A Distance-Weighted Interaction Map Reveals a Previously Uncharacterized Layer of the Bacillus subtilis Spore Coat

SummaryBacillus subtilis spores are encased in a protein assembly called the spore coat that is made up of at least 70 different proteins. Conventional electron microscopy shows the coat to be organized into two distinct layers. Because the coat is about as wide as the theoretical limit of light microscopy, quantitatively measuring the localization of individual coat proteins within the coat is challenging. We used fusions of coat proteins to green fluorescent protein to map genetic dependencies for coat assembly and to define three independent subnetworks of coat proteins. To complement the genetic data, we measured coat protein localization at subpixel resolution and integrated these two data sets to produce a distance-weighted genetic interaction map. Using these data, we predict that the coat comprises at least four spatially distinct layers, including a previously uncharacterized glycoprotein outermost layer that we name the spore crust. We found that crust assembly depends on proteins we predicted to localize to the crust. The crust may be conserved in all Bacillus spores and may play critical functions in the environment

Fabrication of Atomically Precise Nanopores in Hexagonal Boron Nitride

We demonstrate the fabrication of individual nanopores in hexagonal boron nitride (hBN) with atomically precise control of the pore size. Previous methods of pore production in other 2D materials create pores of irregular geometry with imprecise diameters. By taking advantage of the preferential growth of boron vacancies in hBN under electron beam irradiation, we are able to observe the pore growth via transmission electron microscopy, and terminate the process when the pore has reached its desired size. Careful control of beam conditions allows us to nucleate and grow individual triangular and hexagonal pores with diameters ranging from subnanometer to 6nm over a large area of suspended hBN using a conventional TEM. These nanopores could find application in molecular sensing, DNA sequencing, water desalination, and molecular separation. Furthermore, the chemical edge-groups along the hBN pores can be made entirely nitrogen terminated or faceted with boron-terminated edges, opening avenues for tailored functionalization and extending the applications of these hBN nanopores.Comment: 5 pages, 6 figure

Constructing Fluorogenic Bacillus Spores (F-Spores) via Hydrophobic Decoration of Coat Proteins

Background: Bacterial spores are protected by a coat consisting of about 60 different proteins assembled as a biochemically complex structure with intriguing morphological and mechanical properties. Historically, the coat has been considered a static structure providing rigidity and mainly acting as a sieve to exclude exogenous large toxic molecules, such as lytic enzymes. Over recent years, however, new information about the coat’s architecture and function have emerged from experiments using innovative tools such as automated scanning microscopy, and high resolution atomic force microscopy. Principal Findings: Using thin-section electron microscopy, we found that the coat of Bacillus spores has topologically specific proteins forming a layer that is identifiable because it spontaneously becomes decorated with hydrophobic fluorogenic probes from the milieu. Moreover, spores with decorated coat proteins (termed F-spores) have the unexpected attribute of responding to external germination signals by generating intense fluorescence. Fluorescence data from diverse experimental designs, including F-spores constructed from five different Bacilli species, indicated that the fluorogenic ability of F-spores is under control of a putative germination-dependent mechanism. Conclusions: This work uncovers a novel attribute of spore-coat proteins that we exploited to decorate a specific layer imparting germination-dependent fluorogenicity to F-spores. We expect that F-spores will provide a model system to gai

Definition of the σW regulon of Bacillus subtilis in the absence of stress

Bacteria employ extracytoplasmic function (ECF) sigma factors for their responses to environmental stresses. Despite intensive research, the molecular dissection of ECF sigma factor regulons has remained a major challenge due to overlaps in the ECF sigma factor-regulated genes and the stimuli that activate the different ECF sigma factors. Here we have employed tiling arrays to single out the ECF σW regulon of the Gram-positive bacterium Bacillus subtilis from the overlapping ECF σX, σY, and σM regulons. For this purpose, we profiled the transcriptome of a B. subtilis sigW mutant under non-stress conditions to select candidate genes that are strictly σW-regulated. Under these conditions, σW exhibits a basal level of activity. Subsequently, we verified the σW-dependency of candidate genes by comparing their transcript profiles to transcriptome data obtained with the parental B. subtilis strain 168 grown under 104 different conditions, including relevant stress conditions, such as salt shock. In addition, we investigated the transcriptomes of rasP or prsW mutant strains that lack the proteases involved in the degradation of the σW anti-sigma factor RsiW and subsequent activation of the σW-regulon. Taken together, our studies identify 89 genes as being strictly σW-regulated, including several genes for non-coding RNAs. The effects of rasP or prsW mutations on the expression of σW-dependent genes were relatively mild, which implies that σW-dependent transcription under non-stress conditions is not strictly related to RasP and PrsW. Lastly, we show that the pleiotropic phenotype of rasP mutant cells, which have defects in competence development, protein secretion and membrane protein production, is not mirrored in the transcript profile of these cells. This implies that RasP is not only important for transcriptional regulation via σW, but that this membrane protease also exerts other important post-transcriptional regulatory functions

