Spore Germination Mediated by Bacillus megaterium QM B1551 SleL and YpeB

Previous work demonstrated that Bacillus megaterium QM B1551 spores that are null for the sleB and cwlJ genes, which encode cortex-lytic enzymes (CLEs), either of which is required for efficient cortex hydrolysis in Bacillus spores, could germinate efficiently when complemented with a plasmid-borne copy of ypeB plus the nonlytic portion of sleB encoding the N-terminal domain of SleB (sleB^N). The current study demonstrates that the defective germination phenotype of B. megaterium sleB cwlJ spores can partially be restored when they are complemented with plasmid-borne ypeB alone. However, efficient germination in this genetic background requires the presence of sleL, which in this species was suggested previously to encode a nonlytic epimerase. Recombinant B. megaterium SleL showed little, or no, activity against purified spore sacculi, cortical fragments, or decoated spore substrates. However, analysis of muropeptides generated by the combined activities of recombinant SleB and SleL against spore sacculi revealed that B. megaterium SleL is actually an N-acetylglucosaminidase, albeit with apparent reduced activity compared to that of the homologous Bacillus cereus protein. Additionally, decoated spores were induced to release a significant proportion of dipicolinic acid (DPA) from the spore core when incubated with recombinant SleL plus YpeB, although optimal DPA release required the presence of endogenous CLEs. The physiological basis that underpins this newly identified dependency between SleL and YpeB is not clear, since pulldown assays indicated that the proteins do not interact physically in vitro.