The genome of Azorhizobium caulinodans ORS571 encodes two chemotaxis response regulators: CheY1 and CheY2. cheY1 is located in a chemotaxis cluster (cheAWY1BR), while cheY2 is located 37 kb upstream of the cheAWY1BR cluster. To determine the contributions of CheY1 and CheY2, we compared the wild type (WT) and mutants in the free-living state and in symbiosis with the host Sesbania rostrata. Swim plate tests and capillary assays revealed that both CheY1 and CheY2 play roles in chemotaxis, with CheY2 having a more prominent role than CheY1. In an analysis of the swimming paths of free-swimming cells, the ΔcheY1 mutant exhibited decreased frequency of direction reversal, whereas the ΔcheY2 mutant appeared to change direction much more frequently than the WT. Exopolysaccharide (EPS) production in the ΔcheY1 and ΔcheY2 mutants was lower than that in the WT, but the ΔcheY2 mutant had more obvious EPS defects that were similar to those of the ΔcheY1 ΔcheY2 and Δeps1 mutants. During symbiosis, the levels of competitiveness for root colonization and nodule occupation of ΔcheY1 and ΔcheY2 mutants were impaired compared to those of the WT. Moreover, the competitive colonization ability of the ΔcheY2 mutant was severely impaired compared to that of the ΔcheY1 mutant. Taken together, the ΔcheY2 phenotypes are more severe than the ΔcheY1 phenotype in free-living and symbiotic states, and that of the double mutant resembles the ΔcheY2 single-mutant phenotype. These defects of ΔcheY1 and ΔcheY2 mutants were restored to the WT phenotype by complementation. These results suggest that there are different regulatory mechanisms of CheY1 and CheY2 and that CheY2 is a key chemotaxis regulator under free-living and symbiosis conditions. IMPORTANCE Azorhizobium caulinodans ORS571 is a motile soil bacterium that has the dual capacity to fix nitrogen both under free-living conditions and in symbiosis with Sesbania rostrata, forming nitrogen-fixing root and stem nodules. Bacterial chemotaxis to chemoattractants derived from host roots promotes infection and subsequent nodule formation by directing rhizobia to appropriate sites of infection. In this work, we identified and demonstrated that CheY2, a chemotactic response regulator encoded by a gene outside the chemotaxis cluster, is required for chemotaxis and multiple other cell phenotypes. CheY1, encoded by a gene in the chemotaxis cluster, also plays a role in chemotaxis. Two response regulators mediate bacterial chemotaxis and motility in different ways. This work extends the understanding of the role of multiple response regulators in Gram-negative bacteria.

Bioremediation is an attractive strategy of utilizing bacteria to remove crude oil contaminants. In this study, two salt-tolerant crude oil-degrading and biosurfactant-producing bacteria, Dietzia sp. CN-3 and Acinetobacter sp. HC8-3S, were functionally combined to construct a bacterial consortium. The consortium achieved 95.8% degradation efficiency of crude oil in 10 days and various n-alkanes, cycloalkanes, branched alkanes and aromatic hydrocarbons were all depleted more effectively than single strains. Functional optimization of the consortium degraded crude oil efficiently in a wide range of pH (4-10) and salinity (0-120 g L − 1). Furthermore, two alkane hydroxylase genes, alkB in CN-3 and alkM in HC8-3S, were cloned and their expression were examined by real-time quantitative polymerase chain reaction, indicating that alkB was more prominent in long-chain alkanes (C 20 , C 24 and C 26) utilization and alkM played crucial roles in medium-and long-chain alkanes (C 14 , C 16 , C 20 , C 24 and C 26) degradation. In soil microcosms artificially contaminated with crude oil and bioaugmented with the consortium, 58.3% of total petroleum hydrocarbons were depleted after 60 days and the degradation rate (485.8 mg kg − 1 d − 1) was higher than those reported in previous studies. Consequently, the consortium is a promising candidate in crude oil bioremediation.