CLONING, NUCLEOTIDE-SEQUENCE, AND MUTAGENESIS OF THE BACILLUS-SUBTILIS PONA OPERON, WHICH CODES FOR PENICILLIN-BINDING PROTEIN (PBP)-1 AND A PBP-RELATED FACTOR

An oligonucleotide probe designed to hybridize to genes encoding class A high-molecular-weight penicillin-binding proteins (PBPs) was used to identify the ponA gene encoding PBP1a and -1b (PBP1) of Bacillus subtilis. The identity of the ponA product was established by (i) the presence of a sequence coding for a peptide generated from PBP1 and (ii) the disappearance of PBP1 in a ponA mutant. DNA sequence analysis revealed that the amino acid sequence of PBP1 was similar to those of other class A high-molecular-weight PBPs and that ponA appeared to be cotranscribed with an upstream gene (termed prfA) of unknown function. Null mutations in ponA resulted in a slight decrease in growth rate and a change in colony morphology but had no significant effect on cell morphology, cell division, sporulation, spore heat resistance, or spore germination. Mutations in prfA which did not effect ponA expression produced a more significant decrease in growth rate but had no other significant phenotypic effects. Deletion of both prfA and ponA resulted in extremely slow growth and a reduction in sporulation efficiency. Studies of expression of transcriptional fusions of ponA and prfA to lacZ demonstrated that these two genes constitute an operon. Expression of these genes was relatively constant during growth, decreased during sporulation, and was induced approximately 15 min into spore germination. The ponA locus was mapped to the 200 degrees region of the chromosomal physical map.