MesophilicAeromonasspp. constitutively express a single polar flagellum that helps the bacteria move to more favorable environments and is an important virulence and colonization factor. Certain strains can also produce multiple lateral flagella in semisolid media or over surfaces. We have previously reported 16 genes (flgNtoflgL) that constitute region 1 of theAeromonas hydrophilaAH-3 polar flagellum biogenesis gene clusters. We identified 39 new polar flagellum genes distributed in four noncontiguous chromosome regions (regions 2 to 5). Region 2 contained six genes (flaAtomaf-1), including a modification accessory factor gene (maf-1) that has not been previously reported and is thought to be involved in glycosylation of polar flagellum filament. Region 3 contained 29 genes (fliEtoorf29), most of which are involved in flagellum basal body formation and chemotaxis. Region 4 contained a single gene involved in the motor stator formation (motX), and region 5 contained the three master regulatory genes for theA. hydrophilapolar flagella (flrAtoflrC). Mutations in theflaH,maf-1,fliM,flhA,fliA, andflrCgenes, as well as the double mutantflaA flaB, all caused loss of polar flagella and reduction in adherence and biofilm formation. A defined mutation in thepomBstator gene did not affect polar flagellum motility, in contrast to themotXmutant, which was unable to swim even though it expressed a polar flagellum. Mutations in all of these genes did not affect lateral flagellum synthesis or swarming motility, showing that bothA. hydrophilaflagellum systems are entirely distinct.

We have investigated the existence and genetic organization of a functional type III secretion system (TTSS) in a mesophilic Aeromonas strain by initially using the Aeromonas hydrophila strain AH-3. We report for the first time the complete TTSS DNA sequence of an Aeromonas strain that comprises 35 genes organized in a similar disposition as that in Pseudomonas aeruginosa . Using several gene probes, we also determined the presence of a TTSS in clinical or environmental strains of different Aeromonas species: A. hydrophila , A. veronii , and A. caviae . By using one of the TTSS genes ( ascV ), we were able to obtain a defined insertion mutant in strain AH-3 (AH-3AscV), which showed reduced toxicity and virulence in comparison with the wild-type strain. Complementation of the mutant strain with a plasmid vector carrying ascV was fully able to restore the wild-type toxicity and virulence.

MesophilicAeromonasstrains express a polar flagellum in all culture conditions, and certain strains produce lateral flagella on semisolid media or on surfaces. AlthoughAeromonaslateral 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 ofAeromonas hydrophilaAH-3 containing 38 genes, 9 of which (lafA-U) have been reported previously. Among theflgL_LandlafAstructural 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. AllAeromonaslateral flagellar genes were located in a unique chromosomal region, in contrast toVibrio parahaemolyticus, in which the analogous genes are distributed in two different chromosomal regions. InA. hydrophilamutations inflhA_L,lafK,fliJ_L,flgN_L,flgE_L, andmaf-5resulted 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 theA. hydrophilaAH-3 alternative sigma factor σ⁵⁴(rpoN); mutation of this factor suggested that it is involved in expression of both types of flagella.

Bacterial virulence can only be assessed by confronting bacteria with a host. Here, we present a new simple assay to evaluate Aeromonas virulence, making use of Dictyostelium amoebae as an alternative host model. This assay can be modulated to assess virulence of very different Aeromonas species.

The Aeromonas hydrophila type III secretion system (T3SS) has been shown to play a crucial role in this pathogen's interactions with its host. We previously described the genetic organization of the T3SS cluster and the existence of at least one effector, called AexT, in A. hydrophila strain AH-3. In this study, we analyzed the expression of the T3SS regulon by analyzing the activity of the aopN-aopD and aexT promoters (T3SS machinery components and effector, respectively) by means of two different techniques: promoterless gfp fusions and real-time PCR. The expression of the A. hydrophila AH-3 T3SS regulon was induced in response to several environmental factors, of which calcium depletion, a high magnesium concentration, and a high growth temperature were shown to be the major ones. Once the optimal conditions were established, we tested the expression of the T3SS regulon in the background of several virulence determinant knockouts of strain AH-3. The analysis of the data obtained from axsA and aopN mutants, both of which have been described to be T3SS regulators in other species, allowed us to corroborate their function as the major transcription regulator and valve of the T3SS, respectively, in Aeromonas hydrophila . We also demonstrated the existence of a complicated interconnection between the expression of the T3SS and several other different virulence factors, such as the lipopolysaccharide, the PhoPQ two-component system, the ahyIR quorum sensing system, and the enzymatic complex pyruvate deshydrogenase. To our knowledge, this is the first study of the A. hydrophila T3SS regulatory network.