No abstract

An 8,883-bp mini-pXO1 plasmid containing a replicon from Bacillus anthracis pXO1 (181.6 kb) was identified by making large deletions in the original plasmid using a newly developed Cre-loxP system. Portions of the truncated mini-pXO1 were cloned into an Escherichia coli vector unable to replicate in B. anthracis. A 5.95-kb region encompassing three putative genes was identified as the minimal pXO1 fragment required for replication of the resulting recombinant shuttle plasmid (named pMR) in B. anthracis. Deletion analysis showed that the only genes essential for replication were the pXO1-14 and pXO1-16 genes, which are transcribed in opposite directions and encode predicted proteins of 66.5 and 67.1 kDa, respectively. The ORF14 protein contains a helix-turn-helix motif, while the ORF16 upstream region contains attributes of a theta-replicating plasmid origin of replication (Ori), namely, an exclusively A؉T-containing segment, five 9-bp direct repeats, an inverted repeat, and a A -dependent promoter for the putative replication initiator Rep protein (ORF16). Spontaneous mutations generated in the ORF14, ORF16, and Ori regions of pMR during PCR amplification produced a temperature-sensitive plasmid that is unable to replicate in B. anthracis at 37°C. The efficacy of transformation of plasmid-free B. anthracis Ames and Sterne strains by the original pMR was ϳ10 3 CFU/g, while Bacillus cereus strains 569 and ATCC 10987 were transformed with efficiencies of 10 4 and 10 2 CFU/g, respectively. Around 95% of B. anthracis cells retained pMR after one round of sporulation and germination.

The global transcriptional regulator PlcR controls gene expression in Bacillus cereus and Bacillus thuringiensis. Activity of PlcR is regulated by PapR, the product of an open reading frame located immediately downstream of plcR. To be active in B. cereus, PapR must be secreted and then processed to the mature peptide by an unknown protease. This peptide is transported by an oligopeptide permease into the cell where it activates PlcR. In this study, we show that the neutral protease B (NprB) secreted by B. cereus 569 is required for extracellular PapR maturation. Purified recombinant NprB processed the synthetic PapR propeptide to produce a set of peptides derived from the C-terminal domain of PapR. Supplementation of growth media with synthetic PapR-derived C-terminal 5-, 7-, 8-, and 27-amino acid peptides caused activation of intracellular PlcR in a PapR-deficient strain of B. cereus 569 while only the 5- and 7-amino acid peptides activated PlcR in a nprB mutant. The maximum activity was found for the 7-mer peptide. However, even the 7-mer peptide could not activate PlcR with a C-terminal truncation of as few as six amino acids. This indicates that interactions of the C-terminal regions of both PlcR and PapR are important in transcriptional activation of the B. cereus 569 PlcR regulon.