Aeromonas caviae Sch3N possesses a small genomic island that is involved in both flagellin glycosylation and lipopolysaccharide (LPS) O-antigen biosynthesis. This island appears to have been laterally acquired as it is flanked by insertion element-like sequences and has a much lower G؉C content than the average aeromonad G؉C content. Most of the gene products encoded by the island are orthologues of proteins that have been shown to be involved in pseudaminic acid biosynthesis and flagellin glycosylation in both Campylobacter jejuni and Helicobacter pylori. Two of the genes, lst and lsg, are LPS specific as mutation of them results in the loss of only a band for the LPS O-antigen. Lsg encodes a putative Wzx flippase, and mutation of Lsg affects only LPS; this finding supports the notion that flagellin glycosylation occurs within the cell before the flagellins are exported and assembled and not at the surface once the sugar has been exported. The proteins encoded by flmA, flmB, neuA, flmD, and neuB are thought to make up a pseudaminic acid biosynthetic pathway, and mutation of any of these genes resulted in the loss of motility, flagellar expression, and a band for the LPS O-antigen. Furthermore, pseudaminic acid was shown to be present on both flagellin subunits that make up the polar flagellum filament, to be present in the LPS O-antigen of the A. caviae wild-type strain, and to be absent from the A. caviae flmD mutant strain.Mesophilic Aeromonas strains are being increasingly recognized as important bacterial pathogens. They are widely distributed in the environment and cause gastrointestinal and wound infections in healthy humans and, less commonly, septicemia in immunocompromised patients (15). In particular, Aeromonas caviae is reported to be the most prevalent pediatric enteropathogenic species of the genus (30,46). A range of putative virulence factors have been described for the aeromonads, from the hemolytic toxin aerolysin and cytotonic toxins to capsules and extracellular enzymes (44). The process of adherence of aeromonads is still poorly understood, although a number of factors have been implicated, such as long wavy pili, outer membrane proteins, lipopolysaccharide (LPS) Oantigen, and the polar flagellum (1, 44). The mesophilic aeromonads are interesting as most strains express two distinct flagellum systems (10, 34). They have a polar flagellum for swimming in liquid and express separate lateral flagella for swarming over surfaces. Investigations have revealed that both the polar and lateral flagellum systems of the mesophilic aeromonads are involved in adherence to both biotic and abiotic surfaces (20).Previously, we showed that transposon mutations in the flm locus of A. caviae greatly reduced adherence of this organism to the human epithelial cell line HEp-2. In addition, mutation of this locus caused losses of motility, flagella, and the LPS O-antigen (12). In A. caviae Sch3N the flmA and flmB genes were clustered together in a locus with neuA, flmD, and neuB.

Motility in Aeromonas caviae, in a liquid environment (in broth culture), is mediated by a single polar flagellum encoded by the fla genes. The polar flagellum filament of A. caviae is composed of two flagellin subunits, FlaA and FlaB, which undergo O-linked glycosylation with six to eight pseudaminic acid glycans linked to serine and threonine residues in their central region. The flm genetic locus in A. caviae is required for flagellin glycosylation and the addition of pseudaminic acid (Pse) onto the lipopolysaccharide (LPS) O-antigen. However, none of the flm genes appear to encode a candidate glycotransferase that might add the Pse moiety to FlaA/B. The motility-associated factors (Maf proteins) are considered as candidate transferase enzymes, largely due to their conserved proximity to flagellar biosynthesis loci in a number of pathogens. Bioinformatic analysis performed in this study indicated that the genome of A. caviae encodes a single maf gene homologue (maf1). A maf mutant was generated and phenotypic analysis showed it is both nonmotile and lacks polar flagella. In contrast to flm mutants, it had no effect on the LPS O-antigen pattern and has the ability to swarm. Analysis of flaA transcription by reverse transcriptase PCR (RT-PCR) showed that its transcription was unaltered in the maf mutant while a His-tagged version of the FlaA flagellin protein produced from a plasmid was detected in an unglycosylated intracellular form in the maf strain. Complementation of the maf strain in trans partially restored motility, but increased levels of glycosylated flagellin to above wild-type levels. Overexpression of maf inhibited motility, indicating a dominant negative effect, possibly caused by high amounts of glycosylated flagellin inhibiting assembly of the flagellum. These data provide evidence that maf1, a pseudaminyl transferase, is responsible for glycosylation of flagellin and suggest that this event occurs prior to secretion through the flagellar Type III secretion system.