The identification and characterization of genes encoding penicillin-binding proteins (PBPs) in Bacillus subtilis: Vegetative growth and spore outgrowth requirements of strains lacking high-molecular weight PBPs

Bacillus subtilis is a Gram-positive bacterium which sporulates when deprived of essential nutrients. Vegetative growth of B. subtilis involves the synthesis of a peptidoglycan (PG) sacculus which provides shape and resistance to changes in osmotic pressure, while during sporulation a thick PG spore cortex is generated which contributes to dormant spore heat resistance. The terminal steps of PG synthesis are performed by a group of enzymes known as penicillin-binding proteins (PBPs), which polymerize PG strands and regulate PG peptide side chain crosslinking. We have obtained peptide sequence from two of the major PBPs (PBP2a and PBP3) present during vegetative growth and identified the genes encoding these proteins. Transcriptional lacZ fusions were constructed to these genes to examine their expression during growth, sporulation, and spore outgrowth, and primer extension identified putative transcription start sites. Insertional mutagenesis of each pbp gene and examination of PBP profiles from these strains confirmed that we had identified the appropriate gene, after which the morphology and growth properties of these mutant strains were examined along with dormant spore properties. Strains lacking multiple PBPs were constructed and examined similarly. A putative PBP-encoding gene, dacC, identified by the B. subtilis genome sequencing project, was also studied in a similar fashion. While we were unable to identify DacC in B. subtilis, the recombinant protein was expressed in Escherichia coli and its penicillin-binding properties assayed. As spores lacking PBP2a were defective in outgrowth compared to wild type spores, we examined macromolecular synthesis and PG structure during spore outgrowth in a variety of PBP mutant strains. Finally, strains lacking the high molecular weight PBP1 were found to require increased levels of divalent cations for vegetative growth and spore outgrowth, and outgrowth of spores lacking PBP2a was affected by both divalent and monovalent cation levels in the medium. A strain lacking both PBP1 and PBP2a was severely compromised during spore outgrowth compared to either single mutant strain suggesting a redundancy of function between these two proteins.