The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in <em>Bacillus subtilis</em>

The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA

FlgN is required for flagellum based motility by <em>Bacillus subtilis</em>

The assembly of the bacterial flagellum is exquisitely controlled. Flagellar biosynthesis is underpinned by a specialized type III secretion system that allows export of proteins from the cytoplasm to the nascent structure. Bacillus subtilis regulates flagellar assembly using both conserved and species-specific mechanisms. Here, we show that YvyG is essential for flagellar filament assembly. We define YvyG as an orthologue of the Salmonella enterica serovar Typhimurium type III secretion system chaperone, FlgN, which is required for the export of the hook-filament junction proteins, FlgK and FlgL. Deletion of flgN (yvyG) results in a nonmotile phenotype that is attributable to a decrease in hag translation and a complete lack of filament polymerization. Analyses indicate that a flgK-flgL double mutant strain phenocopies deletion of flgN and that overexpression of flgK-flgL cannot complement the motility defect of a ΔflgN strain. Furthermore, in contrast to previous work suggesting that phosphorylation of FlgN alters its subcellular localization, we show that mutation of the identified tyrosine and arginine FlgN phosphorylation sites has no effect on motility. These data emphasize that flagellar biosynthesis is differentially regulated in B. subtilis from classically studied Gram-negative flagellar systems and questions the biological relevance of some posttranslational modifications identified by global proteomic approaches

YuaB Functions Synergistically with the Exopolysaccharide and TasA Amyloid Fibers To Allow Biofilm Formation by <em>Bacillus subtilis</em>

During biofilm formation by Bacillus subtilis, two extracellular matrix components are synthesized, namely, the TasA amyloid fibers and an exopolysaccharide. In addition, a small protein called YuaB has been shown to allow the biofilm to form. The regulatory protein DegU is known to initiate biofilm formation. In this report we show that the main role of DegU during biofilm formation is to indirectly drive the activation of transcription from the yuaB promoter. The N terminus of YuaB constitutes a signal peptide for the Sec transport system. Here we show that the presence of the signal peptide is required for YuaB function. In addition we demonstrate that upon export of YuaB from the cytoplasm, it localizes to the cell wall. We continue with evidence that increased production of TasA and the exopolysaccharide is not sufficient to overcome the effects of a mutation in yuaB, demonstrating the unique involvement of YuaB in forming a biofilm. In line with this, YuaB is not involved in correct synthesis, export, or polymerization of either the TasA amyloid fibers or the exopolysaccharide. Taken together, these findings identify YuaB as a protein that plays a novel role during biofilm formation. We hypothesize that YuaB functions synergistically with the known components of the biofilm matrix to facilitate the assembly of the biofilm matrix

Blast a biofilm:a hands-on activity for school children and members of the public

<p>Microbial biofilms are very common in nature and have both detrimental and beneficial effects on everyday life. Practical and hands-on activities have been shown to achieve greater learning and engagement with science by young people (1, 4, 5). We describe an interactive activity, developed to introduce microbes and biofilms to school age children and members of the public. Biofilms are common in nature and, as the favored mode of growth for microbes, biofilms affect many parts ofeveryday life. This hands-on activity highlights the key  concepts of biofilms by allowing participants to first build, then attempt to ‘blast,’ a biofilm, thus enabling the robust nature of biofilms to become apparent. We developed the blast-a-biofilm activity as part of our two-day Magnificent Microbes event, which took place at the Dundee Science Centre-Sensation in May 2010 (6). This public engagement event was run by scientists from the Division of Molecular Microbiology at the University of Dundee. The purpose of the event was to use fun and interesting activities to make both children and adults think about how fascinating microbes are. Additionally, we aimed to develop interactive resources that could be used in future events and learning environments, of which the blast-a-biofilm activity is one such resource. Scientists and policy makers in the UK believe engaging the public with research ensures that the work of universities and research institutes is relevant to society and wider social concerns and can also help scientists actively contribute to positive social change (2). The activity is aimed at junior school age children (9–11 years) and adults with little or no knowledge of microbiology. The activity is suitable for use at science festivals, science clubs, and also in the classroom, where it can serve as a tool to enrich and enhance the school curriculum.</p