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Polar flagella from mesophilic Aeromonas strains have previously been shown to be modified with a range of glycans. Mass spectrometry studies of purified polar flagellins suggested the glycan typically includes a putative pseudaminic acid like derivative; while some strains are modified with this single monosaccharide, others modified with a heterologous glycan. In the current study, we demonstrate that genes involved in polar flagella glycosylation are clustered in highly polymorphic genomic islands flanked by pseudaminic acid biosynthetic genes (pse). Bioinformatic analysis of mesophilic Aeromonas genomes identified three types of polar flagella glycosylation islands (FGIs), denoted Group I, II and III. FGI Groups I and III are small genomic islands present in Aeromonas strains with flagellins modified with a single monosaccharide pseudaminic acid derivative. Group II were large genomic islands, present in strains found to modify polar flagellins with heterogeneous glycan moieties. Group II, in addition to pse genes, contained numerous glycosyltransferases and other biosynthetic enzymes. All Group II strains shared a common glycosyltransferase downstream of luxC that we named flagella glycosylation island 1, fgi-1 , in A. piscicola AH-3. We demonstrate that Fgi-1 transfers the first sugar of the heterogeneous glycan to the pseudaminic acid derivative linked to polar flagellins and could be used as marker for polysaccharidic glycosylation of Aeromonas polar flagella. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila sodium-driven polar flagellum has a complex stator-motor. Consist of two sets of redundant and non-exchangeable proteins (PomA/PomB and PomA2/PomB2), which are homologs to other sodium-conducting polar flagellum stator motors; and also two essential proteins (MotX and MotY), that they interact with one of those two redundant pairs of proteins and form the T-ring. In this work, we described an essential protein for polar flagellum stability and rotation which is orthologs to Vibrio spp. FlgT and it is encoded outside of the A. hydrophila polar flagellum regions. The flgT was present in all mesophilic Aeromonas strains tested and also in the non- motile Aeromonas salmonicida. The A. hydrophila 1flgT mutant is able to assemble the polar flagellum but is more unstable and released into the culture supernatant from the cell upon completion assembly. Presence of FlgT in purified polar hook-basal bodies (HBB) of wild-type strain was confirmed by Western blotting and electron microscopy observations showed an outer ring of the T-ring (H-ring) which is not present in the ΔflgT mutant. Anchoring and motility of proton-driven lateral flagella was not affected in the ΔflgT mutant and specific antibodies did not detect FlgT in purified lateral HBB of wild type strain. [ABSTRACT FROM AUTHOR]

Plesiomonas shigelloides is the unique member of the Enterobacteriaceae family able to produce polar flagella when grow in liquid medium and lateral flagella when grown in solid or semisolid media. In this study on P. shigelloides 302-73 strain, we found two different gene clusters, one exclusively for the lateral flagella biosynthesis and the other one containing the biosynthetic polar flagella genes with additional putative glycosylation genes. P. shigelloides is the first Enterobacteriaceae were a complete lateral flagella cluster leading to a lateral flagella production is described. We also show that both flagella in P. shigelloides 302-73 strain are glycosylated by a derivative of legionaminic acid (Leg), which explains the presence of Leg pathway genes between the two polar flagella regions in their biosynthetic gene cluster. It is the first bacterium reported with O-glycosylated Leg in both polar and lateral flagella. The flagella O-glycosylation is essential for bacterial flagella formation, either polar or lateral, because gene mutants on the biosynthesis of Leg are non-flagellated. Furthermore, the presence of the lateral flagella cluster and Leg O-flagella glycosylation genes are widely spread features among the P. shigelloides strains tested. [ABSTRACT FROM AUTHOR]

