The spore-forming bacterium Bacillus subtilis forms matrix-enclosed biofilms in response to environmental cues that to date remain poorly defined. Biofilm formation depends on the synthesis of an extracellular matrix, which is indirectly regulated by the transcriptional regulator Spo0A. The activity of Spo0A depends on its phosphorylation state. The level of phosphorylated Spo0A (Spo0A∼P) is controlled by a network of kinases and phosphatases, which respond to environmental and physiological signals. In spite of significant progress in understanding biofilm development, the fundamental question of how cells sense the environmental cues that trigger biofilm formation has largely remained unaddressed. Here, we report that biofilm formation of B. subtilis in LB medium is triggered by a combination of glycerol and manganese (GM). Moreover, LB medium with GM significantly stimulates biofilm-associated sporulation and production of an undefined brown pigment. We further show that transcription of the major operons responsible for matrix production and biofilm formation is dramatically enhanced in response to GM. We also establish that KinD is a principal histidine kinase responsible for sensing the presence of GM exclusively by its extracellular CACHE domain. Finally, we show that GM has a similar biofilm-promoting effect in two related Bacillus species, B. licheniformis and B. cereus , indicating that the biofilm-promoting effect of GM is conserved in Bacillus species.

Streptococcus mutans often adopts a sessile biofilm lifestyle that differs greatly from that of free-living cells. Biofilm formation represents a protected mode of growth that allows cells to survive in hostile environments. In this study, in vitro comparative transcriptome analysis was carried out to identify genes that are differentially expressed in biofilm of S. mutans compared with free-living cells. DNA-microarray analyses indicated that about 12 % of genes showed significant differential expression: 139 were activated and 104 were repressed in biofilm vs the planktonic environment. The differential expression of 20 selected genes was confirmed by real-time RT-PCR. In addition, regulation of expression of these genes during biofilm development was tested in 100 and 400 μm deep biofilms. Direct comparison of optical images consistently demonstrated that changes in biofilm thickness are accompanied by significant shifts in cell viability. From evaluation of gene expression patterns, it was shown that the majority of the genes tested were significantly down-regulated in 400 vs 100 μm deep biofilms. This study provides a genome-scale synopsis and adds important insights into gene expression in biofilm development processes of S. mutans, which are strongly associated with the pathogenesis of dental diseases.

Bacillus subtilisforms biofilms in response to signals that remain poorly defined. We report that biofilm formation is stimulated by sublethal doses of chlorine dioxide (ClO₂), an extremely effective and fast-acting biocide. ClO₂accelerated biofilm formation inB. subtilisas well as in other bacteria, suggesting that biofilm formation is a widely conserved response to sublethal doses of the agent. Biofilm formation depends on the synthesis of an extracellular matrix that holds the constituent cells together. We show that the transcription of the major operons responsible for the matrix production inB. subtilis,epsA-epsOandyqxM-sipW-tasA, was enhanced by ClO₂, in a manner that depended on the membrane-bound kinase KinC. Activation of KinC appeared to be due to the ability of ClO₂to collapse the membrane potential. Importantly, strains unable to make a matrix were hypersensitive to ClO₂, indicating that biofilm formation is a defensive response that helps protect cells from the toxic effects of the biocide.

Streptococcus mutans is known as a primary pathogen of dental caries, one of the most common human infectious diseases. Exopolysaccharide synthesis, adherence to tooth surface and biofilm formation are important physiological and virulence factors of S. mutans. In vitro comparative gene expression analysis was carried out to differentiate 10 selected genes known to be mostly involved in S. mutans biofilm formation by comparing the expression under biofilm and planktonic environments. Real-time RT-PCR analyses indicated that all of the genes tested were upregulated in the biofilm compared to cells grown in planktonic conditions. The influence of simple dietary carbohydrates on gene expression in S. mutans biofilm was tested also. Among the tested genes, in the biofilm phase, the greatest induction was observed for gtf and ftf, which are genes encoding the extracellular polysaccharide-producing enzymes. Biofilm formation was accompanied by a 22-fold induction in the abundance of mRNA encoding glucosyltransferase B (GTFB) and a 14.8 -fold increase in mRNA encoding GTFC. Levels of mRNA encoding fructosyltransferase were induced approximately 11.8-fold in biofilm-derived cells. Another notable finding of this study suggests that glucose affects the expression of S. mutans GS5 biofilm genes. In spite of a significant upregulation in biofilm-associated gene expression in the presence of sucrose, the presence of glucose with sucrose reduced expression of most tested genes. Differential analysis of the transcripts from S. mutans, grown in media with various nutrient contents, revealed significant shifts in the expression of the genes involved in biofilm formation. The results presented here provide new insights at the molecular level regarding gene expression in this bacterium when grown under biofilm conditions, allowing a better understanding of the mechanism of biofilm formation by S. mutans.

Bacillus species present a major concern in the dairy industry as they can form biofilms in pipelines and on surfaces of equipment and machinery used in the entire line of production. These biofilms represent a continuous hygienic problem and can lead to serious economic losses due to food spoilage and equipment impairment. Biofilm formation by Bacillus subtilis is apparently dependent on LuxS quorum sensing (QS) by Autoinducer-2 (AI-2). However, the link between sensing environmental cues and AI-2 induced biofilm formation remains largely unknown. The aim of this study is to investigate the role of lactose, the primary sugar in milk, on biofilm formation by B. subtilis and its possible link to QS processes. Our phenotypic analysis shows that lactose induces formation of biofilm bundles as well as formation of colony type biofilms. Furthermore, using reporter strain assays, we observed an increase in AI-2 production by B. subtilis in response to lactose in a dose dependent manner. Moreover, we found that expression of eps and tapA operons, responsible for extracellular matrix synthesis in B. subtilis, were notably up-regulated in response to lactose. Importantly, we also observed that LuxS is essential for B. subtilis biofilm formation in the presence of lactose. Overall, our results suggest that lactose may induce biofilm formation by B. subtilis through the LuxS pathway.

The objective of this study was to investigate the effect of Mg2+ ions on biofilm formation by Bacillus species, which are considered as problematic microorganisms in the food industry. We found that magnesium ions are capable to inhibit significantly biofilm formation of Bacillus species at 50 mM concentration and higher. We further report that Mg2+ ions don't inhibit bacterial growth at elevated concentrations; hence, the mode of action of Mg2+ ions is apparently specific to inhibition of biofilm formation. Biofilm formation depends on the synthesis of extracellular matrix, whose production in Bacillus subtilis is specified by two major operons: the epsA-O and tapA operons. We analyzed the effect of Mg2+ ions on matrix gene expression using transcriptional fusions of the promoters for eps and tapA to the gene encoding β galactosidase. The expression of the two matrix operons was reduced drastically in response to Mg2+ ions suggesting about their inhibitory effect on expression of the matrix genes in B. subtilis. Since the matrix gene expression is tightly controlled by Spo0A dependent pathway, we conclude that Mg2+ ions could affect the signal transduction for biofilm formation through this pathway.