Work continued to understand the genetic regulation of expression of proteins secreted by Bacillus anthracis and Bacillus cereus that are potential virulence factors. Proteins of particular interest are hemolysins, toxins, and proteases. In B. cereus, expression and secretion of these are controlled by a global regulator, PlcR, which is part of a quorum sensing system that responds to peptides generated from a small protein, PapR. During the current reporting period of 2010, work focused on activation of the latent PlcR regulon in B. anthracis by expression of a PlcR/PapR fusion protein from B. cereus. We found that the activation leads to upregulation of many proteins corresponding to those found in the secretome of B. cereus, including phospholipases and proteases. Contrary to previous reports, activation of the regulon does not alter sporulation or toxin production in strains containing the virulence plasmid pXO1. Additionally, while transcription of major PlcR-dependent hemolysins, sphingomyelinase, and anthrolysin O, is enhanced in response to PlcR activation, in B. anthracis, only anthrolysin O contributes significantly to lysis of human erythrocytes. In contrast, toxicity of bacterial culture supernatants from a PlcR-positive strain to murine macrophages occurred independently of anthrolysin O expression in vitro and in vivo, pointing to the involvement of other toxins and lytic agents. During 2010 we also continued to delete additional secreted proteases from B. anthracis. Consequently, a set of multiple protease mutants were obtained having up to seven proteases inactivated in the same strain. This set of mutant strains is being used to define the contributions of the proteases to virulence. Work is now underway to develop these strains as hosts for the secretion and production of various recombinant proteins, which are expected to be more stable in the absence of the secreted proteases. We and others have often found that genetic manipulation of B. anthracis can lead to the accidental and unwanted selection of mutants that can no longer sporulate. This is a particular problem when the mutant strain being developed is to be tested for virulence in animals, because this is generally done by challenging with spores. By systematic examination of this process, we determined that it occurs more often in certain growth media and on solid medium. By sequencing the gene for the master regulator Spo0A in more than 50 independent spontaneous mutants, we determined that a large fraction of the mutants contained alterations in this gene. The amino acid substitutions that were detected were in residues known to play key roles in binding of this transcriptional regulator to its target DNA. Furthermore, deletions that occurred by recombination between repeated sequences provided insights into the mutational processes. For example, a region in which a 7-nucleotide sequence was duplicated was altered by either duplication or deletion of one of the sets, both of which led to a frameshift mutation. We predicted that the mutant having three tandem duplications of the 7-nucleotide sequence would revert at high frequency, and this was confirmed experimentally. Finally, we sought to identify a convenient method of repairing the unintended sporulation mutants, even when we did not know what specific mutation had occurred. This was achieved using generalized transduction with bacteriophage CP51, followed by selection for production of viable, heat-stable spores. Because of the strength of this selective procedure, we could reliably select sporulation competent transductants, thereby restoring the desired phenotype while retaining the intended mutations in other genes.
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