Protein glycosylation had been considered as an eccentricity of a few bacteria. However, through advances in analytical methods and genome sequencing, it is now established that bacteria possess both N-linked and O-linked glycosylation pathways. Both glycosylation pathways can modify multiple proteins, flagellins from Archaea and Eubacteria being one of these. Flagella O-glycosylation has been demonstrated in many polar flagellins from Gram-negative bacteria and in only the Gram-positive genera Clostridium and Listeria. Furthermore, O-glycosylation has also been demonstrated in a limited number of lateral flagellins. In this work, we revised the current advances in flagellar glycosylation from Gram-negative bacteria, focusing on the structural diversity of glycans, the O-linked pathway and the biological function of flagella glycosylation. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila is the most common Aeromonas species causing infections in human and other animals such as amphibians, reptiles, fish and crustaceans. Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Several mutant strains of A. hydrophila AH-3 were initially used to study their virulence in two animal species, Pacifastacus leniusculus (crayfish) and Tenebrio molitor larvae (mealworm). The AH-3 strains used in this study have mutations in genes involving the synthesis of flagella, LPS structures, secretion systems, and some other factors, which have been reported to be involved in A. hydrophila pathogenicity. Our study shows that the LPS (O-antigen and external core) is the most determinant A. hydrophila AH-3 virulence factor in both animals. Furthermore, we studied the immune responses of these hosts to infection of virulent or non-virulent strains of A. hydrophila AH-3. The AH-3 wild type (WT) containing the complete LPS core is highly virulent and this bacterium strongly stimulated the prophenoloxidase activating system resulting in melanization in both crayfish and mealworm. In contrast, the DwaaE mutant which has LPS without O-antigen and external core was non-virulent and lost ability to stimulate this system and melanization in these two animals. The high phenoloxidase activity found in WT infected crayfish appears to result from a low expression of pacifastin, a prophenoloxidase activating enzyme inhibitor, and this gene expression was not changed in the DwaaE mutant infected animal and consequently phenoloxidase activity was not altered as compared to non-infected animals. Therefore we show that the virulence factors of A. hydrophila are the same regardless whether an insect or a crustacean is infected and the Oantigen and external core is essential for activation of the proPO system and as virulence factors for this bacterium. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Abstract: Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Dsb (Disulfide bond) proteins play an important role in catalyzing disulfide bond formation in proteins within the periplasmic space. An A. hydrophila dsbA mutant with attenuated virulence using Dictyostelium amoebae as an alternative host model was identified. The attenuated virulence was tested in other animal models (by intraperitoneal injection in fish and mice) and was correlated with the presence of a defective type III secretion system for the first time in non enteric bacteria. The dsbA mutation was shown in several enteric bacteria to involve the outer membrane secretin. The defect in Aeromonas also seems to involve the outer membrane secretin homologue named AscC. However, unlike what happen in Escherichia coli, no changes in motility or flagella expression were observed for A. hydrophila dsbA mutants. The loss of E. coli motility caused by deletion of dsbA is likely due to defective disulfide bond formation in FlgI, a component of the flagella. No disulfide bond formation in FlgI homologues in Aeromonas flagella biogenesis, either polar or lateral, could be expected according to their amino acid residues sequences. [Copyright &y& Elsevier]

