Analysis of the lateral flagellar gene system of Aeromonas hydrophila AH-3.

Mesophilic Aeromonas strains express a polar flagellum in all culture conditions, and certain strains produce lateral flagella on semisolid media or on surfaces. Although Aeromonas lateral flagella have been described as a colonization factor, little is known about their organization and expression. Here we characterized the complete lateral flagellar gene cluster of Aeromonas hydrophila AH-3 containing 38 genes, 9 of which (lafA-U) have been reported previously. Among the flgLL and lafA structural genes we found a modification accessory factor gene (maf-5) that is involved in formation of lateral flagella; this is the first time that such a gene has been described for lateral flagellar gene systems. All Aeromonas lateral flagellar genes were located in a unique chromosomal region, in contrast to Vibrio parahaemolyticus, in which the analogous genes are distributed in two different chromosomal regions. In A. hydrophila mutations in flhAL, lafK, fliJL, flgNL, flgEL, and maf-5 resulted in a loss of lateral flagella and reductions in adherence and biofilm formation, but they did not affect polar flagellum synthesis. Furthermore, we also cloned and sequenced the A. hydrophila AH-3 alternative sigma factor sigma54 (rpoN); mutation of this factor suggested that it is involved in expression of both types of flagella.

Lateral flagella are required for increased cell adherence, invasion and biofilm formation by Aeromonas spp.

Two types of flagella are responsible for motility in mesophilic Aeromonas strains. A polar unsheathed flagellum is expressed constitutively that allows the bacterium to swim in liquid environments and, in media where the polar flagellum is unable to propel the cell, Aeromonas express peritrichous lateral flagella. Recently, Southern blot analysis using a DNA probe based on the Aeromonas caviae Sch3N lateral flagellin gene sequence showed a good correlation between strains positive for the DNA probe, swarming motility and the presence of lateral flagella by microscopy. Here, we conclude that the easiest method for the detection of the lateral flagellin gene(s) is by PCR (polymerase chain reaction); this showed good correlation with swarming motility and the presence of lateral flagella. This was despite the high degree of DNA heterogeneity found in Aeromonas gene sequences. Furthermore, by reintroducing the laf (lateral flagella) genes into several mesophilic lateral-flagella-negative Aeromonas wild-type strains, we demonstrate that this surface structure enhances the adhesion to and invasion of HEp-2 cells and the capacity for biofilm formation in vitro. These results, together with previous data obtained using Laf- mutants, demonstrate that lateral flagella production is a pathogenic feature due to its enhancement of the interaction with eukaryotic cell surfaces.

A polar flagella operon (flg) of Aeromonas hydrophila contains genes required for lateral flagella expression.

Aeromonas spp. are pathogens of both humans and poikilothermic animals, causing a variety of diseases. Certain strains are able to produce two distinct types of flagella; polar flagella for swimming in liquid and lateral flagella for swarming over surfaces. Although, both types of flagella have been associated as colonisation factors, little is known about their organisation and expression. Here we characterised a complete flagellar locus of Aeromonas hydrophila (flg) containing 16 genes, this was analogous to region 1 of the Vibrio parahaemolyticus polar flagellum, with the difference that no flagellin genes were found on A. hydrophila while V. parahaemolyticus showed three flagellin genes. The flg region was present in all Aeromonas strain tested. Defined insertion mutants in flgL, were unable to swim, had a drastic reduction in swarming, lateral flagella, HEp-2 cell adhesion and biofilm formation. Mutations in flgN caused a drastic reduction in lateral flagella, inability to swarm, but these strains were still able to swim. Whereas the cheV mutants still produced both types of flagella and were able to swim and swarm. These results suggest that FlgN is required for lateral flagella formation and swarming motility, but not for polar flagellum-mediated swimming.