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.

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An Aeromonas hydrophila AH-3 miniTn5 mutant unable to produce polar and lateral flagella was isolated, in which the transposon was inserted into a gene whose encoded protein was an orthologue of the Campylobacter jejuni motility accessory factor (Maf) protein. In addition to this gene, several other related genes were found in this cluster that was adjacent to the region 2 genes of the polar flagellum. Mutation of the A. hydrophila AH-3 maf-2, neuB-like, flmD or neuA-like genes resulted in non-motile cells that were unable to swim or swarm due to the absence of both polar and lateral flagella. However, both polar and lateral flagellins were present but were unglycosylated. Although the A. hydrophila AH-3 or Aeromonas caviae Sch3N genes did not hybridize with each other at the nucleotide level, the gene products were able to fully complement the mutations in either bacterium. Furthermore, well-characterized C. jejuni genes involved in flagella glycosylation (Cj1293, -1294 and -1317) were fully able to complement A. hydrophila mutants in the corresponding genes (flmA, flmB and neuB-like). It was concluded that the maf-2, neuB-like, flmD and neuA-like genes are involved in the glycosylation of both the polar and the lateral flagella in Aeromonas strains.

Motility is an essential characteristic for mesophilic Aeromonas strains. We identified a new polar flagellum region (region 6) in the A. hydrophila AH-3 (serotype O34) chromosome that contained two additional polar stator genes, named pomA 2 and pomB 2. A. hydrophila PomA 2 and PomB 2 are highly homologous to other sodium-conducting polar flagellum stator motors as well as to the previously described A. hydrophila AH-3 PomA and PomB. pomAB and pomA 2 B 2 were present in all the mesophilic Aeromonas strains tested and were independent of the strains' ability to produce lateral flagella. Unlike MotX, which is a stator protein that is essential for polar flagellum rotation, here we demonstrate that PomAB and PomA 2 B 2 are redundant sets of proteins, as neither set on its own is essential for polar flagellum motility in either aqueous or high-viscosity environments. Both PomAB and PomA 2 B 2 are sodium-coupled stator complexes, although PomA 2 B 2 is more sensitive to low concentrations of sodium than PomAB. Furthermore, the level of transcription in aqueous and high-viscosity environments of pomA 2 B 2 is reduced compared to that of pomAB. The A. hydrophila AH-3 polar flagellum is the first case described in which two redundant sodium-driven stator motor proteins (PomAB and PomA 2 B 2 ) are found.The bacterial flagellum usually is powered by a reversible rotator motor at the base of the flagellum structure, which uses energy from either the proton or sodium ion gradient to drive the rotation of the flagellum filament. The flagellum motor is divided into two substructures: the stator and the rotor. The rotor is composed of the FliM and FliN proteins, which form the C ring structure, and the FliG protein. These three proteins act as a switch that controls the direction of flagellum rotation, clockwise or counterclockwise. The stator is the stationary component of the motor and consists of different proteins surrounding the rotor, which constitute proton or sodium ion channels that couple the flow of ions to flagellum rotation (8,10

A mutation in galU that causes the lack of O34-antigen lipopolysaccharide (LPS) in Aeromonas hydrophila strain AH-3 was identified. It was proved that A. hydrophila GalU is a UDP-glucose pyrophosphorylase responsible for synthesis of UDP-glucose from glucose 1-phosphate and UTP. The galU mutant from this strain showed two types of LPS structures, represented by two bands on LPS gels. The first one (slow-migrating band in gels) corresponds to a rough strain having the complete core, with two significant differences: it lacks the terminal galactose residue from the LPS-core and 4-amino-4-deoxyarabinose residues from phosphate groups in lipid A. The second one (fast-migrating band in gels) corresponds to a deeply truncated structure with the LPS-core restricted to one 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and three L-glycero-Dmanno-heptose residues. galU mutants in several motile mesophilic Aeromonas strains from serotypes O1, O2, O11, O18, O21 and O44 were also devoid of the O-antigen LPS. The galU mutation reduced to less than 1 % the survival of these Aeromonas strains in serum, decreased the ability of these strains to adhere and reduced by 1.5 or 2 log units the virulence of Aeromonas serotype O34 strains in a septicaemia model in either fish or mice. All the changes observed in the galU mutants were rescued by the introduction of the corresponding single wild-type gene.