Aeromonas dhakensis possesses dual flagellar systems for motility under different environments. Flagella-mediated motility is necessary for biofilm formation through an initial attachment of bacteria to the surface, but this has not been elucidated in A. dhakensis. This study investigates the role of polar (flaH , maf1) and lateral (lafB , lafK and lafS) flagellar genes in the biofilm formation of a clinical A. dhakensis strain WT187 isolated from burn wound infection. Five deletion mutants and corresponding complemented strains were constructed using pDM4 and pBAD33 vectors, respectively, and analyzed for motility and biofilm formation using crystal violet staining and real-time impedance-based assays. All mutants were significantly reduced in swimming (p < 0.0001), swarming (p < 0.0001) and biofilm formation using crystal violet assay (p < 0.05). Real-time impedance-based analysis revealed WT187 biofilm was formed between 6 to 21 h, consisting of early (6–10 h), middle (11–18 h), and late (19–21 h) stages. The highest cell index of 0.0746 was recorded at 22–23 h and biofilms began to disperse starting from 24 h. Mutants Δ maf1 , Δ lafB , Δ lafK and Δ lafS exhibited reduced cell index values at 6–48 h when compared to WT187 which indicates less biofilm formation. Two complemented strains c maf1 and c lafB exhibited full restoration to wild-type level in swimming, swarming, and biofilm formation using crystal violet assay, hence suggesting that both maf1 and lafB genes are involved in biofilm formation through flagella-mediated motility and surface attachment. Our study shows the role of flagella in A. dhakensis biofilm formation warrants further investigations. • Flagella-mediated motility involved in Aeromonas dhakensis biofilm formation. • Polar Δ flaH and Δ maf1 were reduced 64–67% in swimming. • Lateral Δ lafB , Δ lafK and Δ lafS were abolished 85–86% in swarming. • Biofilm formation of all mutants was reduced 21–40% using crystal violet assay. • RTCA: maf1 and lafB genes involved in A. dhakensis biofilm formation. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila AH-3 lateral flagella are not assembled when bacteria grow in liquid media; however, lateral flagellar genes are transcribed. Our results indicate that A. hydrophila lateral flagellar genes are transcribed at three levels (class I to III genes) and share some similarities with, but have many important differences from, genes of Vibrio parahaemolyticus. A. hydrophila lateral flagellum class I gene transcription is σ70 dependent, which is consistent with the fact that lateral flagellum is constitutively transcribed, in contrast to the characteristics of V. parahaemolyticus. The fact that multiple genes are included in class I highlights that lateral flagellar genes are less hierarchically transcribed than polar flagellum genes. The A. hydrophila lafK-fliEJL gene cluster (where the subscript L distinguishes genes for lateral flagella from those for polar flagella) is exclusively from class I and is in V. parahaemolyticus class I and II. Furthermore, the A. hydrophila flgAMNL cluster is not transcribed from the σ54/LafK-dependent promoter and does not contain class II genes. Here, we propose a gene transcriptional hierarchy for the A. hydrophila lateral flagella. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila polar-flagellum class I gene transcription is σ70 dependent, which is consistent with the fact that the A. hydrophila polar flagellum is constitutively expressed. In contrast to other bacteria with dual flagellar systems such as Vibrio parahaemolyticus, the A. hydrophila LafK protein does not compensate for the lack of the polar-flagellum regulator FlrA (V. parahaemolyticus FlaK homologue). This is consistent with the fact that the A. hydrophila FlrA mutation abolishes polar-flagellum formation in liquid and on solid surfaces but does not affect inducible lateral-flagellum formation. The results highlight that the polar- and lateral-flagellum interconnections and control networks are specific and that there are differences between the dual flagellar systems in A. hydrophila and V. parahaemolyticus. Furthermore, our results indicate that the A. hydrophila polar-flagellum transcriptional hierarchy (also in class II, III, and IV genes) shares some similarities with but has many important differences from the transcriptional hierarchies of Vibrio cholerae and Pseudomonas aeruginosa. The A. hydrophila flhF and flhG genes are essential for the assembly of a functional polar flagellum because in-frame mutants fail to swim in liquid medium and lack the polar flagellum. In Vibrio and Pseudomonas flhG disruption increases the number of polar flagella per cell, and Pseudomonas flhF disruption gives an aberrant placement of flagellum. Here, we propose the gene transcriptional hierarchy for the A. hydrophila polar flagellum. [ABSTRACT FROM AUTHOR]

The article identifies several genes involved in glycosylation of polar and lateral flagella in mesophilic Aeromonas. The genes are not present in the complete sequenced genome of Vibrio parahaemolyticus, a bacterium also able to express two flagella systems, polar and lateral. The article reports on the isolation of an Aeromonas hydrophila AH-3 miniTn5 mutant unable to produce polar and lateral flagella.

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 σ54 (rpoN); mutation of this factor suggested that it is involved in expression of both types of flagella. [ABSTRACT FROM AUTHOR]

Mesophilic Aeromonas spp. 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 (flgN to flgL) that constitute region 1 of the Aeromonas hydrophila AH-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 (flaA to maf-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 (fliE to orf29), 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 the A. hydrophila polar flagella (flrA to flrC). Mutations in the flaH, maf-1, fliM, flhA, fliA, and flrC genes, as well as the double mutant flaA flaB, all caused loss of polar flagella and reduction in adherence and biofilm formation. A defined mutation in the pomB stator gene did not affect polar flagellum motility, in contrast to the motX mutant, 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 both A. hydrophila flagellum systems are entirely distinct. [ABSTRACT FROM AUTHOR]