Molecular tiling on the surface of a bacterial spore - the exosporium of the Bacillus anthracis/cereus/thuringiensis group.

Bacteria of the genera Bacillus and Clostridium form highly resistant spores, which in the case of some pathogens act as the infectious agents. An exosporium forms the outermost layer of some spores; it plays roles in protection, adhesion, dissemination, host targeting in pathogens and germination control. The exosporium of the Bacillus cereus group, including the anthrax pathogen, contains a 2D-crystalline basal layer, overlaid by a hairy nap. BclA and related proteins form the hairy nap, and require ExsFA (BxpB) for their localization on the basal layer. Until now, the identity of the main structural protein components of the basal layer was unknown. We demonstrate here that ExsY forms one of the essential components. Through heterologous expression in Escherichia coli, we also demonstrate that ExsY can self-assemble into ordered 2D arrays that mimic the structure of the exosporium basal layer. Self-assembly is likely to play an important role in the construction of the exosporium. The ExsY array is stable to heat and chemical denaturants, forming a robust layer that would contribute to overall spore resistance. Our structural analysis also provides novel insight into the location of other molecular components anchored onto the exosporium, such as BclA and ExsFA.

Proteomic analysis of Bacillus subtilis strains engineered for improved production of heterologous proteins.

The use of bacterial systems for recombinant protein production has advantages of simplicity, time and cost over competing systems. However, widely used bacterial expression systems (e.g. Escherichia coli, Pseudomonas fluorescens) are not able to secrete soluble proteins directly into the culture medium. This limits yields and increases downstream processing time and costs. In contrast, Bacillus spp. secrete native enzymes directly into the culture medium at grams-per-litre quantities, although the yields of some recombinant proteins are severely limited. We have engineered the Bacillus subtilis genome to generate novel strains with precise deletions in the genes encoding ten extracytoplasmic proteases that affect recombinant protein secretion, which lack chromosomal antibiotic resistance genes. The deletion sites and presence of single nucleotide polymorphisms were confirmed by sequencing. The strains are stable and were used in industrial-scale fermenters for the production of the Bacillus anthracis vaccine protein, protective antigen, the productivity of which is extremely low in the unmodified strain. We also show that the deletion of so-called quality control proteases appears to influence cell-wall synthesis, resulting in the induction of the cell-wall stress regulon that encodes another quality control protease.

CopZ from Bacillus subtilis interacts in vivo with a copper exporting CPx-type ATPase CopA.

The structure of the hypothetical copper-metallochaperone CopZ from Bacillus subtilis and its predicted partner CopA have been studied but their respective contributions to copper export, -import, -sequestration and -supply are unknown. DeltacopA was hypersensitive to copper and contained more copper atoms cell(-1) than wild-type. Expression from the copA operator-promoter increased in elevated copper (not other metals), consistent with a role in copper export. A bacterial two-hybrid assay revealed in vivo interaction between CopZ and the N-terminal domain of CopA but not that of a related transporter, YvgW, involved in cadmium-resistance. Activity of copper-requiring cytochrome caa(3) oxidase was retained in deltacopZ and deltacopA. DeltacopZ was only slightly copper-hypersensitive but deltacopZ/deltacopA was more sensitive than deltacopA, implying some action of CopZ that is independent of CopA. Significantly, deltacopZ contained fewer copper atoms cell(-1) than wild-type under these conditions. CopZ makes a net contribution to copper sequestration and/or recycling exceeding any donation to CopA for export.

Recombinant Bacillus subtilis spores expressing MPT64 evaluated as a vaccine against tuberculosis in the murine model.

Recombinant Bacillus subtilis spores expressing a TB antigen, MPT64, were tested for their ability to protect mice against tuberculosis challenge. A chimeric gene consisting of the spore coat gene cotB fused to mpt64 was constructed, and expression of a stable CotB-MPT64 hybrid protein of the spore coat verified. Spores were evaluated as a live vaccine and also formaldehyde inactivated. Mice were given three doses of spores or alternatively used in a prime-boost regimen with BCG. The results showed that inactivated recombinant spores were able to reduce the bacterial burden in the lungs of mice to comparable levels to that of BCG. In the prime-boost regimen, both live and inactivated spores showed a reduction in bacterial load in comparison with BCG. ELISPOT and polyfunctional T-cell analysis were performed to examine cellular responses and showed that antigen-specific secretion of Th1 cytokines was stimulated after immunisation with inactive recombinant spores and BCG. In summary, recombinant spores can elicit Th1 responses, which are important for protection against TB disease.

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.

Oral delivery of a DNA vaccine against tuberculosis using operator-repressor titration in a Salmonella enterica vector.

Attenuated Salmonella enterica offers a vaccine delivery route that has the benefits of enhanced immunogenicity and oral delivery. The majority of immunization studies have been conducted to deliver recombinant proteins, expressed from a gene that is either chromosomally integrated or carried on a low- or medium-copy number plasmid. There are, however, an increasing number of reports demonstrating the delivery of DNA vaccines, but the high-copy number plasmids that are preferentially used for this application are unstable in Salmonella. Here, we use the Operator-Repressor Titration (ORT) plasmid maintenance system in Salmonella enterica serovar Typhimurium to deliver a high-copy number plasmid expressing the Mycobacterium tuberculosis gene mpt64 to mice. MPT64 expression was detected in phagocytes using immunofluorescence microscopy following Salmonella-mediated delivery of the DNA vaccine. The indicative CD8+ responses measured by antigen-specific IFN-γ were higher from the live bacterial vector than from injected plasmid DNA, and a reduction in the pulmonary bacterial load was seen following an aerogenic challenge. This illustrates the potential of live attenuated Salmonella as oral tuberculosis vaccine vectors.

Predicting metals sensed by ArsR-SmtB repressors: allosteric interference by a non-effector metal.

Many bacterial genomes encode multiple metal-sensing ArsR-SmtB transcriptional repressors. There is interest in understanding and predicting their metal specificities. Here we analyse two arsR-smtB genes, ydeT and yozA (now aseR and czrA) from Bacillus subtilis. Purified AseR and CzrA formed complexes in gel-retardation and fluorescence-anisotropy assays with fragments of promoters that were derepressed in DeltaaseR and DeltaczrA cells. Candidate (i) partly thiolate, alpha3-helix (for AseR) and (ii) tetrahedral, non-thiolate, alpha5-helix (for CzrA) metal binding sites were predicted then tested in vitro and/or in vivo. The precedents are for such sites to sense arsenite/antimonite (alpha3) and zinc (alpha5). This correlated with the respective metal inducers of AseR and CzrA repressed promoters in B. subtilis and matched the metals that impaired formation of protein-DNA complexes in vitro. The putative sensory sites of 1024 ArsR-SmtB homologues are reported. Although AseR did not sense zinc in vivo, it bound zinc in vitro exploiting alpha3 thiols, but AseR DNA binding was not impaired by zinc. If selectivity relies on discriminatory triggering of allostery not just selective metal binding, then tight non-effector metal complexes could theoretically inhibit metal sensing. AseR remained arsenite-sensitive in equimolar zinc, while CzrA remained zinc-sensitive in equimolar arsenite in vitro. However, cupric ions did not impair CzrA-DNA complex formation but did inhibit zinc-mediated allostery in vitro and prevent zinc binding. Access to copper must be controlled in vivo to avoid formation of cupric CzrA.