The succinyl and acetyl modifications of succinoglycan influence susceptibility of succinoglycan to cleavage by the Rhizobium meliloti glycanases ExoK and ExsH

In Rhizobium meliloti (Sinorhizobium meliloti) cultures, the endo-1,3-1,4-beta-glycanases ExoK and ExsH depolymerize nascent high-molecular-weight (HMW) succinoglycan to yield low-molecular-weight (LMW) succinoglycan. We report here that the succinyl and acetyl modifications of succinoglycan influence the susceptibility of succinoglycan to cleavage by these glycanases. It was previously shown that exoH mutants, which are blocked in the succinylation of succinoglycan, exhibit a defect in the production of LMW succinoglycan. We have determined that exoZ mutants, which are blocked in the acetylation of succinoglycan, exhibit an increase in production of LMW succinoglycan. For both wild-type and exoZ mutant strains, production of LMW succinoglycan is dependent on the exoK(+) and exsH(+) genes, implying that the ExoK and ExsH glycanases cleave HMW succinoglycan to yield LMW succinoglycan. By supplementing cultures of glycanase-deficient strains with exogenously added ExoK or ExsH, we have demonstrated directly that the absence of the acetyl group increases the susceptibility of succinoglycan to cleavage by ExoK and ExsH, that the absence of the succinyl group decreases the susceptibility of succinoglycan to cleavage, and that the succinyl effect outweighs the acetyl effect for succinoglycan lacking both modifications. Strikingly, nonsuccinylated succinoglycan actually can be cleaved by ExoK and ExsH to yield LMW succinoglycan, but only when the glycanases are added to cultures at greater than physiologically relevant concentrations. Thus, we conclude that the molecular weight distribution of succinoglycan in R. meliloti cultures is determined by both the levels of ExoK and ExsH glycanase expression and the susceptibility of succinoglycan to cleavage.