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. vcopA was hypersensitive to copper and contained more copper atoms cell 31 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 copperrequiring cytochrome caa 3 oxidase was retained in vcopZ and vcopA. vcopZ was only slightly copper-hypersensitive but vcopZ/vcopA was more sensitive than vcopA, implying some action of CopZ that is independent of CopA. Significantly, vcopZ contained fewer copper atoms cell 31 than wild-type under these conditions. CopZ makes a net contribution to copper sequestration and/or recycling exceeding any donation to CopA for export.

SummaryBacteria 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.

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 zincmediated allostery in vitro and prevent zinc binding. Access to copper must be controlled in vivo to avoid formation of cupric CzrA.

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.

A new implicit time-stepping scheme which uses Runge-Kutta time-stepping and Krylov methods as a smoother inside FAS-cycle multigrid acceleration is proposed to stabilise the flow solver and its discrete adjoint counterpart. The algorithm can fully converge the discrete adjoint solver in a wide range of cases where conventional point-implicit methods fail due to either physical or numerical instability. This enables the discrete adjoint to be applied to a much wider range of flow regimes. In addition, the new algorithm offers improved efficiency when applied to stable cases for which the conventional Block-Jacobi solver can fully converge. Both stable and unstable cases are presented to demonstrate the improved robustness and performance of the new scheme. Eigen-analysis is presented to outline the mechanism of the adjoint stabilisation effect.