Abstract
Bacillus subtilis is intensively studied as a model organism for the development of bacterial biofilms or pellicles. A key component is currently undefined exopolysaccharides produced from proteins encoded by genes within the eps locus. Within this locus are four genes, epsHIJK, known to be essential for pellicle formation. We show they encode proteins synthesizing the broadly expressed microbial carbohydrate poly-N-acetylglucosamine (PNAG). PNAG was present in both pellicle and planktonic wild-type B. subtilis cells and in strains with deletions in the epsA-G and -L-O genes but not in strains deleted for epsH-K. Cloning of the B. subtilis epsH-K genes into Escherichia coli with in-frame deletions in the PNAG biosynthetic genes pgaA-D, respectively, restored PNAG production in E. coli. Cloning the entire B. subtilis epsHIJK locus into pga-deleted E. coli, Klebsiella pneumoniae, or alginate-negative Pseudomonas aeruginosa restored or conferred PNAG production. Bioinformatic and structural predictions of the EpsHIJK proteins suggest EpsH and EpsJ are glycosyltransferases (GT) with a GT-A fold; EpsI is a GT with a GT-B fold, and EpsK is an α-helical membrane transporter. B. subtilis, E. coli, and pga-deleted E. coli carrying the epsHIJK genes on a plasmid were all susceptible to opsonic killing by antibodies to PNAG. The immunochemical and genetic data identify the genes and proteins used by B. subtilis to produce PNAG as a significant carbohydrate factor essential for pellicle formation.
Highlights
Bacillus subtilis is a model organism for analyzing bacterial biofilms, but the carbohydrate components are undescribed
We found that the predicted multiple antimicrobial extrusion protein (MATE) function of the B. subtilis EpsK protein had similarity to PelG and PslK in P. aeruginosa used for the synthesis of the PEL and PSL polysaccharides, respectively
P. aeruginosa, unlike E. coli and K. pneumoniae, does not normally synthesize PNAG, so we introduced the pUCP18Tc-epsHIJK plasmid into WT and algF-interrupted (Tn::algF) P. aeruginosa strain FRD1 to ascertain whether PNAG could be synthesized
Summary
Bacillus subtilis is a model organism for analyzing bacterial biofilms, but the carbohydrate components are undescribed. Full biofilm formation by some strains of B. subtilis such as NCIB 3610 (3610) is dependent on 15 proteins encoded by genes in the eps locus (epsA– epsO) that are associated with the carbohydrate content and complexity of a surface pellicle [16] involved in the overall biofilm structure. Given the ubiquity of PNAG synthesis among a range of microbial organisms, we evaluated B. subtilis biofilms for PNAG production and further examined the B. subtilis eps locus for genes potentially encoding PNAG biosynthetic proteins Both biofilm and planktonic cells produced PNAG, and within the available annotated genome, we identified four genes, epsH–K, as potentially being responsible for PNAG synthesis. B. subtilis lacking epsH–K genes are unable to form biofilms, indicating PNAG is essential for biofilm/pellicle formation by this organism
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