are required for these systems. Many other fimbrial systems use AraC-like transcriptional activators to regulate gene expression and often the regulator is immediately adjacent to the fimbrial operon, as is found for the sef operon. For example, the 987P fimbria in enterotoxigenic E. coli utilizes a homologous regulator (FasH) to activate gene transcription (Edwards & Schifferli, 1997
). The similarity and location of these regulators and the similar organization of the fimbrial operons as a whole [in general they are organized major subunit-chaperone-usher-(optional minor subunits)-regulator] suggests that many fimbrial operons have evolved from some common ancestor.
All of the AraC-like family of transcriptional activators involved in regulation of fimbrial gene expression have similar C-terminal domains. This domain with conserved motifs is the DNA-binding domain. In contrast the N-terminal domains are extremely different. This domain with no conserved motifs is presumably the sensor domain that regulates DNA binding in response to environmental cues. This suggests as practical experience has shown (Edwards & Puente, 1998 ; Gallegos et al., 1997
; Jordi, 1992
; Martinez-Laguna et al., 1999
) that each regulator may respond to different environmental stimuli, and hence each fimbria is expressed in different conditions and perhaps in a different location in the host.
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ACKNOWLEDGEMENTS |
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Received 9 March 2001;
revised 5 June 2001;
accepted 12 June 2001.
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