A mutation in pssE affects exopolysaccharide synthesis by Rhizobium leguminosarum bv. trifolii, its surface properties, and symbiosis with clover

Abstract

Background and aimsA considerable majority of the proteins involved in exopolysaccharide synthesis in Rhizobium leguminosarum bv. trifolii are encoded by pss genes located in a large chromosomal region named Pss-I. The aim of this work was to characterize the phenotypic and symbiotic properties of strain Rt1933, which has a mutation in pssE. This gene encodes an enzyme responsible for the second step of EPS unit assembly.MethodsThe EPS-deficient Rt1933pssE strain was obtained via random mutagenesis using the mTn5SSgusA40 transposon. The mutation site in the Rt1933 genome was identified using hybridization, PCR amplification, and sequence analysis. Complementation of the pssE mutation was performed using biparental conjugation and a set of plasmids containing different fragments of the Pss-I region. The phenotypic properties of this mutant were established in growth kinetics experiments, as well as motility, sensitivity and hydrophobicity assays. The symbiotic proficiency of the Rt1933 strain in interaction with red clover (Trifolium pratense) was determined in plant tests, whereas occupation of root nodules by this mutant was investigated using light microscopy and bacteria harboring gusA for β-glucuronidase.ResultsAn exo99 mutation in Rt1933 was identified at 3′-end of the pssE gene located in region Pss-I, which resulted in a lack of 16 amino acids at the C-end of PssE. This mutation totally abolished EPS synthesis in R. leguminosarum bv. trifolii. Strain Rt1933 was characterized by considerably decreased growth kinetics and motility, and an increased sensitivity to some stress factors. Also, the hydrophobicity of the mutant cells differed significantly from that of the wild-type Rt24.2 and the complemented Rt1933 cells. Moreover, the pssE mutant showed strong disturbances in symbiosis with clover; it induced much fewer nodules on clover roots at a later time than normal, and the mass of the plants inoculated with the mutant was significantly lower than that of the plants inoculated with the wild-type strain.ConclusionsThe pssE gene plays a crucial role in EPS synthesis in R. leguminosarum bv. trifolii, and the presence of this polysaccharide affects the cell-surface properties of the bacterium and is required for both adaptation to stress conditions and the establishment of effective symbiosis with clover plants.