Identification of the Wzx flippase, Wzy polymerase and sugar‐modifying enzymes for spore coat polysaccharide biosynthesis in Myxococcus xanthus

Abstract

The rod‐shaped cells of Myxococcus xanthus, a Gram‐negative deltaproteobacterium, differentiate to environmentally resistant spores upon starvation or chemical stress. The environmental resistance depends on a spore coat polysaccharide that is synthesised by the ExoA‐I proteins, some of which are part of a Wzx/Wzy‐dependent pathway for polysaccharide synthesis and export; however, key components of this pathway have remained unidentified. Here, we identify and characterise two additional loci encoding proteins with homology to enzymes involved in polysaccharide synthesis and export, as well as sugar modification and show that six of the proteins encoded by these loci are essential for the formation of environmentally resistant spores. Our data support that MXAN_3260, renamed ExoM and MXAN_3026, renamed ExoJ, are the Wzx flippase and Wzy polymerase, respectively, responsible for translocation and polymerisation of the repeat unit of the spore coat polysaccharide. Moreover, we provide evidence that three glycosyltransferases (MXAN_3027/ExoK, MXAN_3262/ExoO and MXAN_3263/ExoP) and a polysaccharide deacetylase (MXAN_3259/ExoL) are important for formation of the intact spore coat, while ExoE is the polyisoprenyl‐phosphate hexose‐1‐phosphate transferase responsible for initiating repeat unit synthesis, likely by transferring N‐acetylgalactosamine‐1‐P to undecaprenyl‐phosphate. Together, our data generate a more complete model of the Exo pathway for spore coat polysaccharide biosynthesis and export.

Characterization of the exopolysaccharide biosynthesis pathway inMyxococcus xanthus

“…EpsZ is the predicted PHPT of the EPS biosynthesis pathway. Similar to WcaJ Ec from E. coli and WbaP Se from Salmonella enterica (41, 59, 60), we identified a PF13727 (CoA_binding _3) domain, a C-terminal PF02397 (Bac_transf) domain and five transmembrane regions in EpsZ (Fig. 5A), all features of PHPT proteins.…”

“…Third, blocking translocation of a specific sugar unit across the IM can cause sequestration of Und-P and, thereby, result in pleiotropic effects on the synthesis of other polysaccharides (47-51). Consistently, a Δ MXAN_1035 mutation was previously shown to cause a reduction in glycerol-induced sporulation (see below) likely by interfering with spore coat polysaccharide biosynthesis (39); however, MXAN_3260 (ExoM) was recently shown to be the flippase involved in spore coat polysaccharide synthesis (41). Together, these considerations support that it is unlikely that MXAN_1035 is part of the EPS biosynthesis machinery.…”

“…1B). Several of these homologs were previously shown to be important for LPS synthesis or spore coat polysaccharide biosynthesis (37-41) (Fig. 1B).…”

“…In a bioinformatics approach searching for orthologs of the proteins encoded by the entire eps locus in all fully sequenced Myxococcales genomes using a reciprocal best BlastP hit method as in (41), we found that the two gene clusters encoding proteins for polysaccharide synthesis are largely conserved in closely related Cystobacterineae (Fig. 2B).…”

“…4A). Cells of the Δ exoE mutant, which lacks the PHPT for initiating spore coat polysaccharide biosynthesis and were used as a negative control (39, 41), remained rod-shaped and did not form phase-bright spores. Interestingly, the sporulation efficiency of all five Δeps mutants was increased compared to WT (Fig.…”