Complete spore-cortex hydrolysis during germination of Bacillus subtilis 168 requires SleB and YpeB

The role of the sleB gene of Bacillus subtilis, which encodes a putative sp ore-cortex-lytic enzyme, and the downstream ypeB gene were investigated. Bo th SleB and YpeB were required for normal germination to occur. The corresp onding mutants formed phase-bright, heal-resistant spores with no apparent defects in dormancy. However, mutant spore suspensions lost optical density slower than the wild-type and spores were phase-grey even 12 h after the t riggering of germination. Since the loss of heat resistance and release of dipicolinic acid was similar to the wild-type, these mutants were blocked i n the later stages of germination. The mutants were nevertheless capable of outgrowth on rich agar to form colonies, indicating that other spore compo nents can compensate for their function sufficiently to allow outgrowth. Th e expression and regulation of the operon was examined using a lacZ transcr iptional fusion. Expression of the operon began 2 h after the onset of spor ulation and was under the control of RNA polymerase containing the forespor e-specific sigma factor, sigma(G). The application of reverse phase HPLC re vealed that the mutants do not have any structural defect in the dormant sp ore cortex and therefore these genes are not required for normal spore-cort ex synthesis. The analysis of peptidoglycan dynamics during germination sho wed, however, that the cortex was only partially hydrolysed in both mutants . This analysis also revealed that the likely hydrolytic bond specificity o f SleB is likely to be that of a lytic transglycosylase.