https://www.cell.com/trends/microbiology/pdf/S0966-842X(04)00264-1.pdf

Survival strategies of infectious biofilms.

The endospore-forming rhizobacterium Bacillus subtilis- the model system for Gram-positive organisms, is able to produce more than two dozen antibiotics with an amazing variety of structures. The produced anti-microbial active compounds include predominantly peptides that are either ribosomally synthesized and post-translationally modified (lantibiotics and lantibiotic-like peptides) or non-ribosomally generated, as well as a couple of non-peptidic compounds such as polyketides, an aminosugar, and a phospholipid. Here I summarize the structures of all known B. subtilis antibiotics, their biochemistry and genetic analysis of their biosyntheses. An updated summary of well-studied antibiotic regulation pathways is given. Furthermore, current findings are resumed that show roles for distinct B. subtilis antibiotics beyond the "pure" anti-microbial action: Non-ribosomally produced lipopeptides are involved in biofilm and swarming development, lantibiotics function as pheromones in quorum-sensing, and a "killing factor" effectuates programmed cell death in sister cells. A discussion of how these antibiotics may contribute to the survival of B. subtilis in its natural environment is given.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2005.04587.x

Bacillus subtilis antibiotics: structures, syntheses and specific functions

Biofilms are communities of microorganisms that live attached to surfaces. Biofilm formation has received much attention in the last decade, as it has become clear that virtually all types of bacteria can form biofilms and that this may be the preferred mode of bacterial existence in nature. Our current understanding of biofilm formation is based on numerous studies of myriad bacterial species. Here, we review a portion of this large body of work including the environmental signals and signaling pathways that regulate biofilm formation, the components of the biofilm matrix, and the mechanisms and regulation of biofilm dispersal.

Signals, regulatory networks, and materials that build and break bacterial biofilms

Relatively little is known about the exact mechanisms used by Bacillus subtilis in its behavior as a biocontrol agent on plants. Here, we report the development of a sensitive plant infection model demonstrating that the bacterial pathogen Pseudomonas syringae pv tomato DC3000 is capable of infecting Arabidopsis roots both in vitro and in soil. Using this infection model, we demonstrated the biocontrol ability of a wild-type B. subtilis strain 6051 against P. syringae. Arabidopsis root surfaces treated with B. subtilis were analyzed with confocal scanning laser microscopy to reveal a three-dimensional B. subtilis biofilm. It is known that formation of biofilms by B. subtilis is a complex process that includes secretion of surfactin, a lipopeptide antimicrobial agent. To determine the role of surfactin in biocontrol by B. subtilis, we tested a mutant strain, M1, with a deletion in a surfactin synthase gene and, thus, deficient in surfactin production. B. subtilis M1 was ineffective as a biocontrol agent against P. syringae infectivity in Arabidopsis and also failed to form robust biofilms on either roots or inert surfaces. The antibacterial activity of surfactin against P. syringae was determined in both broth and agar cultures and also by live-dead staining methods. Although the minimum inhibitory concentrations determined were relatively high (25 μg mL-1), the levels of the lipopeptide in roots colonized by B. subtilis are likely to be sufficient to kill P. syringae. Our results collectively indicate that upon root colonization, B. subtilis 6051 forms a stable, extensive biofilm and secretes surfactin, which act together to protect plants against attack by pathogenic bacteria.

Biocontrol of Bacillus subtilis against Infection of Arabidopsis Roots by Pseudomonas syringae Is Facilitated by Biofilm Formation and Surfactin Production

Biofilms are ubiquitous communities of tightly associated bacteria encased in an extracellular matrix. Bacillus subtilis has long served as a robust model organism to examine the molecular mechanisms of biofilm formation, and a number of studies have revealed that this process is regulated by several integrated pathways. In this Review, we focus on the molecular mechanisms that control B. subtilis biofilm assembly, and then briefly summarize the current state of knowledge regarding biofilm disassembly. We also discuss recent progress that has expanded our understanding of B. subtilis biofilm formation on plant roots, which are a natural habitat for this soil bacterium.

/pdf/sticking-together-building-a-biofilm-the-bacillus-subtilis-59fj6f3u5v.pdf

Sticking together: building a biofilm the Bacillus subtilis way

In nature, bacteria often exist as biofilms. Here, we discuss the environmental signals and regulatory proteins that affect both the initiation of bacterial biofilm formation and the nature of the mature biofilm structure. Current research suggests that the environmental signals regulating whether bacterial cells will initiate a biofilm differ from one bacterial species to another. This may allow each bacterial species to colonize its preferred environment efficiently. In contrast, some of the environmental signals that have currently been identified to regulate the structure of a mature biofilm are nutrient availability and quorum sensing, and are not species specific. These environmental signals evoke changes in the nature of the mature biofilm that may ensure optimal nutrient acquisition. Nutrient availability regulates the depth of the biofilm in such a way that the maximal number of cells in a biofilm appears to occur at suboptimal nutrient concentrations. At either extreme, nutrient-rich or very nutrient-poor conditions, greater numbers of cells are in the planktonic phase where they have greater access to the local nutrients or can be distributed to a new environment. Similarly, quorum-sensing control of the formation of channels and pillar-like structures may ensure efficient nutrient delivery to cells in a biofilm.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2004.04036.x

