Production of muramic delta-lactam in Bacillus subtilis spore peptidoglycan.

Bacterial spore heat resistance is primarily dependent upon dehydration of the spore cytoplasm, a state that is maintained by the spore peptidoglycan wall, the spore cortex. A peptidoglycan structural modification found uniquely in spores is the formation of muramic delta-lactam. Production of muramic delta-lactam in Bacillus subtilis requires removal of a peptide side chain from the N-acetylmuramic acid residue by a cwlD-encoded muramoyl-L-Alanine amidase. Expression of cwlD takes place in both the mother cell and forespore compartments of sporulating cells, though expression is expected to be required only in the mother cell, from which cortex synthesis derives. Expression of cwlD in the forespore is in a bicistronic message with the upstream gene ybaK. We show that ybaK plays no apparent role in spore peptidoglycan synthesis and that expression of cwlD in the forespore plays no significant role in spore peptidoglycan formation. Peptide cleavage by CwlD is apparently followed by deacetylation of muramic acid and lactam ring formation. The product of pdaA (yfjS), which encodes a putative deacetylase, has recently been shown to also be required for muramic delta-lactam formation. Expression of CwlD in Escherichia coli results in muramoyl L-Alanine amidase activity but no muramic delta-lactam formation. Expression of PdaA alone in E. coli had no effect on E. coli peptidoglycan structure, whereas expression of CwlD and PdaA together resulted in the formation of muramic delta-lactam. CwlD and PdaA are necessary and sufficient for muramic delta-lactam production, and no other B. subtilis gene product is required. PdaA probably carries out both deacetylation and lactam ring formation and requires the product of CwlD activity as a substrate.

Coordinate regulation of bacterial virulence genes by a novel adenylate cyclase-dependent signaling pathway.

Type III secretion systems (TTSSs) are utilized by numerous bacterial pathogens to inject effector proteins directly into host cells. Using a whole-genome microarray, we investigated the conditions and regulatory factors that control the expression of the Pseudomonas aeruginosa TTSS. The transcriptional response of known TTSS genes indicates a hierarchical pattern of expression in which a set of secretion apparatus and regulatory genes is constitutively expressed. Further analysis of genes coordinately regulated with those encoding the TTSS led to the identification of a signaling pathway that originates from a membrane-associated adenylate cyclase and controls TTSS gene expression. Transcriptome analysis of mutants lacking the ability to synthesize cAMP or the cAMP binding protein Vfr implicated this pathway in the global regulation of host-directed virulence determinants, including the TTSS.