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

Abstract

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.

FIG. 1

Western blots measuring the effect of the exoZ and exoH mutations on the extracellular accumulation of ExsH (by use of anti-ExsH polyclonal antibodies) (A) and ExoK (by use of anti-ExoK polyclonal antibodies) (B and C). The exoY, exoY exoZ, exoY exoH, and exoY exoH exoZ strains were cultivated in GMS medium (A, B) or in MGS medium (C) for a total of 5 days. Each lane contains a 5-μl aliquot of cell-free culture supernatant from day 1, day 3, or day 5 cultures. For blots A and C, the control lane corresponds to the negative control exoY exoK exsH strain. For blot B, the control lane corresponds to the exoY strain cultivated in MGS medium, which serves as a positive control for detection of extracellular ExoK. Each control sample corresponds to cell-free supernatants of a day 5 culture. Arrows indicate expected positions of ExsH and ExoK. Lines indicate positions of molecular weight markers (in kilodaltons).

FIG. 2

Graph of LMW carbohydrate (expressed as percentage of total extracellular carbohydrate in culture) generated by the addition of ExsH to cultures of glycanase-deficient strains versus the total amount of extracellular carbohydrate that had accumulated in cultures of these strains after 4 days of incubation. Note that approximately 97% of the total extracellular carbohydrate is succinoglycan. exoK exsH strain, open squares; exoZ exoK exsH strain, solid squares. ExsH was added to cultures gradually over the course of 4 days to the final, physiologically relevant concentration of 200 ng/ml.

FIG. 3

Degree of polymerization of HMW succinoglycan samples, expressed relative to degree of polymerization of wild-type HMW succinoglycan (data from Table 5), plotted versus the percentage of total extracellular carbohydrate in cultures that is in HMW forms (data from Table 2). Note that approximately 97% of total extracellular carbohydrate in cultures is succinoglycan.