Biofilm formation by Bacillus subtilis: new insights into regulatory strategies and assembly mechanisms

Biofilm formation is a social behaviour that generates favourable conditions for sustained survival in the natural environment. For the Gram-positive bacterium Bacillus subtilis the process involves the differentiation of cell fate within an isogenic population and the production of communal goods that form the biofilm matrix. Here we review recent progress in understanding the regulatory pathways that control biofilm formation and highlight developments in understanding the composition, function and structure of the biofilm matrix.

Phosphorylated DegU Manipulates Cell Fate Differentiation in the Bacillus subtilis Biofilm

Cell differentiation is ubiquitous and facilitates division of labor and development. Bacteria are capable of multicellular behaviors that benefit the bacterial community as a whole. A striking example of bacterial differentiation occurs throughout the formation of a biofilm. During Bacillus subtilis biofilm formation, a subpopulation of cells differentiates into a specialized population that synthesizes the exopolysaccharide and the TasA amyloid components of the extracellular matrix. The differentiation process is indirectly controlled by the transcription factor Spo0A that facilitates transcription of the eps and tapA (tasA) operons. DegU is a transcription factor involved in regulating biofilm formation. Here, using a combination of genetics and live single-cell cytological techniques, we define the mechanism of biofilm inhibition at high levels of phosphorylated DegU (DegU∼P) by showing that transcription from the eps and tapA promoter regions is inhibited. Data demonstrating that this is not a direct regulatory event are presented. We demonstrate that DegU∼P controls the frequency with which cells activate transcription from the operons needed for matrix biosynthesis in favor of an off state. Subsequent experimental analysis led us to conclude that DegU∼P functions to increase the level of Spo0A∼P, driving cell fate differentiation toward the terminal developmental process of sporulation.

BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm

Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-positive soil bacterium that functions as an effective plant growth-promoting agent. The biofilm matrix is composed of an exopolysaccharide and an amyloid fiber-forming protein, TasA, and assembles with the aid of a small secreted protein, BslA. Here we show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophysical analysis demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Molecular function is revealed from analysis of the crystal structure of BslA, which consists of an Ig-type fold with the addition of an unusual, extremely hydrophobic "cap" region. A combination of in vivo biofilm formation and in vitro biophysical analysis demonstrates that the central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. The hydrophobic cap exhibits physiochemical properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. We suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community.

Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis

A commercial Bacillus anthracis (Anthrax) whole genome protein microarray has been used to identify immunogenic Anthrax proteins (IAP) using sera from groups of donors with (a) confirmed B. anthracis naturally acquired cutaneous infection, (b) confirmed B. anthracis intravenous drug use-acquired infection, (c) occupational exposure in a wool-sorters factory, (d) humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups. Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However, a number of other chromosomally-located and plasmid encoded open reading frame proteins were also recognized by infected or exposed groups in comparison to controls. Some of these antigens e.g., BA4182 are not recognized by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo that are not currently found in the UK licensed Anthrax Vaccine (AVP). These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis "infectome." These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesized, tested in mouse immunogenicity studies and validated in parallel using human sera from the same study.

CD4+ T Cells Targeting Dominant and Cryptic Epitopes from Bacillus anthracis Lethal Factor

Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called 'cryptic' or 'subdominant' epitopes. We analyzed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISpot assays we characterized epitopes that elicited a response following immunization with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, as a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 transgenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were influenced by the specific HLA alleles presenting the peptide, and imply that construction of future epitope string vaccines which are immunogenic across a wide range of HLA alleles could benefit from a combination of both cryptic and immunodominant anthrax epitopes.

For host factors weddings and a Koch’s bacillus funeral: actin, lipids, phagosome maturation and inflammasome activation

The chapter describes the 10 years research of Anes Laboratory on the interactions of M. tuberculosis with macrophages namely with the actin. modulation by lipids, phagosome maturation and inflammasome activation

Isolamento e caracterização de uma proteína tóxica produzida por um isolado açoriano de Bacillus weihenstephanensis

Dissertação de Mestrado, Biotecnologia em Controlo Biológico, 27 de Junho de 2013, Universidade dos Açores.

Estudo de atividades antimicrobianas em bactérias do género Bacillus isoladas nos Açores : otimização das condições de produção, identificação e caracterização de bacteriocinas

Dissertação de Mestrado, Ciências Biomédicas, 18 de Novembro de 2015, Universidade dos Açores.

