Sequencing and phylogenetic analysis of the spoIIA operon from diverse Bacillus and Paenibacillus species.

In order to clone the spoIIA operon from three different Bacillus and Paenibacillus species, we designed two sets of PCR primers based on three previously published Bacillus spoIIA sequences. One set of primers corresponded to the C-terminal region of SpoIIAB and a region near the middle of SpoIIAC. These primers were used to amplify the corresponding region of spoIIA from Bacillus stearothermophilus and Paenibacillus polymyxa (previously called Bacillus polymyxa [see Ash, C., Priest, F.G., Collins, M.D., 1993. Molecular identification of ribosomal-RNA group 3 bacilli using a PCR probe test - proposal for the creation of a new genus Paenibacillus. Antonie van Leeuwenhoek Int. J. Gen. Mol. Microbiol. 64, 253-260]. The other set of primers, corresponding to an N-terminal and a C-terminal region of SpoIIAC, was used for B. sphaericus. The PCR products were used as probes for Southern blotting of homologous chromosomal DNA. DNA corresponding to spoIIA from the three organisms was identified by screening chromosomal DNA libraries, and cloned. Sequence analysis showed that all spoIIA sequences were conserved, but conservation was strongest in SpoIIAC and least strong in SpoIIAA. In the promoter the -35 region was conserved well but the -10 region rather poorly. Within the proteins, certain regions were particularly strongly conserved, suggesting that they are essential to the function of the protein. Phylogenetic analysis of spoIIA suggested that B. stearothermophilus is close to B. subtilis and B. licheniformis, but that P. polymyxa and B. sphaericus are remote from B. subtilis.