Chemotaxis and Wsp-like pathways affect biofilm formation in Halomonas titanicae KHS3

Abstract

Halomonas titanicae KHS3 is a moderately halophilic bacteria isolated from seawater of Mar del Plata harbour. During the analysis of its genomic sequence, two chemosensory clusters were identified. Cluster 1 includes genes and organization similar to those of the canonical Escherichia coli chemotaxis gene cluster and is involved in Halomonas chemotaxis. Cluster 2 encodes a Wsp-like pathway, whose genes and organization resemble those of the homonymous Pseudomonas aeruginosa cluster. In this pathway, a chemoreceptor-controlled histidine kinase activates a diguanylate cyclase (DGC) by phosphorylation, and the downstream response includes higher levels of biofilm. In this work, the participation of both chemosensory pathways in motility and biofilm formation was analyzed. Cluster 1 function was disrupted by a deletion in its histidine kinase gene, cheA1 (che1- mutant). The wsp-like pathway was targeted in two different ways. On one hand, Htc10 (cluster 2 chemoreceptor) was inactivated by a plasmid insertion (che2- mutant). On the other, the methylesterase gene cheB2 was deleted in order to assess the effect of an overmethylation (and presumably over-activation) of the pathway on the phenotype (che2++ mutant). Both che1- and che2++ mutants showed a significantly exacerbated biofilm formation when compared to wild-type strain when using the crystal violet assay. However, only the che2++ cells had a wrinkly aspect in agar medium, suggesting that the increased ability to form biofilm of the two strains was due to different mechanisms. Chemotaxis behavior, as assessed in soft agar plates, was severely affected in both hyperbiofilm mutants. However, when compared by video tracking analysis using SMT software, the motility of che1- mutant was indistinguishable from the wild-type strain, whereas che2++ showed a remarkable decrease in the number of motile cells. Substrate adherence after a short centrifugation was significantly increased in che2++ cells, and long-term biofilm assays also showed increased persistence of adhered cells in this mutant strain. Likewise, Congo Red staining of macrocolonies revealed an increased production of exopolysaccharides in this strain. All these features are consistent with a role of cluster 2 in biofilm formation as described for the Pseudomonas wsp pathway. Consistently, the che2-mutant showed a reduced ability to form biofilm under the same circumstances. The hyperbiofilm phenotype of the che1- mutant remains intriguing: complementation with very low levels of CheA1 restores the wild-type biofilm behavior even though chemotaxis is not fully restored. Up to now, we cannot find the mechanism underlying the increased biofilm in the absence of the chemotaxis kinase. Disruption of the cluster 2 chemoreceptor gene in the che1- mutant will help to elucidate whether or not the hyperbiofilm phenotype is dependent on the presence of cluster 2.