Abstract
In the environment, bacteria mainly exist in multicellular, surface-adhered communities called biofilms. One key feature of the biofilms is a self-produced extracellular matrix that embeds cells within a structured community and contributes to their adhesion, organization, and retention. The matrix of most characterized biofilms contains three primary components: polysaccharides, protein, and extracellular DNA (eDNA). Upon biofilm induction,
Bacillus subtilis
produces and secretes a polysaccharide known as exopolysaccharide (EPS). The 15 genes of the
epsA-O
operon encode proteins that coordinate the assembly, export, and polymerization of EPS oligosaccharide (glycan) subunits. While much progress has been made to elucidate the chemical structure of EPS, further work is needed to determine the contributions of some eps genes to EPS biosynthesis experimentally. Here, we generated a collection of marker-less, in-frame deletions targeting each eps gene in the
epsA-O
operon in
B. subtilis
strain NCIB 3610 using the Cre system. Biofilm phenotypes of resulting mutants were used to assess each gene's importance to biofilm development.
Impact
The
epsA-O
single-gene deletion collection was designed to enable systematic assessment of the contribution of each eps gene to EPS production and biofilm-associated phenotypes. When combined with current knowledge of EPS structure, this collection will help clarify the roles of individual genes in EPS biosynthesis. This approach can also be easily adapted for the systematic analyses of other large, complex operons in diverse bacterial species.