Genetic characterization of the Pss region and the role of PssS in exopolysaccharide production and symbiosis of Rhizobium leguminosarum bv. trifolii with clover

BACKGROUND AND AIMS: In the symbiotic bacterium Rhizobium leguminosarum bv. trifolii, a majority of proteins involved in exopolysaccharide (EPS) synthesis are encoded by genes located in a large polysaccharide synthesis cluster (Pss). The aim of this study was genetic characterization of the Pss region in the Rt24.2 strain in the context of EPS production and symbiosis with red clover (Trifolium pratense). METHODS: The expression of genes located in the Pss cluster was determined using constructed pss-lacZ transcriptional fusions. The role of transcriptional regulator RosR in pss transcription was confirmed using a rosR mutant and the Rt24.2(pBR1) strain carrying multiple rosR copies. An EPS-deficient mutant, Rt770 was obtained using a random mutagenesis and mTn5SSgusA40 transposon. Symbiotic properties of the Rt770 strain in interaction with clover were characterized in inoculation experiments. Infection of host roots and nodule occupancy by this mutant were investigated using both light and electron microscopy. RESULTS: Transcriptional levels of particular pss genes differed significantly; the genes encoding glycosyltransferases and enzymes modifying EPS have promoters of weak activities, whereas those encoding proteins involved in EPS polymerization and export possess stronger promoters. Furthermore, RosR affected expression of some pss genes. A mutation in Rt24.2 pssS encoding glucosyltransferase totally abolished EPS synthesis, decreased motility, and increased sensitivity to some stressors. The pssS mutant Rt770 induced formation of nodules on clover roots, which were ineffective in nitrogen fixation. CONCLUSION: EPS secreted by Rt24.2 is required for both adaptation to soil conditions and the establishment of effective symbiosis with clover plants.