The glycosyltransferase encoded by the lpcC gene affects biofilm formation and nodulation performance in Bradyrhizobium japonicum

FERRARI, W.; ROSSI, F.; MEDEOT, D.B.; FISCHER, S.E.; LAGARES, A.; JOFRE, E.

Workshop; II Workshop Latinoamericano sobre Rizobacterias Promotoras del Desarrollo Vegetal; 2014

The soil bacterium Bradyrhizobium japonicum can establish a nitrogen-fixing symbiosis with the leguminous plant soybean, resulting in the formation of nodules where atmospheric nitrogen is fixed. Bacterial cell surface components play an important role during the establishment of symbiosis. Among these surface components the lipopolysaccharide (LPS) composed by a lipid A, a core oligosaccharide, and a polysaccharide chain known as O-antigen is required for a functional symbiosis by certain rhizobial species. The lpcC gene encodes a glycosyltransferase involved in the LPS biosynthesis. Previous reports demonstrated that lpcC mutants affected in the LPS-inner core from Rhizobium leguminosarum, are unable to form functional nodules on Pisumsativum. Several studies reported that B. japonicum with an altered LPS were unable to establish an effective symbiosis with Glycine max. Here, we show that the lack of lpcC produced a truncated LPS, altered the surface hydrophobicity, and affected the symbiosis with G. max. Inactivation of lpcC gene was performed by site-directed mutagenesis. For heterologous complementation, the orthologous genes were cloned into pFAJ1708 plasmid. LPS profiles were analyzed by DOC-PAGE. Motility assays were determined on PSY medium supplemented with 0.3 % agar. The relative surface hydrophobicity was measured by the xylene method according to Rosenberg et al. (1980). The biofilm-forming abilities of the B. japonicum strains were quantitatively determined by measuring the amount of cells attached to polystyrene micro wells using the crystal violet staining method. To investigate the effect of lpcC mutation on the ability to establish a functional symbiosis with Glycine max, nodulation assays were performed and nodule numbers were counted after 35 days post inoculation. Nodules were excised from the plants, transversally cut and then analyzed by optic and electronic microscopy. DOC-PAGE analysis showed that the LPS from the B. japonicum lpcC mutant strain lacks the multiple bands of lower electrophoretic mobility that are present in the wild-type LPS. This result suggests that the B. japonicum lpcC mutant strain produces an abnormal LPS lacking the outer core and the O-antigen. Motility was also affected in the mutant strain when compared with the wild type strain. As a results of the alterations in the LPS an enhancement of the surface hydrophobicity and biofilm formation ability were observed. The lpcC mutant strain was unable to induce the formation of functional nodules; only a few pseudonodules were observed. When analyzed by electronic microscopy, no bacteroids were observed inside of the pseudonodules. Lack of the O-polysaccharide of the LPS increased the biofilm-forming ability, presumably through a significant increment of the cell surface hydrophobicity. Decrease in motility may also be the consequence of the increased hydrophobicity observed in the lpcC mutant strain. Our findings support previous studies emphasizing the role of the LPS during the nodulation of G. max roots and demonstrate the requirement of the O-antigen of this polysaccharide to establish a functional symbiosis.