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.

Aims: To determine the effect of sporulation temperature on Bacillus subtilis spore resistance and spore composition. Methods and Results: Bacillus subtilis spores prepared at temperatures from 22 to 48°C had identical amounts of dipicolinic acid and small, acid-soluble proteins but the core water content was lower in spores prepared at higher temperatures. As expected from this latter finding, spores prepared at higher temperatures were more resistant to wet heat than were spores prepared at lower temperatures. Spores prepared at higher temperatures were also more resistant to hydrogen peroxide, Betadine, formaldehyde, glutaraldehyde and a superoxidized water, Sterilox. However, spores prepared at high and low temperatures exhibited nearly identical resistance to u.v. radiation and dry heat. The cortex peptidoglycan in spores prepared at different temperatures showed very little difference in structure with only a small, albeit significant, increase in the percentage of muramic acid with a crosslink in spores prepared at higher temperatures. In contrast, there were readily detectable differences in the levels of coat proteins in spores prepared at different temperatures and the levels of at least one coat protein, CotA, fell significantly as the sporulation temperature increased. However, this latter change was not due to a reduction in cotA gene expression at higher temperatures. Conclusions: The temperature of sporulation affects a number of spore properties, including resistance to many different stress factors, and also results in significant alterations in the spore coat and cortex composition. Significance and Impact of the Study: The precise conditions for the formation of B. subtilis spores have a large effect on many spore properties.

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 ␦-lactam. Production of muramic ␦-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 ␦-lactam formation. Expression of CwlD in Escherichia coli results in muramoyl L-Alanine amidase activity but no muramic ␦-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 ␦-lactam. CwlD and PdaA are necessary and sufficient for muramic ␦-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.Bacterial endospores can maintain a dormant, highly resistant state for long periods and then, under favorable conditions, rapidly germinate to produce vegetative cells. Spore dormancy and heat resistance are dependent on the relative dehydration of the spore core (6, 24, 28). Spore core dehydration requires the integrity of a thick spore peptidoglycan wall, and peptidoglycan hydrolysis is required for rehydration and resumption of spore core metabolism during spore germination (27,31).The spore peptidoglycan is composed of two contiguous layers that are synthesized between the two membranes surrounding the developing forespore. The inner layer, the germ cell wall, makes up only 10 to 15% of the total spore peptidoglycan (23) and is apparently synthesized by proteins expressed on the surface of the inner forespore membrane (22, 35). The germ cell wall has a structure resembling the peptidoglycan of the vegetative cell wall (23), is maintained during spore germination to serve as the initial wall of the outgrowing spore (5), and appears to function as a template for proper synthesis of the outer spore peptidoglycan layers, the cortex (22). The cortex makes up Ͼ80% of the spore peptidoglycan (23), is synthesized by proteins present on the surface of the outer forespore membrane (10, 35), is rapidly degraded...

The peptidoglycan cortex of endospores of Bacillusspecies is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted byd,d-carboxypeptidases. We report here the construction of mutant B. subtilis strains lacking all combinations of two and three of the four apparentd,d-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains. The data indicate that while thedacA and dacC products have no significant role in spore peptidoglycan formation, the dacB anddacF products both function in regulating the degree of cross-linking of spore peptidoglycan. The spore peptidoglycan of adacB dacF double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance. A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.