The YmdB Phosphodiesterase Is a Global Regulator of Late Adaptive Responses in Bacillus subtilis

Bacillus subtilis mutants lacking ymdB are unable to form biofilms, exhibit a strong overexpression of the flagellin gene hag, and are deficient in SlrR, a SinR antagonist. Here, we report the functional and structural characterization of YmdB, and we find that YmdB is a phosphodiesterase with activity against 2′,3′- and 3′,5′-cyclic nucleotide monophosphates. The structure of YmdB reveals that the enzyme adopts a conserved phosphodiesterase fold with a binuclear metal center. Mutagenesis of a catalytically crucial residue demonstrates that the enzymatic activity of YmdB is essential for biofilm formation. The deletion of ymdB affects the expression of more than 800 genes; the levels of the σ^D-dependent motility regulon and several sporulation genes are increased, and the levels of the SinR-repressed biofilm genes are decreased, confirming the role of YmdB in regulating late adaptive responses of B. subtilis.     

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria ^1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors ^7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells ^11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm ^18-21.Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation ^11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis^15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin ²⁵. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers ¹⁵.We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B.subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.     

The Biofilm Regulatory Network from Bacillus subtilis: A Structure-Function Analysis

Bacterial biofilms are notorious for their ability to protect bacteria from environmental challenges, most importantly the action of antibiotics. Bacillus subtilis is an extensively studied model organism used to understand the process of biofilm formation. A complex network of principal regulatory proteins including Spo0A, AbrB, AbbA, Abh, SinR, SinI, SlrR, and RemA, work in concert to transition B. subtilis from the free-swimming planktonic state to the biofilm state. In this review, we explore, connect, and summarize decades worth of structural and biochemical studies that have elucidated this protein signaling network. Since structure dictates function, unraveling aspects of protein molecular mechanisms will allow us to devise ways to exploit critical features of the biofilm regulatory pathway, such as possible therapeutic intervention. This review pools our current knowledge base of B. subtilis biofilm regulatory proteins and highlights potential therapeutic intervention points.     

Chemical shift assignments and secondary structure prediction of the master biofilm regulator,SinR, from Bacillus subtilis

Bacillus subtilis is a soil-dwelling Gram-positive bacterial species that has been extensively studied as a model of biofilm formation and stress-induced cellular differentiation. The tetrameric protein, SinR, has been identified as a master regulator for biofilm formation and linked to the regulation of the early transition states during cellular stress response, such as motility and biofilm-linked biosynthetic genes. SinR is a 111-residue protein that is active as a dimer of dimers, composed of two distinct domains, a DNA-binding helix-turn-helix N-terminus domain and a C-terminal multimerization domain. In order for biofilm formation to proceed, the antagonist, SinI, must inactivate SinR. This interaction results in a dramatic structural rearrangement of both proteins. Here we report the full-length backbone and side chain chemical shift values in addition to the experimentally derived secondary structure predictions as the first step towards directly studying the complex interaction dynamics between SinR and SinI.     

Spo0A positively regulates epr expression by negating the repressive effect of co-repressors, SinR and ScoC, in Bacillus subtilis

Bacillus subtilis under nutritional deprivation exhibits several physiological responses such as synthesis of degradative enzymes, motility, competence, sporulation, etc. At the onset of post-exponential phase the global response regulator, Spo0A, directly or indirectly activates the expression of genes involved in the above processes. These genes are repressed during the exponential phase by a group of proteins called transition state regulators, e.g. AbrB, ScoC and SinR. One such post-exponentially expressed gene is epr, which encodes a minor extracellular serine protease and is involved in the swarming motility of B. subtilis. Deletion studies of the upstream region of epr promoter revealed that epr is co-repressed by transition state regulators, SinR and ScoC. Our study shows that Spo0A positively regulates epr expression by nullifying the repressive effect of co-repressors, SinR and ScoC. We demonstrate via in vitro mobility shift assays that Spo0A binds to the upstream region of epr promoter and in turn occludes the binding site of one of the co-repressor, SinR. This explains the mechanism behind the positive regulatory effect of Spo0A on epr expression.     

