Characterisation of genes involved in exopolysaccharide biosynthesis and transport in the Mesorhizobium loti strain R7A

Abstract

Rhizobia are bacteria that are important in fixing gaseous nitrogen into a plant-available form via a symbiotic interaction with leguminous plants. In order for this to occur, the legume and the rhizobia must communicate with each other using molecular signals. One molecule that rhizobia produce that has been implicated as a signal is exopolysaccharide (EPS). Mesorhizobium loti strain R7A EPS consists of an octasaccharide repeating subunit, comprising a backbone that contains one galactose and three glucose (Glc) residues and a side-branch containing two Glc, one glucuronic acid and a terminal riburonic acid residue. The biosynthesis of EPS is carried out by proteins encoded by genes termed exo genes that are arranged in a cluster. The EPS is believed to play a role in promoting infection thread formation by signalling a suppression of the host immune defences. The purified low molecular weight (LMW) form of EPS comprising a single subunit has been shown to bind strongly to the Lotus japonicus receptor EPR3. Mutants of M. loti R7A affected in the synthesis of the EPS backbone, e.g. exoYF and exoB mutants, induce effective nodules, albeit with reduced efficiency, while most mutants in genes affecting synthesis of the branch portion of the EPS, e.g. exoU mutants, induce uninfected nodules. It has been hypothesised that the latter mutants secrete a truncated form of EPS that is recognised by EPR3 to induce a defence response. The aims of this study were to test these hypotheses by determining whether EPS transport genes were required for the negative phenotype exoU mutants and by determining the phenotype of exoK mutants. The exoK gene encodes a glycanase thought to be necessary for the production of the EPS subunit monomers. In past experiments, obtaining an in-frame markerless deletion exoK mutant in R7A has proven difficult, although polar mutants containing mutations with potential negative effects on downstream genes were acquired using insertion duplication mutagenesis (IDM). In this study, further attempts were made to generate R7A mutants deficient in the production of ExoK as well as attempts to generate a ΔexoK mutant in an EPS-deficient background in order to investigate the possibility of lethality caused by the deletion of exoK. The results demonstrated that deletion of exoK had a lethal effect on R7A when the strain was expressing exoYF and growth inhibition was relieved on deletion of the exoYF genes. A polar exoK mutant obtained by Kelly (PhD thesis) was partially complemented with the exoK gene, confirming that exoK was deleted and expression of downstream genes was affected in the mutant.