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Cloning of an Azorhizobium caulinodans endoglucanase gene and analysis of its role in symbiosis.

ABSTRACT: Azorhizobium caulinodans ORS571, a symbiont of the tropical leguminous plant Sesbania rostrata, showed low, constitutive levels of endoglucanase (Egl) activity. A clone carrying the gene responsible for this phenotype was isolated via introduction of a genomic library into the wild-type strain and screening for transconjugants with enhanced Egl activity. By subcloning and expression in Escherichia coli, the Egl phenotype was allocated to a 3-kb EcoRI-BamHI fragment. However, sequence analysis showed the egl gene to be much larger, consisting of an open reading frame of 1,836 amino acids. Within the deduced polypeptide, three kinds of putative domains were identified: a catalytic domain, two cellulose-binding domains, and an eightfold reiterated motif. The catalytic domain belongs to the family A of cellulases. A C-terminal stretch of 100 amino acids was similar to family II cellulose-binding domains. A second copy of this domain occurred near the middle of the polypeptide, flanked by reiterated motifs. ORS571 mutants carrying a Tn5 insertion in the egl gene had lost the Egl activity. These mutants as well as Egl-overproducing strains showed a normal nodulation behavior, indistinguishable from wild-type nodulation on Sesbania rostrata under laboratory conditions.

SUBMITTER: Geelen D

PROVIDER: S-EPMC167611 | biostudies-other | 1995 Sep

REPOSITORIES: biostudies-other

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Cloning of an Azorhizobium caulinodans endoglucanase gene and analysis of its role in symbiosis.

Geelen D D van Montagu M M Holsters M M

Applied and environmental microbiology 19950901 9

Azorhizobium caulinodans ORS571, a symbiont of the tropical leguminous plant Sesbania rostrata, showed low, constitutive levels of endoglucanase (Egl) activity. A clone carrying the gene responsible for this phenotype was isolated via introduction of a genomic library into the wild-type strain and screening for transconjugants with enhanced Egl activity. By subcloning and expression in Escherichia coli, the Egl phenotype was allocated to a 3-kb EcoRI-BamHI fragment. However, sequence analysis sh ...[more]

PMID: 7574641

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Project description:UnlabelledAzorhizobium caulinodans ORS571 is a free-living nitrogen-fixing bacterium which can induce nitrogen-fixing nodules both on the root and the stem of its legume host Sesbania rostrata This bacterium, which is an obligate aerobe that moves by means of a polar flagellum, possesses a single chemotaxis signal transduction pathway. The objective of this work was to examine the role that chemotaxis and aerotaxis play in the lifestyle of the bacterium in free-living and symbiotic conditions. In bacterial chemotaxis, chemoreceptors sense environmental changes and transmit this information to the chemotactic machinery to guide motile bacteria to preferred niches. Here, we characterized a chemoreceptor of A. caulinodans containing an N-terminal PAS domain, named IcpB. IcpB is a soluble heme-binding protein that localized at the cell poles. An icpB mutant strain was impaired in sensing oxygen gradients and in chemotaxis response to organic acids. Compared to the wild-type strain, the icpB mutant strain was also affected in the production of extracellular polysaccharides and impaired in flocculation. When inoculated alone, the icpB mutant induced nodules on S. rostrata, but the nodules formed were smaller and had reduced N2-fixing activity. The icpB mutant failed to nodulate its host when inoculated competitively with the wild-type strain. Together, the results identify chemotaxis and sensing of oxygen by IcpB as key regulators of the A. caulinodans-S. rostrata symbiosis.ImportanceBacterial chemotaxis has been implicated in the establishment of various plant-microbe associations, including that of rhizobial symbionts with their legume host. The exact signal(s) detected by the motile bacteria that guide them to their plant hosts remain poorly characterized. Azorhizobium caulinodans ORS571 is a diazotroph that is a motile and chemotactic rhizobial symbiont of Sesbania rostrata, where it forms nitrogen-fixing nodules on both the roots and the stems of the legume host. We identify here a chemotaxis receptor sensing oxygen in A. caulinodans that is critical for nodulation and nitrogen fixation on the stems and roots of S. rostrata These results identify oxygen sensing and chemotaxis as key regulators of the A. caulinodans-S. rostrata symbiosis.

Project description:BackgroundA wide variety of bacterial adaptative responses to environmental conditions are mediated by signal transduction pathways. Two-component signal transduction systems are one of the predominant means used by bacteria to sense the signals of the host plant and adjust their interaction behaviour. A total of seven open reading frames have been identified as putative two-component response regulators in the gram-negative nitrogen-fixing bacteria Azorhizobium caulinodans ORS571. However, the biological functions of these response regulators in the symbiotic interactions between A. caulinodans ORS571 and the host plant Sesbania rostrata have not been elucidated to date.ResultsIn this study, we identified and investigated a two-component response regulator, AcfR, with a phosphorylatable N-terminal REC (receiver) domain and a C-terminal HTH (helix-turn-helix) LuxR DNA-binding domain in A. caulinodans ORS571. Phylogenetic analysis showed that AcfR possessed close evolutionary relationships with NarL/FixJ family regulators. In addition, six histidine kinases containing HATPase_c and HisKA domains were predicted to interact with AcfR. Furthermore, the biological function of AcfR in free-living and symbiotic conditions was elucidated by comparing the wild-type strain and the ΔacfR mutant strain. In the free-living state, the cell motility behaviour and exopolysaccharide production of the ΔacfR mutant were significantly reduced compared to those of the wild-type strain. In the symbiotic state, the ΔacfR mutant showed a competitive nodule defect on the stems and roots of the host plant, suggesting that AcfR can provide A. caulinodans with an effective competitive ability for symbiotic nodulation.ConclusionsOur results showed that AcfR, as a response regulator, regulates numerous phenotypes of A. caulinodans under the free-living conditions and in symbiosis with the host plant. The results of this study help to elucidate the involvement of a REC + HTH_LuxR two-component response regulator in the Rhizobium-host plant interaction.

Dataset Information

Cloning of an Azorhizobium caulinodans endoglucanase gene and analysis of its role in symbiosis.

Publications

Cloning of an Azorhizobium caulinodans endoglucanase gene and analysis of its role in symbiosis.

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