Environmental signals and regulatory pathways that influence biofilm formation

Biofilms are structured communities of cells that are encased in a self-produced polymeric matrix and are adherent to a surface. Many biofilms have a significant impact in medical and industrial settings. The model gram-positive bacterium Bacillus subtilis has recently been shown to form biofilms. To gain insight into the genes involved in biofilm formation by this bacterium, we used DNA microarrays representing >99% of the annotated B. subtilis open reading frames to follow the temporal changes in gene expression that occurred as cells transitioned from a planktonic to a biofilm state. We identified 519 genes that were differentially expressed at one or more time points as cells transitioned to a biofilm. Approximately 6% of the genes of B. subtilis were differentially expressed at a time when 98% of the cells in the population were in a biofilm. These genes were involved in motility, phage-related functions, and metabolism. By comparing the genes differentially expressed during biofilm formation with those identified in other genomewide transcriptional-profiling studies, we were able to identify several transcription factors whose activities appeared to be altered during the transition from a planktonic state to a biofilm. Two of these transcription factors were Spo0A and sigma-H, which had previously been shown to affect biofilm formation by B. subtilis. A third signal that appeared to be affecting gene expression during biofilm formation was glucose depletion. Through quantitative biofilm assays and confocal scanning laser microscopy, we observed that glucose inhibited biofilm formation through the catabolite control protein CcpA.

Identification of Catabolite Repression as a Physiological Regulator of Biofilm Formation by Bacillus subtilis by Use of DNA Microarrays

Biofilms are communities of microbial cells that are encased in a self-produced, polymeric matrix and are adherent to a surface. For several species of bacteria, an enhanced ability to form biofilms has been linked with an increased capability to produce exopolymers. To identify exopolymers of Bacillus subtilis that can contribute to biofilm formation, we transferred the genetic determinants that control exopolymer production from a wild, exopolymer-positive strain to a domesticated, exopolymer-negative strain. Mapping these genetic determinants led to the identification of gamma-poly-dl-glutamic acid (gamma-PGA) as an exopolymer that increases biofilm formation, possibly through enhancing cell-surface interactions. Production of gamma-PGA by Bacillus subtilis was known to be dependent on the two-component regulator ComPA; this study highlighted the additional dependence on the DegS-DegU, DegQ and SwrA regulator proteins. The inability of the domestic strain of B. subtilis to produce gamma-PGA was mapped to two base pairs; a single base pair change in the promoter region of degQ and a single base pair insertion in the coding region of swrA. Introduction of alleles of degQ and swrA from the wild strain into the domestic strain was sufficient to allow gamma-PGA production. In addition to controlling gamma-PGA production, ComPA and DegSU were also shown to activate biofilm formation through an as yet undefined pathway. The identification of these regulators as affecting gamma-PGA production and biofilm formation suggests that these processes are regulated by osmolarity, high cell density and phase variation.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2005.04746.x

Defining the genetic differences between wild and domestic strains of Bacillus subtilis that affect poly-gamma-DL-glutamic acid production and biofilm formation

Bacillus subtilis is a ubiquitous soil bacterium that forms biofilms in a process that is negatively controlled by the transcription factor AbrB. To identify the AbrB-regulated genes required for biofilm formation by B. subtilis, genome-wide expression profiling studies of biofilms formed by spo0A abrB and sigH abrB mutant strains were performed. These data, in concert with previously published DNA microarray analysis of spo0A and sigH mutant strains, led to the identification of 39 operons that appear to be repressed by AbrB. Eight of these operons had previously been shown to be repressed by AbrB, and we confirmed AbrB repression for a further six operons by reverse transcription-PCR. The AbrB-repressed genes identified in this study are involved in processes known to be regulated by AbrB, such as extracellular degradative enzyme production and amino acid metabolism, and processes not previously known to be regulated by AbrB, such as membrane bioenergetics and cell wall functions. To determine whether any of these AbrB-regulated genes had a role in biofilm formation, we tested 23 mutants, each with a disruption in a different AbrB-regulated operon, for the ability to form biofilms. Two mutants had a greater than twofold defect in biofilm formation. A yoaW mutant exhibited a biofilm structure with reduced depth, and a sipW mutant exhibited only surface-attached cells and did not form a mature biofilm. YoaW is a putative secreted protein, and SipW is a signal peptidase. This is the first evidence that secreted proteins have a role in biofilm formation by Bacillus subtilis.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2004.04023.x

Nicola R. Stanley

Papers

Environmental signals and regulatory pathways that influence biofilm formation

Identification of Catabolite Repression as a Physiological Regulator of Biofilm Formation by Bacillus subtilis by Use of DNA Microarrays

Defining the genetic differences between wild and domestic strains of Bacillus subtilis that affect poly-gamma-DL-glutamic acid production and biofilm formation

Identification of AbrB-regulated genes involved in biofilm formation by Bacillus subtilis