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An ahpC mutant derivative of Azospirillum brasilense Sp245 (strain SK586) that encodes an alkyl hydroperoxide reductase was found to be more sensitive to oxidative stress caused by organic hydroperoxides compared with the wild-type. In addition, the ahpC mutant strain had multiple defects in a large array of cellular functions that were consistent with alteration of cell-surface properties, such as cell morphology in stationary phase, Calcofluor White-, Congo Red- and lectin-binding abilities, as well as cell-to-cell aggregation and flocculation. All phenotypes of the ahpC mutant were complemented by in trans expression of AhpC, and overexpression of AhpC in the wild-type strain was found to affect the same set of phenotypes, suggesting that the pleiotropic effects were caused by the ahpC mutation. SK586 was also found to be fully motile, but it lost motility at a higher rate than the wild-type during growth, such that most SK586 cells were non-motile in stationary phase. Despite these defects, the mutant did not differ from the wild-type in short-term colonization of sterile wheat roots when inoculated alone, and in competition with the wild-type strain; this implied that AhpC activity may not endow the cells with a competitive advantage in colonization under these conditions. Although the exact function of AhpC in affecting these phenotypes remains to be determined, changes in cell morphology, surface properties, cell-to-cell aggregation and flocculation are common adaptive responses to various stresses in bacteria, and the data obtained here suggest that AhpC contributes to modulating such stress responses in A. brasilense.

A chemotaxis signal transduction pathway (hereafter called Che1) has been previously identified in the alphaproteobacterium Azospirillum brasilense. Previous experiments have demonstrated that although mutants lacking CheB and/or CheR homologs from this pathway are defective in chemotaxis, a mutant in which the entire chemotaxis pathway has been mutated displayed a chemotaxis phenotype mostly similar to that of the parent strain, suggesting that the primary function of this Che1 pathway is not the control of motility behavior. Here, we report that mutants carrying defined mutations in the cheA1 (strain AB101) and the cheY1 (strain AB102) genes and a newly constructed mutant lacking the entire operon [[DELTA](cheA1-cheR1)::Cm] (strain AB103) were defective, but not null, for chemotaxis and aerotaxis and had a minor defect in swimming pattern. We found that mutations in genes of the Che1 pathway affected the cell length of actively growing cells but not their growth rate. Cells of a mutant lacking functional cheB1 and cheR1 genes (strain BS104) were significantly longer than wild-type cells, whereas cells of mutants impaired in the cheA1 or cheY1 genes, as well as a mutant lacking a functional Che1 pathway, were significantly shorter than wild-type cells. Both the modest chemotaxis defects and the observed differences in cell length could be complemented by expressing the wild-type genes from a plasmid. In addition, under conditions of high aeration, cells of mutants lacking functional cheA1 or cheY1 genes or the Che1 operon formed clumps due to cell-to-cell aggregation, whereas the mutant lacking functional CheB1 and CheR1 (BS104) clumped poorly, if at all. Further analysis suggested that the nature of the exopolysaccharide produced, rather than the amount, may be involved in this behavior. Interestingly, mutants that displayed clumping behavior (lacking cheA1 or cheY1 genes or the Che1 operon) also flocculated earlier and quantitatively more than the wild-type cells, whereas the mutant lacking both CheB1 and CheR1 was delayed in flocculation. We propose that the Che1 chemotaxis-like pathway modulates the cell length as well as clumping behavior, suggesting a link between these two processes. Our data are consistent with a model in which the function of the Che1 pathway in regulating these cellular functions directly affects flocculation, a cellular differentiation process initiated under conditions of nutritional imbalance.

To compete in complex microbial communities, bacteria must sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the regulation of multiple behaviors in response to changes in the environment, including motility patterns, exopolysaccharide production, and cell-to-cell interactions. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting flocculation. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Whereas the wild-type strain produces a smooth mucosal extracellular matrix after 24 h, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition, lectin-binding assays, and comparison of lipopolysaccharides profiles suggest that the extracellular matrix differs between the cheA1 and the cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition. [ABSTRACT FROM AUTHOR]