The Aeromonas hydrophila wb * _O34 Gene Cluster: Genetics and Temperature Regulation

Abstract

The Aeromonas hydrophila wb* O34 gene cluster of strain AH-3 (serotype O34) was cloned and sequenced. This cluster contains genes necessary for the production of O34-antigen lipopolysaccharide (LPS) in A. hydrophila. We determined, using either mutation or sequence homology, roles for the majority of genes in the cluster by using the chemical O34-antigen LPS structure obtained for strain AH-3. The O34-antigen LPS export system has been shown to be a Wzy-dependent pathway typical of heteropolysaccharide pathways. Furthermore, the production of A. hydrophila O34-antigen LPS in Escherichia coli K-12 strains is dependent on incorporation of the Gne enzyme (UDP-N-acetylgalactosamine 4-epimerase) necessary for the formation of UDP-galactosamine in these strains. By using rapid amplification of cDNA ends we were able to identify a transcription start site upstream of the terminal wzz gene, which showed differential transcription depending on the growth temperature of the strain. The Wzz protein is able to regulate the O34-antigen LPS chain length. The differential expression of this protein at different temperatures, which was substantially greater at 20°C than at 37°C, explains the previously observed differential production of O34-antigen LPS and its correlation with the virulence of A. hydrophila serotype O34 strains.In gram-negative bacteria lipopolysaccharide (LPS) is one of the major structural and immunodominant molecules of the outer membrane. LPS consists of three moieties: lipid A, core oligosaccharide, and O-specific antigen or O side chain. The O antigen is the external component of LPS, and it consists of a polymer of oligosaccharide repeating units. Another interesting feature is the high chemical variability shown by the O-antigen LPS, which leads to similar genetic variation in the genes involved in LPS biosynthesis, the so-called wb* cluster (for a review, see reference 45). The genetics of O-antigen biosynthesis have been intensively studied in the Enterobacteriaceae, and it has been shown that the wb* clusters usually contain genes involved in biosynthesis of activated sugars, glycosyl transferases, and O-antigen polymerases and in O-antigen export. Despite heterogeneity in the structures of the O antigens, only three pathways for assembly of O antigens have been recognized: the Wzy-dependent pathway, the ABC transporter-dependent pathway, and the synthase-dependent pathway (45).Mesophilic motile Aeromonas species are opportunistic and primary pathogens of a variety of aquatic and terrestrial animals, including humans; the clinical manifestations range from gastroenteritis to soft tissue infections, including septicemia and meningitis (5, 25). Mesophilic Aeromonas sp. serotype O34 strains have been recovered from moribund fish (33) and from clinical specimens (23); serotype O34 is the single most common Aeromonas serotype (24), accounting for 26.4% of all infections. Previous investigations have documented that serotype O34 strains are important causes of infections in humans (23,24). The varied clin...

A UDP-HexNAc:Polyprenol-P GalNAc-1-P Transferase (WecP) Representing a New Subgroup of the Enzyme Family

“…However, the sugar analysis of LPS from AH-3⌬wecP mutant with plasmid pBAD33-WecA Ec grown under expressing conditions (with arabinose) showed the same monosaccharides as described above plus 6-deoxy-talose (L-6dTal) and mannose (D-Man), in agreement with the reported O34-antigen LPS (Fig. 1) (20,23), but with GlcNAc instead of N-acetyl-galactosamine (GalNAc), which is the other sugar from the O34-antigen LPS in the wild-type strain (Fig. 1).…”

“…As we previously published, the AH-3⌬wecP mutant was completely unable to biosynthesize the O34-antigen LPS (20). The mutant harboring plasmid pBAD33-WecP Ah (carrying the gene from strain AH-3) showed an LPS profile identical to that of the wild-type strain, while no changes could be observed when the mutant carried the plasmid vector alone or plasmid pBAD33-WbaP Se carrying the Salmonella enterica LT2 wbaP (Fig.…”

“…The AH-3⌬wecP mutant was complemented by different pBAD33 plasmids with several Aeromonas wecP homologues from strains like A. hydrophila PPD134/91 (20), A. caviae Sch3N (12), A. veronii biovar Sobria AH-41 (34), and A. salmonicida subsp. salmonicida A449 (21), according to their LPS profiles (data not shown).…”

“…The Aeromonas hydrophila wb O34 gene cluster contains genes necessary for the production of O34-antigen LPS, and a role for the majority of these genes was previously attributed according to the chemical O34-antigen LPS structure obtained from strain AH-3 ( Fig. 1) (20). By mutation and sequence homology, ORF15 from the A. hydrophila wb O34 gene cluster was putatively annotated as an undecaprenol-sugar-P-transferase (WecP, formerly WecA) able to initiate the assembly of the O34-antigen units, and the assembly of the O34 antigen was characterized as a Wzy-dependent pathway (20).…”

“…1) (20). By mutation and sequence homology, ORF15 from the A. hydrophila wb O34 gene cluster was putatively annotated as an undecaprenol-sugar-P-transferase (WecP, formerly WecA) able to initiate the assembly of the O34-antigen units, and the assembly of the O34 antigen was characterized as a Wzy-dependent pathway (20). In this study, we report the characterization of Aeromonas WecP, which represents a new class of UDPHexNAc:polyprenol-P HexNAc-1-P transferases, according to its specific substrate preference.…”