Metabolic and morphogenetic engineering of Bacillus subtilis: biotechnology for industry

Dissertation presented to obtain the Ph.D. degree in “Biology” at the Institute of Chemical and Biological Technology of the New University of Lisbon

Inoculation with Metal-Mobilizing Plant-GrowthPromoting Rhizobacterium Bacillus sp. SC2b and Its Role in Rhizoremediation

A plant growth-promoting bacterial (PGPB) strain SC2b was isolated from the rhizosphere of Sedum plumbizincicola grown in lead (Pb)/zinc (Zn) mine soils and characterized as Bacillus sp. based on (1) morphological and biochemical characteristics and (2) partial 16S ribosomal DNA sequencing analysis. Strain SC2b exhibited high levels of resistance to cadmium (Cd) (300 mg/L), Zn (730 mg/L), and Pb (1400 mg/L). This strain also showed various plant growth-promoting (PGP) features such as utilization of 1-aminocyclopropane-1-carboxylate, solubilization of phosphate, and production of indole-3-acetic acid and siderophore. The strain mobilized high concentration of heavy metals from soils and exhibited different biosorption capacity toward the tested metal ions. Strain SC2b was further assessed for PGP activity by phytagar assay with a model plant Brassica napus. Inoculation of SC2b increased the biomass and vigor index of B. napus. Considering such potential, a pot experiment was conducted to assess the effects of inoculating the metal-resistant PGPB SC2b on growth and uptake of Cd, Zn and Pb by S. plumbizincicola in metal-contaminated agricultural soils. Inoculation with SC2b elevated the shoot and root biomass and leaf chlorophyll content of S. plumbizincicola. Similarly, plants inoculated with SC2b demonstrated markedly higher Cd and Zn accumulation in the root and shoot system, indicating that SC2b enhanced Cd and Zn uptake by S. plumbizincicola through metal mobilization or plant-microbial mediated changes in chemical or biological soil properties. Data demonstrated that the PGPB Bacillus sp. SC2b might serve as a future biofertilizer and an effective metal mobilizing bioinoculant for rhizoremediation of metal polluted soils.

Determinants for the subcellular localization of the inner and outer spore coat hubs in Bacillus subtilis

Endospores, or spores for simplicity, are a highly resistant cell type produced by some bacterial species under adverse conditions. Two main protective layers contribute to the resilience of spores: the cortex, composed of peptidoglycan, and the outermost proteinaceous coat. In Bacillus subtilis, the coat comprises up to 80 different proteins, organized into four sublayers: the basement layer, the inner coat, the outer coat and the crust. These proteins are synthesized at different times during sporulation and deposited at the spore surface in multiple coordinated waves. Central to coat formation is a group of morphogenetic proteins that guide the assembly of the coat components. Targeting of the coat proteins to the surface of the developing spore is mainly controlled by the SpoIVA morphogenetic ATPase. In a second stage, the coat proteins fully encircle the spore, a process termed encasement that requires the morphogenetic protein SpoVID. Assembly of the inner coat requires SafA, whereas formation of the outer coat and the crust requires CotE. SafA interacts directly with the N terminus of SpoVID. (...)

Comparative study of bovine heart and bacillus subtilis cytochrome c oxidase vesicles and the influence of bulk pH on cytochrome c oxidase components

Studies on the steady state behavior of soluble cytochrome c oxidase are extensive. These studies have examined the influence of ionic strength and pH and may provide answers to questions such as the link between proton translocation and charge separation. The present study examined the influence of external bulk pH on ApH formation, biphasic kinetics, and steady state reduction of cytochromes c and a of cytochrome c oxidase in proteoliposomes. Bulk pH has an appreciable effect on ApH formation and steady state reduction levels of cytochromes c and 8. Bulk pH affected total Vmax and Km at the low affinity binding site of cytochrome c. This study also examined the influence of bovine serum albumin and free fatty acids on proton pumping activity in bovine heart proteoliposomes. Proton pumping activity decreased after treatment with BSA, and was subsequently reinstated after further treatment with FFA. Much study in the superfamily of haem/copper oxidases has recently been devoted to the bacterial oxidases. The present study has examined some protein composition characteristics and bioenergetic features of Bacillus subtilis cytochrome caa3 oxidase. Results provide evidence for the structural composition of the enzyme in relation to the covalently bound cytochrome c to the oxidas~. Bioenergetically, caa3 COV showed appreciable proton pumping activity. Steady state analysis of the caa3 COV showed significantly different cytochrome c and a reduction characteristics compared to the bovine enzyme.