The Rhizobium meliloti ExoK and ExsH glycanases specifically depolymerize nascent succinoglycan chains

Abstract

The Rhizobium meliloti ExoK and ExsH glycanases have been proposed to contribute to production of low molecular weight (LMW) succinoglycan by depolymerizing high molecular weight succinoglycan chains in R. meliloti cultures. We expressed and purified ExoK and ExsH and determined that neither enzyme can extensively cleave succinoglycan prepared from R. meliloti cultures, although neutral͞ heat treatment and acid͞heat treatment convert succinoglycan to forms that can be cleaved efficiently by both enzymes. These results were somewhat surprising, given that the exoK ؉ and exsH ؉ genes play a crucial role in production of LMW succinoglycan in R. meliloti cultures. We demonstrated by Western blot analyses that R. meliloti expresses ExoK and ExsH, that both proteins can be detected extracellularly, and that ExsH secretion depends on the prsD ؉ ͞prsE ؉ genes, consistent with previous predictions based on mutant analyses. Furthermore, we determined that the depolymerization activities associated with purified ExoK and ExsH are comparable with exoK ؉ and exsH ؉ -dependent depolymerization activities expressed in R. meliloti cultures. We resolved the apparent contradiction between the results of our previous genetic analyses and depolymerization assays by determining that ExoK and ExsH can cleave high molecular weight succinoglycan that is being produced actively by R. meliloti, but not succinoglycan that has accumulated in cultures, to yield LMW succinoglycan. We propose that ExoK and ExsH dynamically regulate the molecular weight distribution of succinoglycan by cleaving nascent succinoglycan only during a limited period after its synthesis, perhaps before it undergoes a timedependent change in its conformation or aggregation state.

Characterization of the Pseudomonas aeruginosa Glycoside Hydrolase PslG Reveals That Its Levels Are Critical for Psl Polysaccharide Biosynthesis and Biofilm Formation

“…This depressed activity may be a consequence of the incompatibility between the intrinsically compact structure of long polysaccharides and the requirement to bind in a thermodynamically disfavored extended conformation across an active site groove. The existence of polysaccharide aggregates has been previously suggested to result in low hydrolysis for the endo-acting GHs ExoK and ExsH from Rhizobium meliloti and BcsZ (16,66). In support of this hypothesis, BcsZ was more efficient at hydrolyzing in vitro synthesized cellulose in situ (67).…”

“…An obvious candidate for the production of cell-free low molecular weight Psl is the hydrolysis of the high molecular weight Psl by the action of PslG. Such a mechanism would be similar to the biosynthesis of the low molecular weight succinoglycan, which occurs via the hydrolysis of the high molecular weight polysaccharide by the non-essential extracellular endoglycases (GH16) ExoK and ExsH (66). This low molecular weight form is beneficial but not absolutely required for this rhizobial species to establish symbiosis with its host plants (75,76) and serves as a signaling molecule to facilitate root nodulation and bacterial update into these nodules (76).…”

The ExpR/Sin Quorum-Sensing System Controls Succinoglycan Production in Sinorhizobium meliloti

“…essary for the depolymerization of succinoglycan, as well as the endo-1,3-1,4-␤-glycanase, exoK (32,(74)(75)(76). Expression of exsH, the other endoglycanase responsible for LMW succinoglycan production, appears to be maximally expressed in an intact ExpR/Sin quorum-sensing system in mid-logarithmic and early stationary phase.…”

Extracellular Glycanases of Rhizobium leguminosarum Are Activated on the Cell Surface by an Exopolysaccharide-Related Component

“…Recent work with R. meliloti is pertinent to this point. The mature succinoglycan of R. meliloti is not cleaved by the secreted glycanases ExoK and ExsH (52). It was recently demonstrated that the levels of succinylation and acetylation strongly influence the susceptibility of nascent succinoglycan to glycanases (51).…”

“…A type I secretion system (encoded by the prsDE genes) is required for the secretion of the EPS-cleaving enzymes ExoK and ExsH (50), and PlyA and PlyB of R. leguminosarum are secreted similarly (14,15). The mature EPS of R. meliloti is not cleaved by the ExoK and ExsH glycanases; these enzymes cleave nascent EPS chains (52), and the absence of the succinyl group decreased the susceptibility of succinoglycan to cleavage (51). It is not known if modification of the R. leguminosarum EPS confers resistance to cleavage by PlyA and/or PlyB.…”