Characterization of Important Carbohydrate Polymers of Myxococcus xanthus Involved In S-motility and Osmoprotection.

Abstract

Myxococcus xanthus, a soil bacterium, serves as a model for studying complex social behaviours in bacteria, such as swarming and S-motility. Research highlighted the crucial role of the cell's exopolysaccharide (EPS), known as "fibrils," in initiating type IV pili (T4P) retraction for S-motility and promoting cell-to-cell cohesion during swarming. Intriguingly, it has been reported that the artificial carbohydrate derivative methylcellulose and chitin are able to stimulate S-motility even in isolated single cells. Therefore, it was hypothesized that a modified version of cellulose may be the main component of EPS fibrils, supported by specific staining with Calcofluor White (CFW). To study the structure and chemistry of the EPS fibrils, a protocol was developed for isolating and purifying the fibrils using CFW-staining to monitor the purification process. The purified material resembled cotton fibres and underwent solid-state NMR analysis. The spectrum revealed peaks indicating cellulose and presence of series of unknown peaks. Cellulase treatment in M. Xanthus DK1622 cells disrupted alignment and swarming. Similarly, deleting the epsD gene, a putative cellulose synthase, disrupted cell alignment, abolished CFW-binding, S-motility, and fruiting body formation. In the same operon, epsC and epsB are annotated as serine acetyltransferase and cellulase, respectively. This operon involved in synthesis, modification, and degradation of identified EPS cellulose polymer. The deletion of epsC results in abolished of CFW-binding and S-motility. Interestingly, the deletion of epsB results in the entire colony being stained with CFW, whereas in the WT, only the edge of the colony is stained. Therefore, the proposed model suggests that the cooperation between EspD, the cellulose synthase, EpsC, modifier, and EspB, the cellulase, would have a vectoral effect on S-motility of the expanding swarm. Bioinformatics identified putative cellulose synthases, but disrupting these genes did not affect fibril production, underscoring EpsD's unique role. The epsD-A gene cluster within the eps locus appears crucial for modified cellulose production, initiating T4P retraction. The thesis's second part aimed to identify genes for a novel osmoregulated periplasmic glucan (OPG) polymer in M. xanthus. Mutants lacking known carbohydrate polymers (CPS, BPS, and EPS) were studied for OPG synthesis, but no involvement was found. Bioinformatics-guided disruption of carbohydrate-synthesizing enzymes, particularly periplasmic ones, yielded no candidates for OPG production.