Biofilm Building:A Simple Board Game to Reinforce Knowledge of Biofilm Formation

The increasing risk of antibiotic resistance and correlating decrease in effective clinical treatments is a serious issue worldwide. One source antibiotic tolerance derives from biofilm infections: collectives of bacteria surrounded by an extracellular matrix that adhere to surfaces. Here we outline a board game designed to raise awareness and knowledge of biofilms in young learners.

The intertwined roles of specialized metabolites within the Bacillus subtilis biofilm

Bacteria produce specialized metabolites with a range of functions. In this issue of the Journal of Bacteriology, Schoenborn et al. study the production and role of secondary metabolites during biofilm development and sporulation in Bacillus subtilis (A. A. Schoenborn, S. M. Yannarell, E. D. Wallace, H. Clapper, et al., J Bacteriol 203:e00337-21, 2021, https://doi.org/https://doi.org/10.1128/JB.00337-21). Most metabolites studied are produced during differentiation, and six are required for the development of biofilms and/or spores. The authors propose a model for the timing of production and role in differentiation exerted by each secondary metabolite

Biofilm dispersal for spore release in <i>Bacillus subtilis</i>

The dispersal of bacterial cells from a matured biofilm can be mediated either by active or passive mechanisms. In this issue of the Journal of Bacteriology, Nishikawa and Kobayashi demonstrate that the presence of calcium influences the dispersal of spores from the pellicle biofilm of Bacillus subtilis (M. Nishikawa and K. Kobayashi, J Bacteriol 203:e00114-21, 2021, https://doi.org/10.1128/JB.00114-21). The authors propose that temporal heterogeneity in matrix production and chelation of calcium by dipicolinic acid in spores weakens the biofilm matrix and causes passive dispersal

The role of the phosphopantetheinyltransferase enzyme, PswP, in the biosynthesis of antimicrobial secondary metabolites by <em>Serratia marcescens </em>Db10

Phosphopantetheinyltransferase (PPTase) enzymes fulfil essential roles in primary and secondary metabolism in prokaryotes, archaea and eukaryotes. PPTase enzymes catalyse the essential modification of the carrier protein domain of fatty acid synthases, polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs). In bacteria and fungi, NRPS and PKS enzymes are often responsible for the biosynthesis of secondary metabolites with clinically relevant properties; these secondary metabolites include a variety of antimicrobial peptides. We have previously shown that in the Gram-negative bacterium Serratia marcescens Db10, the PPTase enzyme PswP is essential for the biosynthesis of an NRPS-PKS dependent antibiotic called althiomycin. In this work we utilize bioinformatic analyses to classify PswP as belonging to the F/KES subfamily of Sfp type PPTases and to putatively identify additional NRPS substrates of PswP, in addition to the althiomycin NRPS-PKS, in Ser. marcescens Db10. We show that PswP is required for the production of three diffusible metabolites by this organism, each possessing antimicrobial activity against Staphylococcus aureus. Genetic analyses identify the three metabolites as althiomycin, serrawettin W2 and an as-yet-uncharacterized siderophore, which may be related to enterobactin. Our results highlight the use of an individual PPTase enzyme in multiple biosynthetic pathways, each contributing to the ability of Ser. marcescens to inhibit competitor bacteria by the production of antimicrobial secondary metabolites

The insect pathogen Serratia marcescens Db10 uses a hybrid non-ribosomal peptide synthetase-polyketide synthase to produce the antibiotic althiomycin

There is a continuing need to discover new bioactive natural products, such as antibiotics, in genetically-amenable micro-organisms. We observed that the enteric insect pathogen, Serratia marcescens Db10, produced a diffusible compound that inhibited the growth of Bacillis subtilis and Staphyloccocus aureus. Mapping the genetic locus required for this activity revealed a putative natural product biosynthetic gene cluster, further defined to a six-gene operon named alb1-alb6. Bioinformatic analysis of the proteins encoded by alb1-6 predicted a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line (Alb4/5/6), tailoring enzymes (Alb2/3) and an export/resistance protein (Alb1), and suggested that the machinery assembled althiomycin or a related molecule. Althiomycin is a ribosome-inhibiting antibiotic whose biosynthetic machinery had been elusive for decades. Chromatographic and spectroscopic analyses confirmed that wild type S. marcescens produced althiomycin and that production was eliminated on disruption of the alb gene cluster. Construction of mutants with in-frame deletions of specific alb genes demonstrated that Alb2-Alb5 were essential for althiomycin production, whereas Alb6 was required for maximal production of the antibiotic. A phosphopantetheinyl transferase enzyme required for althiomycin biosynthesis was also identified. Expression of Alb1, a predicted major facilitator superfamily efflux pump, conferred althiomycin resistance on another, sensitive, strain of S. marcescens. This is the first report of althiomycin production outside of the Myxobacteria or Streptomyces and paves the way for future exploitation of the biosynthetic machinery, since S. marcescens represents a convenient and tractable producing organism