EXOPOLYSACCHARIDE (EPS) SYNTHESIS IN BRADYRHIZOBIUM-JAPONICUM - SEQUENCE, OPERON STRUCTURE AND MUTATIONAL ANALYSIS OF AN EXO GENE-CLUSTER

The nucleotide sequence of a 8330-bp DNA fragment from Bradyrhizobium japonicum 110spc4 was determined. Sequence analysis revealed that six ORFs were present and the deduced amino acid sequences were homologous to enzymes involved in exopolysaccharide (EPS) biosynthesis. The gene s appear to be organized into at least four different operons. One gen e was found to be homologous to exoB, which encodes a UDP-galactose 4' -epimerase. Other ORFs were homologous to UDP-hexose transferases and one ORF showed similarity to Sinorhizobium (Rhizobium) meliloti ExoP, which has been suggested to be involved in EPS chain-length determinat ion. A set of deletion and insertion mutants was constructed and the r esulting B. japonicum strains were tested for their symbiotic traits. Deletion mutant Delta P22, which lacks the C-terminal part of ExoP, th e UDP-hexose transferase ExoT and the N-terminal part of ExoB, shows a delayed nodulation phenotype and induces symptoms of plant defense re actions; its EPS does not contain galactose and no high molecular weig ht fraction is synthesized. In contrast, insertion mutant EH3, which e xpresses an exoP gene product that is truncated in its putative peripl asmic domain, produced an EPS containing both HMW and LMW fractions. H owever, the interaction of EH3 with soybeans was severely perturbed. A s a rule, only the initial steps of nodule formation were observed.