Characterization of an rpoN mutant of Mesorhizobium ciceri

Abstract

Aims: To study the genetic basis of C4‐dicarboxylate transport (Dct) in relation to symbiotic nitrogen fixation in Mesorhizobium ciceri. Methods and Results: A Tn5‐induced mutant strain (TL16) of M. ciceri, unable to grow on C4‐dicarboxylates, was isolated from the wild‐type strain TAL 620. The mutant lacked activities of the enzymes, which use C4‐dicarboxylates as substrate. The sequencing of the 3·2kb EcoRI fragment, which was the site of Tn5 insertion, revealed three complete and two partial open reading frames. In the mutant, Tn5 interrupted the rpoN gene, of which only one copy was there. Complementation and biochemical studies suggest that the M. ciceri rpoN activity is required for C4‐Dct, maturation of bacteroids and symbiotic nitrogen fixation. The fine structure of the ineffective nodules produced by TL16 on Cicer arietinum L changed in comparison with those produced by the wild type. Conclusions: The mutant strain TL16 suffered a disruption in the rpoN gene. Only one copy of rpoN gene is present in M. ciceri. The mutation abolishes Dct activity. It additionally abolishes the symbiotic nitrogen fixation activity of the bacteroids in the nodules. Significance and Impact of the Study: This first document in M. ciceri shows that a functional rpoN gene is essential for the transport of dicarboxylic acids and symbiotic nitrogen fixation.

Genome-wide analysis of the RpoN regulon in Geobacter sulfurreducens

“…RpoN was first identified in Escherichia coli and was reported to regulate the transcription initiation of nitrogen assimilation genes [4]. Since then, RpoN homologs have been identified in bacteria from different phylogenetic origins and are involved in regulation of genes related to a diverse functional categories [5], including genes for pili and flagella biosynthesis and quorum sensing in Pseudomonas aeruginosa [6-8], C 4 -dicarboxylate transport in Mesorhizobium ciceri [9] and carbon metabolism in Gram positive bacteria, Bacillus subtilis and Listeria monocytogenes [10-13]. …”

Characterization of a (pckA) mutant of the non-nodulating bacterium Rhizobium pusense NRCPB10 induced by transposon Tn5 mutagenesis

“…R. pusense NRCPB100 grown in YM for 2 days at 28 °C was mutagenised by mating with the donor E. coli WA803 (Wood 1996) harboring the suicide plasmid pGS9:: Tn5 (Selvaraj and Iyer 1983) grown in Luria broth for 12 h at 37 °C as described (Gautam et al 2007). Transconjugants were selected by plating dilutions of the bacteria on RMM containing glucose plus succinate as the carbon sources and supplemented with 2,3,5-triphenyl tetrazolium chloride (30 μg/ml), rifampicin (50 μg/ml) and neomycin (100 μg/ml).…”