Structure and organisation of SinR, the master regulator of biofilm formation in Bacillus subtilis

sinR encodes a tetrameric repressor of genes required for biofilm formation in Bacillus subtilis. sinI, which is transcribed under Spo0A control, encodes a dimeric protein that binds to SinR to form a SinR-SinI heterodimer in which the DNA-binding functions of SinR are abrogated and repression of biofilm genes is relieved. The heterodimer-forming surface comprises residues conserved between SinR and SinI. Each forms a pair of α-helices that hook together to form an intermolecular four-helix bundle. Here, we are interested in the assembly of the SinR tetramer and its binding to DNA. Size-exclusion chromatography with multi-angle laser light scattering and crystallographic analysis reveal that a DNA-binding fragment of SinR (residues 1-69) is a monomer, while a SinI-binding fragment (residues 74-111) is a tetramer arranged as a dimer of dimers. The SinR(74-111) chain forms two α-helices with the organisation of the dimer similar to that observed in the SinR-SinI complex. The tetramer is formed through interactions of residues at the C-termini of the four chains. A model of the intact SinR tetramer in which the DNA binding domains surround the tetramerisation core was built. Fluorescence anisotropy and surface plasmon resonance experiments showed that SinR binds to an oligonucleotide duplex, 5′-TTTGTTCTCTAAAGAGAACTTA-3′, containing a pair of SinR consensus sequences in inverted orientation with a K_d of 300 nM. The implications of these data for promoter binding and the curious quaternary structural transitions of SinR upon binding to (i) SinI and (ii) the SinR-like protein SlrR, which “repurposes” SinR as a repressor of autolysin and motility genes, are discussed.     

Cyclic diguanylate regulation of Bacillus cereus group biofilm formation

Biofilm formation can be considered a bacterial virulence mechanism. In a range of Gram‐negatives, increased levels of the second messenger cyclic diguanylate (c‐di‐GMP) promotes biofilm formation and reduces motility. Other bacterial processes known to be regulated by c‐di‐GMP include cell division, differentiation and virulence. Among Gram‐positive bacteria, where the function of c‐di‐GMP signalling is less well characterized, c‐di‐GMP was reported to regulate swarming motility in Bacillus subtilis while having very limited or no effect on biofilm formation. In contrast, we show that in the Bacillus cereus group c‐di‐GMP signalling is linked to biofilm formation, and to several other phenotypes important to the lifestyle of these bacteria. The Bacillus thuringiensis 407 genome encodes eleven predicted proteins containing domains (GGDEF/EAL) related to c‐di‐GMP synthesis or breakdown, ten of which are conserved through the majority of clades of the B. cereus group, including Bacillus anthracis. Several of the genes were shown to affect biofilm formation, motility, enterotoxin synthesis and/or sporulation. Among these, cdgF appeared to encode a master diguanylate cyclase essential for biofilm formation in an oxygenated environment. Only two cdg genes (cdgA, cdgJ) had orthologs in B. subtilis, highlighting differences in c‐di‐GMP signalling between B. subtilis and B. cereus group bacteria.     

Evolved Biofilm: Review on the Experimental Evolution Studies of Bacillus subtilis Pellicles

For several decades, laboratory evolution has served as a powerful method to manipulate microorganisms and to explore long-term dynamics in microbial populations. Next to canonical Escherichia coli planktonic cultures, experimental evolution has expanded into alternative cultivation methods and species, opening the doors to new research questions. Bacillus subtilis, the spore-forming and root-colonizing bacterium, can easily develop in the laboratory as a liquid–air interface colonizing pellicle biofilm. Here, we summarize recent findings derived from this tractable experimental model. Clonal pellicle biofilms of B. subtilis can rapidly undergo morphological and genetic diversification creating new ecological interactions, for example, exploitation by biofilm non-producers. Moreover, long-term exposure to such matrix non-producers can modulate cooperation in biofilms, leading to different phenotypic heterogeneity pattern of matrix production with larger subpopulation of “ON” cells. Alternatively, complementary variants of biofilm non-producers, each lacking a distinct matrix component, can engage in a genetic division of labor, resulting in superior biofilm productivity compared to the “generalist” wild type. Nevertheless, inter-genetic cooperation appears to be evanescent and rapidly vanquished by individual biofilm formation strategies altering the amount or the properties of the remaining matrix component. Finally, fast-evolving mobile genetic elements can unpredictably shift intra-species interactions in B. subtilis biofilms. Understanding evolution in clonal biofilm populations will facilitate future studies in complex multispecies biofilms that are more representative of nature.     

