ABSTRACT
We report sequencing of the O antigen encoded by the rfb gene cluster of Salmonella enterica serotype Jangwani (O17) and Salmonella serotype Cerro (O18). We developed serogroup O17- and O18-specific PCR assays based on rfb gene targets and found them to be sensitive and specific for rapid identification of Salmonella serogroups O17 and O18.
Serotyping of Salmonella enterica based on O and H surface antigens has proven to be extremely useful for understanding the host range and disease spectrum of this pathogen (20) and has been an invaluable typing method for epidemiological investigations (2, 3, 19, 20). It does, however, have limitations; production and quality control of the hundreds of antisera required for serotyping is difficult and time consuming, and not all Salmonella strains are typeable. Many of the enzymes involved in O-antigen biosynthesis are encoded by an rfb gene cluster which varies in size for each serogroup depending on the sugar composition and complexity of the O antigen (24). rfb gene clusters have been characterized from a growing number of gram-negative bacteria (23), including 13 Salmonella serogroups (9, 22, 26, 28). The use of PCR targeting regions within rfb gene clusters for O-antigen identification of various organisms (6, 7, 11, 27), including Salmonella (9, 10, 16), has been reported as an alternative to O-group detection by traditional serotyping. The rfb gene clusters of the more common Salmonella serogroups have been the most extensively studied at the molecular level to date. Little is known about the rfb gene clusters of other Salmonella O serogroups. As part of a larger project to develop a DNA-based approach for serotyping Salmonella, we report here the sequence analysis of the rfb gene cluster of two serogroups, O17 from Salmonella enterica serotype Jangwani and O18 from Salmonella serotype Cerro, and the development of serogroup-specific PCR detection assays based on the rfb gene targets wzy and wzx, respectively.
Salmonella isolates 00-0073 (Salmonella serotype Jangwani, 17:a:1,5) and 99-0087 (Salmonella serotype Cerro, 18:z4,z23:[1,5]) were selected for rfb sequence analysis. Serotyping was performed according to the Kaufmann-White scheme (2, 3). For evaluation of the serogroup O17- and O18-specific PCR assays, 443 strains representing all of the subspecies/serogroup combinations recognized in the Kaufmann-White scheme were evaluated. The rfb regions of O17 and O18 were amplified by PCR using previously described primers (14). A combination of subcloning and primer walking was used to sequence the rfb regions in both directions on an automated DNA sequencer (PE Applied Biosystems, California). DNA sequence data were assembled and analyzed using Lasergene 99 (DNAStar) software.
O17 rfb gene cluster.
The O17-antigen structure is unknown. Chemical analyses suggest that the O17 antigen contains a polysaccharide core comprised of galactose, glucose, glucosamine, heptose, and 3-deoxy-oct-2-ulosonic acid but has no reported additional sugars in its O17-antigen-specific side chain (15). Analysis of the DNA sequence of the rfb gene cluster from Salmonella serotype Jangwani revealed 9.130 kbp flanked by JUMPstart and gnd and eight open reading frames (ORFs) (Fig. 1A). All ORFs were analyzed using BLAST, and the genes were named in accordance with Bacterial Polysaccharide Gene Database (BPGD) nomenclature (21). All genes had the same transcriptional direction and have low G+C content (Table 1).
Organization and G+C content of (A) O17 and (B) O18, O6,14, and C1 rfb gene clusters. The percent G+C ratios were calculated and plotted for every 100 bases. Putative ORFs are represented by arrows, with the corresponding assignment of the gene name.
S. enterica serogroup O17 antigen biosynthetic proteins
We identified the gene mnaA, encoding a UDP-N-acetylglucosamine (GlcNAc)-2-epimerase. When analyzed using Pfam protein analysis, the predicted protein exhibited significant identity to the UDP-N-acetylglucosamine-2-epimerase (PN 00235) protein family. This enzyme catalyzes the reversible epimerization of UDP-N-acetylglucosamine (UDP-GlcNAc) and thereby provides the cell with UDP-N-acetylmannosamine (UDP-ManNAc), the activated donor of ManNAc residues. The presence of this gene in the O17 rfb gene cluster suggests that ManNAc may be one of the sugars in the Salmonella O17 antigen. orf7 showed the highest identity (65%) at the amino acid level with Glf, a UDP-galactopyranose mutase from Streptococcus suis. Salmonella serogroup B and D isolates have also been shown to have genes with identity to glf (Table 1), but they are not located in the rfb gene cluster. These enzymes catalyze the interconversion of the pyranose (UDP-Galp) and furanose (UDP-Galf) forms of galactose (18). The presence of this gene in the O17 rfb gene cluster suggests that galactofuranose is one of the sugars in the O17 antigen. The O17 rfb gene cluster contains four ORFs predicted to encode putative transferases. orf3, -5, -6, and -8 shared various degrees of similarity with glycosyltransferases from a range of other organisms (Table 1) and have been named wfbQ, wfbR, wfbS, and wfbU. Three of four putative O17 transferases (wfbQ, wfbR, and wfbS) are part of the GT4 family of retaining glycosyltransferases, and wfbU is a putative inverting GT2 family glycosyltransferase based on the carbohydrate-active enzyme database (CAZy) (http://www.cazy.org) classification (4, 5). We identified two ORFs encoding hydrophobic proteins with 10 and 11 predicted transmembrane domains. These are characteristic features for the O-antigen polymerase and transporter proteins, and we named these genes wzy and wzx. The putative O17 Wzx protein had similarities to several putative polysaccharide repeat unit transporters (Table 1).
O18 rfb gene cluster.
