Characterization of the mbl determinant and cloning of the spoIIID gene from Bacillus cereus ATCC 10987

To examine the transcriptional start site of the putative Bacillus cereus mbl gene, we have cloned and sequenced the upstream region of the previously reported B. cereus mbl determinant, which showed sequence similarity to the mreB morphogene from Escherichia coli. Primer extension analysis revealed the transcriptional start site of mbl to 259 bp upstream of the putative translational start site and showed that the mbl gene was expressed at 3, 6, and 10 h of growth after inoculation. Antibodies against B. cereus Mbl showed that even though mbl mRNA was present in amounts detectable with Northern blot analysis exclusively in the early logarithmic growth phase, the amount of protein was constant during the cell cycle. Immunogold labeling cryo-transmission electron microscopy indicates that B. cereus Mbl is a cytoplasmic protein. The upstream sequence of mbl revealed two open reading frames, spoIIID and orf1. The amino acid sequence of B. cereus SpoIIID is identical to the SpoIIID sequence from Bacillus thuringiensis. Primer extension showed that mbl is not cotranscribed with spoIIID.