Differentiation of Bacillus pumilus and Bacillus safensis using MALDI-TOF-MS

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) despite being increasingly used as a method for microbial identification, still present limitations in which concerns the differentiation of closely related species. Bacillus pumillus and Bacillus safensis, are species of biotechnological and pharmaceutical significance, difficult to differentiate by conventional methodologies. In this study, using a well-characterized collection of B. pumillus and B. safensis isolates, we demonstrated the suitability of MALDI-TOF-MS combined with chemometrics to accurately and rapidly identify them. Moreover, characteristic species-specific ion masses were tentatively assigned, using UniProtKB/Swiss-Prot and UniProtKB/TrEMBL databases and primary literature. Delineation of B. pumilus (ions at m/z 5271 and 6122) and B. safensis (ions at m/z 5288, 5568 and 6413) species were supported by a congruent characteristic protein pattern. Moreover, using a chemometric approach, the score plot created by partial least square discriminant analysis (PLSDA) of mass spectra demonstrated the presence of two individualized clusters, each one enclosing isolates belonging to a species-specific spectral group. The generated pool of species-specific proteins comprised mostly ribosomal and SASPs proteins. Therefore, in B. pumilus the specific ion at m/z 5271 was associated with a small acid-soluble spore protein (SASP O) or with 50S protein L35, whereas in B. safensis specific ions at m/z 5288 and 5568 were associated with SASP J and P, respectively, and an ion at m/z 6413 with 50S protein L32. Thus, the resulting unique protein profile combined with chemometric analysis, proved to be valuable tools for B. pumilus and B. safensis discrimination, allowing their reliable, reproducible and rapid identification.Dr. Kasthuri Venkateswaran, Dr. Irene Ouoba, Dr. Joseph W. Kloepper, Dr. Cecilie From and Dr. Maria Morea are gratefully acknowledged for providing isolates FO-36bT, SAFN-027, SAFN-037, KL-052, 51-3C and 82-2C; Bs31; SE 49 (AP3) and SE 52 (AP7); FEL 55, UNG22 and MIL46, respectively. Raquel Branquinho was supported by a PhD fellowship (Ref. SFRH/BD/61410/2009) and Clara Sousa by a post-doctoral fellowship (Ref. SFRH/BPD/70548/2010), from FCT (Fundacao para a Ciencia e Tecnologia, Portugal). Hugo Osorio acknowledges the funding from QREN-FEDER through the Operational Program ON. 2 - O Novo Norte. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by the Portuguese Foundation for Science and Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competin

YwdL in Bacillus cereus: Its Role in Germination and Exosporium Structure

In members of the Bacillus cereus group the outermost layer of the spore is the exosporium, which interacts with hosts and the environment. Efforts have been made to identify proteins of the exosporium but only a few have so far been characterised and their role in determining spore architecture and spore function is still poorly understood. We have characterised the exosporium protein, YwdL. ΔywdL spores have a more fragile exosporium, subject to damage on repeated freeze-thawing, although there is no evidence of altered resistance properties, and coats appear intact. Immunogold labelling and Western blotting with anti-YwdL antibodies identified YwdL to be located exclusively on the inner surface of the exosporium of B. cereus and B. thuringiensis. We conclude that YwdL is important for formation of a robust exosporium but is not required to maintain the crystalline assembly within the basal layer or for attachment of the hairy nap structure. ΔywdL spores are unable to germinate in response to CaDPA, and have altered germination properties, a phenotype that confirms the expected defect in localization of the cortex lytic enzyme CwlJ in the coat

Curing of Plasmid pXO1 from Bacillus anthracis Using Plasmid Incompatibility

The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures) can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome

Hypervirulent Clostridium difficile PCR-Ribotypes Exhibit Resistance to Widely Used Disinfectants

The increased prevalence of Clostridium difficile infection (CDI) has coincided with enhanced transmissibility and severity of disease, which is often linked to two distinct clonal lineages designated PCR-ribotype 027 and 017 responsible for CDI outbreaks in the USA, Europe and Asia. We assessed sporulation and susceptibility of three PCR-ribotypes; 012, 017 and 027 to four classes of disinfectants; chlorine releasing agents (CRAs), peroxygens, quaternary ammonium compounds (QAC) and biguanides. The 017 PCR-ribotype, showed the highest sporulation frequency under these test conditions. The oxidizing biocides and CRAs were the most efficacious in decontamination of C. difficile vegetative cells and spores, the efficacy of the CRAs were concentration dependent irrespective of PCR-ribotype. However, there were differences observed in the susceptibility of the PCR-ribotypes, independent of the concentrations tested for Virkon®, Newgenn®, Proceine 40® and Hibiscrub®. Whereas, for Steri7® and Biocleanse® the difference observed between the disinfectants were dependent on both PCR-ribotype and concentration. The oxidizing agent Perasafe® was consistently efficacious across all three PCR ribotypes at varying concentrations; with a consistent five Log10 reduction in spore titre. The PCR-ribotype and concentration dependent differences in the efficacy of the disinfectants in this study indicate that disinfectant choice is a factor for llimiting the survival and transmission of C. difficile spores in healthcare settings