The O18-antigen-specific O-subunit structure has been determined using nuclear magnetic resonance and chemicals methods (25). Analysis of the DNA sequence from the Salmonella enterica O18 rfb gene cluster revealed 9.018 kbp flanked by JUMPstart and the gnd gene and contained seven open reading frames (Table 2). Five of the seven genes had a low G+C content ranging from 28.27% to 31.21% (Fig. 1B), a feature characteristic of other Salmonella rfb gene clusters. The two genes, manC and manB, had G+C contents of 47.46% and 55.29%, respectively. All of the genes have the same transcriptional direction. Since the O18 antigen contains mannose, it was anticipated that the rfb cluster would contain manB, encoding phosphomannomutase, and manC, encoding mannose-1-phosphate guanylytransferase, the two genes responsible for the biosynthesis of GDP-mannose from mannose-6-phosphate (12). orf3 and orf4 were identified as manB and manC, respectively, based on their high levels of identity to other GDP-mannose biosynthesis genes (12). The deduced gene products of orf1, orf2, and orf7 shared identity to glycosyltransferases, and we have named these genes wfbV, wfbW, and wfbX. wfbV and wfbW have specific homology to mannosyltransferases and retain glycosyltransferases from family GT4 (5). The remaining O:18 transferase, encoded by wfbV, could not be assigned to 1 of the 65 different glycosyltransferase families previously described and was left unclassified (5). A Pfam search (1) suggests it to be related to an acyltransferase family (PF01757). orf5 and orf6 are predicted to encode hydrophobic integral membrane proteins with 12 and 11 transmembrane domains, respectively, and have a 45% and 48% amino acid identity to the Wzy and Wzx proteins of Salmonella enterica O6,14 (9). We have named these genes wzy and wzx, respectively.
S. enterica serogroup O18 antigen biosynthetic proteins
O17- and O18-specific PCR.
A total of 46 pools of genomic DNA (50 ng/μl per isolate) were made, corresponding to the 46 O serogroups; each pool contained DNA from 1 to 12 isolates. The O17- and O18-specific primers (O17, 5′-GGC TGG GTT GTG GCT TTT T-3′ and 5′-CTT CCG AAA TCA ATA GAA AAA TCA A-3′; and O18, 5′-CTC TAG GAT CAA CTG AAG GTG GTC-3′ and 5′-CAA CCC AGC AAT AAA GCA GAA-3′) were derived from sequences within wzy and wzx to generate a 565-bp and a 370-bp PCR amplicon. PCR amplification was carried out with the following parameters: 96°C for 2 min, 25 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 45 s, and 72°C for 10 min. When a positive PCR result was seen for a particular genomic DNA pool, individual DNA samples from isolates represented in that pool were individually retested by PCR. A 565-bp amplicon was generated only with the pool containing DNA from isolates from different serotypes belonging to serogroup O17, and a 370-bp amplicon was generated only with the pool containing DNA from isolates from different serotypes belonging to serogroup O18. All other pools, containing DNA from 398 strains representing the other 44 serogroups, were negative in the PCR assays. DNA samples from the 12 strains in each of the O17 and O18 pools were individually tested in the PCR, and all generated a PCR product of the expected size (Fig. 2). An additional 19 O17 strains and 20 O18 strains were tested, and all generated the expected 565- and 370-bp PCR products, respectively.
(A) PCR amplification products with serogroup O17-specific primers using template DNA from different S. enterica serogroups. Lanes 1 and 5, serogroup O17 strains; lane 2, serogroup C1; lane 3, serogroup B; lane 4, serogroup D1; lane 6, 100-bp ladder (Gibco). (B) PCR amplification products with serogroup O18-specific primers using template DNA from different S. enterica serogroups. Lanes 1 and 7, 100-bp ladders (Gibco); lanes 2, 4, 5, and 6, serogroup O18 strains; lane 3, serogroup B strain.
Conclusions.
DNA sequence analysis of the rfb gene cluster from Salmonella serogroup O17 had limited identity to previously characterized rfb gene clusters and appears to have been acquired by lateral transfer from a low-G+C-content species. Evolution of most of the O18 rfb gene cluster also occurred within a low-G+C-content species prior to transfer to S. enterica, but the cluster appears to have been assembled from more than one source as the manC and manB genes at least appear to have been derived from the Salmonella cps cluster. The O18 rfb gene cluster is most related to Salmonella serogroups O6,14 and C1. The PCR assays described here form the basis for the development of molecular detection assays for the identification of Salmonella serogroups O17 and O18 based on genes directly involved in the formation of the O antigens. Together with the serogroup PCR assays that have previously been described for serogroups A/D, B, C2, and D (16) and, more recently, O6,14 (9), we intend to identify additional rfb targets specific for other Salmonella O antigens associated with the top 100 serotypes of human Salmonella infection. Our overall goal is to combine this serogroup identification system with DNA targets specific for H antigens (17) to establish a comprehensive DNA-based scheme for the identification of the major Salmonella serotypes (8). Currently, we are exploring the utility of additional technologies, including Luminex, as a potential platform to combine the O- and H-antigen molecular assays on a single platform. This will allow a rapid and convenient alternative for the definitive identification of Salmonella serotypes attainable by nonspecialized laboratories.
Nucleotide sequence accession numbers.
The DNA sequences have been deposited in GenBank and assigned accession numbers EF032634 and EF032635.
ACKNOWLEDGMENTS
We thank Bernard Henrissat (University of Marseille, France) for classifying the glycosyltransferases from Salmonella serogroups O17 and O18 using the CAZy scheme and Kate Wilson and Peter Reeves (University of Sydney, Australia) for assigning BPGD gene names for the Salmonella O17 and O18 rfb gene clusters.
The use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.
FOOTNOTES
- Received 5 May 2006.
- Accepted 9 October 2006.
↵▿ Published ahead of print on 20 October 2006.
