Function of a chemotaxis-like signal transduction pathway in modulating motility, cell clumping, and cell length in the alphaproteobacterium Azospirillum 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.

Role of CheB and CheR in the complex chemotactic and aerotactic pathway of Azospirillum brasilense.

It has previously been reported that the alpha-proteobacterium Azospirillum brasilense undergoes methylation-independent chemotaxis; however, a recent study revealed cheB and cheR genes in this organism. We have constructed cheB, cheR, and cheBR mutants of A. brasilense and determined that the CheB and CheR proteins under study significantly influence chemotaxis and aerotaxis but are not essential for these behaviors to occur. First, we found that although cells lacking CheB, CheR, or both were no longer capable of responding to the addition of most chemoattractants in a temporal gradient assay, they did show a chemotactic response (albeit reduced) in a spatial gradient assay. Second, in comparison to the wild type, cheB and cheR mutants under steady-state conditions exhibited an altered swimming bias, whereas the cheBR mutant and the che operon mutant did not. Third, cheB and cheR mutants were null for aerotaxis, whereas the cheBR mutant showed reduced aerotaxis. In contrast to the swimming bias for the model organism Escherichia coli, the swimming bias in A. brasilense cells was dependent on the carbon source present and cells released methanol upon addition of some attractants and upon removal of other attractants. In comparison to the wild type, the cheB, cheR, and cheBR mutants showed various altered patterns of methanol release upon exposure to attractants. This study reveals a significant difference between the chemotaxis adaptation system of A. brasilense and that of the model organism E. coli and suggests that multiple chemotaxis systems are present and contribute to chemotaxis and aerotaxis in A. brasilense.

Lon protease of the alpha-proteobacterium Agrobacterium tumefaciens is required for normal growth, cellular morphology and full virulence.

The ATP-dependent Lon (La) protease is ubiquitous in nature and regulates a diverse set of physiological responses in bacteria. In this paper a lon mutant of the alpha-proteobacterium Agrobacterium tumefaciens C58 has been characterized. Unlike lon mutants of Escherichia coli, the lon mutant of A. tumefaciens grows very slowly, is not filamentous and exhibits normal resistance to UV irradiation. The mutant retains motility and chemotaxis, produces apparently normal amounts of exopolysacchride, but displays severe defects in cell morphology, with 80 % of the mutant cells appearing Y-shaped. Lon protease of A. tumefaciens shares high homology with its counterparts in E. coli and in Sinorhizobium meliloti, and functionally complements an E. coli lon mutant for defects in morphology and RcsA-mediated regulation of capsular polysaccharide production. Mutations at sites of Lon(At) corresponding to the ATP-binding site and the active site serine of the E. coli Lon protease abolish complementation of phenotypes of the A. tumefaciens and E. coli lon mutants. The nucleotide sequence upstream of A. tumefaciens lon contains an element similar to the consensus sigma(32) heat-shock promoter of E. coli. Northern and Western blot analyses indicated that expression of lon is induced by elevated temperature, albeit to a much lower level than that of groEL. The lon mutant is highly attenuated for virulence, suggesting that Lon may be required for the proper expression, assembly or function of the VirB/D4-mediated T-DNA transfer system.

An energy taxis transducer promotes root colonization by Azospirillum brasilense.

Motility responses triggered by changes in the electron transport system are collectively known as energy taxis. In Azospirillum brasilense, energy taxis was shown to be the principal form of locomotor control. In the present study, we have identified a novel chemoreceptor-like protein, named Tlp1, which serves as an energy taxis transducer. The Tlp1 protein is predicted to have an N-terminal periplasmic region and a cytoplasmic C-terminal signaling module homologous to those of other chemoreceptors. The predicted periplasmic region of Tlp1 comprises a conserved domain that is found in two types of microbial sensory receptors: chemotaxis transducers and histidine kinases. However, the function of this domain is currently unknown. We characterized the behavior of a tlp1 mutant by a series of spatial and temporal gradient assays. The tlp1 mutant is deficient in (i) chemotaxis to several rapidly oxidizable substrates, (ii) taxis to terminal electron acceptors (oxygen and nitrate), and (iii) redox taxis. Taken together, the data strongly suggest that Tlp1 mediates energy taxis in A. brasilense. Using qualitative and quantitative assays, we have also demonstrated that the tlp1 mutant is impaired in colonization of plant roots. This finding supports the hypothesis that energy taxis and therefore bacterial metabolism might be key factors in determining host specificity in Azospirillum-grass associations.