Characterization of the exopolysaccharide biosynthesis pathway inMyxococcus xanthus

Abstract

18Myxococcus xanthus arranges into two morphologically distinct biofilms depending on its 19 nutritional status, i.e. coordinately spreading colonies in the presence of nutrients and spore-20 filled fruiting bodies in the absence of nutrients. A secreted polysaccharide referred to as 21 exopolysaccharide (EPS) is a structural component of both biofilms and is also important for 22Here, we characterized the EPS biosynthetic machinery and show that it makes up a 45 Wzx/Wzy-dependent pathway for polysaccharide biosynthesis. Mutants lacking a component 46 of this pathway had reduced type IV pili-dependent motility and a conditional defect in 47 development. Also, these analysis suggest that EPS and/or the EPS biosynthetic machinery 48 is important for type IV pili formation. 49 50 51

Bacterial glycocalyx integrity drives multicellular swarm biofilm dynamism

“…Originally referred to as "slime fibrils/fibers", sinewy structures connecting the surfaces of clustered M. xanthus cells have been known for >40 years (88,89), with the only known requirement being the presence of the cell-surface EPS glycocalyx (55,63,74,75,78). Given the phenotypic, biochemical, and biophysical data presented herein, we propose that it is not use "EPS" to specifically denote the principal matrix polysaccharide assembled and secreted via the WzxX-WzyX-WzcX-WzeX-WzaX proteins (32,33,65). However, "EPS" has also been used to non-specifically refer to diverse secreted polysaccharides (i.e.…”

“…4) (49). These proteins are now known to be a part of the MASC and BPS assembly pathways, respectively (32,33,47). Wzb-deficient cells also exhibited faster auto-aggregation in cuvettes compared to WT cells, and were able to aggregate earlier in development resulting in faster fruiting body formation (49).…”

“…Each of EPS, BPS, and MASC is synthesized by a separate Wzx/Wzy-dependent pathway, with each respective component given the suffix X (exopolysaccharide), B (biosurfactant), or S (spore coat) (32)(33)(34)(35). Individual polysaccharide repeat units in such pathways are assembled on the lipid carrier undecaprenyl pyrophosphate (UndPP) at the cytoplasmic leaflet of the inner membrane (IM), followed by processing via a suite of integral IM proteins (36).…”

“…However, the developmental transition to fruiting bodies for BPS − swarms could take place at lower initial cell densities compared to WT swarms, suggesting that cells may aggregate more efficiently in BPS − swarms (32). To specifically probe aggregative differences among M. xanthus cell-surface polysaccharide mutants, the real-time ability of cells to auto-aggregate in liquid media was compared, a phenomenon known to be dependent on the presence of cell-surface EPS and an extendable T4P (22,26,30,33,55,56,(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68). While both EPS − and T4P − cells remained in suspension, BPS − cells rapidly auto-aggregated by the first post-mixing time point (10 min), resulting in faster initial unaided sedimentation relative to WT cells; after ~40 min, WT and BPS − cells in rich media continued to sediment, but at comparable rates, consistent with continued metabolism ( Fig.…”

“…Retention of Trypan Blue dye is used as a readout for M. xanthus cell-surface EPS levels (30,58,69). Intriguingly, despite compromised T4P-dependent swarm spreading (32,33), BPSpathway mutants ΔwzcB, ΔwzcBBYK, ΔwzaB, and ΔwzaB ΔwzcBin which periplasmic BPS polymerization should be permitted but secretion compromisedreproducibly bound more Trypan Blue than WT cells (combined interquartile range of 102-133% of WT) ( Fig. 2D) (32).…”