Influence of Lysogeny of Tectiviruses GIL01 and GIL16 on Bacillus thuringiensis Growth,Biofilm Formation,and Swarming Motility

Bacillus thuringiensis is an entomopathogenic bacterium that has been used as an efficient biopesticide worldwide. Despite the fact that this bacterium is usually described as an insect pathogen, its life cycle in the environment is still largely unknown. B. thuringiensis belongs to the Bacillus cereus group of bacteria, which has been associated with many mobile genetic elements, such as species-specific temperate or virulent bacteriophages (phages). Temperate (lysogenic) phages are able to establish a long-term relationship with their host, providing, in some cases, novel ecological traits to the bacterial lysogens. Therefore, this work focuses on evaluating the potential influence of temperate tectiviruses GIL01 and GIL16 on the development of different life traits of B. thuringiensis. For this purpose, a B. thuringiensis serovar israelensis plasmid-cured (nonlysogenic) strain was used to establish bacterial lysogens for phages GIL01 and GIL16, and, subsequently, the following life traits were compared among the strains: kinetics of growth, metabolic profiles, antibiotics susceptibility, biofilm formation, swarming motility, and sporulation. The results revealed that GIL01 and GIL16 lysogeny has a significant influence on the bacterial growth, sporulation rate, biofilm formation, and swarming motility of B. thuringiensis. No changes in metabolic profiles or antibiotic susceptibilities were detected. These findings provide evidence that tectiviruses have a putative role in the B. thuringiensis life cycle as adapters of life traits with ecological advantages.     

Biofilm Formation and Cell Surface Properties among Pathogenic and Nonpathogenic Strains of the Bacillus cereus Group

Biofilm formation by 102 Bacillus cereus and B. thuringiensis strains was determined. Strains isolated from soil or involved in digestive tract infections were efficient biofilm formers, whereas strains isolated from other diseases were poor biofilm formers. Cell surface hydrophobicity, the presence of an S layer, and adhesion to epithelial cells were also examined.The Bacillus cereus group includes B. cereus sensu stricto, B. anthracis, and B. thuringiensis, three genetically close pathogenic species. Based on genetic evidence, it has been suggested that they could represent one species (7). B. cereus sensu stricto is itself an opportunistic human pathogen occasionally found to cause various diseases such as endophthalmitis or periodontitis but is more frequently involved in gastrointestinal diseases with diarrheal or emetic syndromes (4, 12). Emetic syndromes result from the presence of cereulide, a heat-stable toxin produced in food before ingestion, whereas diarrheal syndromes require survival of the bacterium in the host digestive tract. B. thuringiensis is an insect pathogen, and B. anthracis causes anthrax, a lethal human disease.The persistent contamination of industrial food processing systems by B. cereus (12) may facilitate its involvement in gastroenteritis. This persistence is due to spores, which may survive pasteurization, heating, and gamma-ray irradiation (9, 13), and to biofilms, which have been shown to be highly resistant to cleaning procedures (18). Biofilms are also suspected to be involved in bacterial pathogenicity, as they may form on host epithelia (15).In this study, we wanted to test whether biofilm formation by species of the B. cereus group could be connected to the pathogenicity of the bacterium. For this purpose, we screened a collection of 102 pathogenic (diarrheal, emetic, and oral diseases) and nonpathogenic strains of B. cereus and B. thuringiensis for their capability to form biofilms. As adhesion to inert or living surfaces is a prerequisite for biofilm formation, we have investigated relationships within our collection of strains between biofilm formation and cell surface hydrophobicity, the presence of an S-layer, or adhesion to epithelial cells.     

d-Amino Acids Do Not Inhibit Biofilm Formation in Staphylococcus aureus

Bacteria can either exist in the planktonic (free floating) state or in the biofilm (encased within an organic framework) state. Bacteria biofilms cause industrial concerns and medical complications and there has been a great deal of interest in the discovery of small molecule agents that can inhibit the formation of biofilms or disperse existing structures. Herein we show that, contrary to previously published reports, d-amino acids do not inhibit biofilm formation of Bacillus subtilis (B. subtilis), Staphylococcus aureus (S. aureus), and Staphylococcus epidermis (S. epidermis) at millimolar concentrations. We evaluated a diverse set of natural and unnatural d-amino acids and observed no activity from these compounds in inhibiting biofilm formation.     

Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.     

A novel factor controlling bistability in Bacillus subtilis: the YmdB protein affects flagellin expression and biofilm formation

Cells of Bacillus subtilis can either be motile or sessile, depending on the expression of mutually exclusive sets of genes that are required for flagellum or biofilm formation, respectively. Both activities are coordinated by the master regulator SinR. We have analyzed the role of the previously uncharacterized ymdB gene for bistable gene expression in B. subtilis. We observed a strong overexpression of the hag gene encoding flagellin and of other genes of the σ(D)-dependent motility regulon in the ymdB mutant, whereas the two major operons for biofilm formation, tapA-sipW-tasA and epsA-O, were not expressed. As a result, the ymdB mutant is unable to form biofilms. An analysis of the individual cells of a population revealed that the ymdB mutant no longer exhibited bistable behavior; instead, all cells are short and motile. The inability of the ymdB mutant to form biofilms is suppressed by the deletion of the sinR gene encoding the master regulator of biofilm formation, indicating that SinR-dependent repression of biofilm genes cannot be relieved in a ymdB mutant. Our studies demonstrate that lack of expression of SlrR, an antagonist of SinR, is responsible for the observed phenotypes. Overexpression of SlrR suppresses the effects of a ymdB mutation.     

Nonribosomal Peptide Synthase Gene Clusters for Lipopeptide Biosynthesis in Bacillus subtilis 916 and Their Phenotypic Functions

Bacillus cyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features of Bacillus strains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology. Bacillus subtilis 916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome of B. subtilis 916 contains four nonribosomal peptide synthase (NRPS) gene clusters, srf, bmy, fen, and loc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studying B. subtilis 916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activity in vitro, the strain mutated in srfAA had significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other than fen resulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion, B. subtilis 916 coproduces four families of LPs which contribute to the phenotypic features of B. subtilis 916 in an intricate way.     

d-Amino Acids Indirectly Inhibit Biofilm Formation in Bacillus subtilis by Interfering with Protein Synthesis

The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine and was reported to inhibit biofilm formation via the incorporation of these d-amino acids into the cell wall. Here, we show that l-amino acids were able to specifically reverse the inhibitory effects of their cognate d-amino acids. We also show that d-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding d-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of d-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of d-amino acids without losing the ability to incorporate at least one noncanonical d-amino acid, d-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of d-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis.     

Ecological Advantages of Autolysis during the Development and Dispersal of Pseudoalteromonas tunicata Biofilms

In the ubiquitous marine bacterium Pseudoalteromonas tunicata, subpopulations of cells are killed by the production of an autocidal protein, AlpP, during biofilm development. Our data demonstrate an involvement of this process in two parameters, dispersal and phenotypic diversification, which are of importance for the ecology of this organism and for its survival within the environment. Cell death in P. tunicata wild-type biofilms led to a major reproducible dispersal event after 192 h of biofilm development. The dispersal was not observed with a ΔAlpP mutant strain. Using flow cytometry and the fluorescent dye DiBAC₄(3), we also show that P. tunicata wild-type cells that disperse from biofilms have enhanced metabolic activity compared to those cells that disperse from ΔAlpP mutant biofilms, possibly due to nutrients released from dead cells. Furthermore, we report that there was considerable phenotypic variation among cells dispersing from wild-type biofilms but not from the ΔAlpP mutant. Wild-type cells that dispersed from biofilms showed significantly increased variations in growth, motility, and biofilm formation, which may be important for successful colonization of new surfaces. These findings suggest for the first time that the autocidal events mediated by an antibacterial protein can confer ecological advantages to the species by generating a metabolically active and phenotypically diverse subpopulation of dispersal cells.