Cleavage of an engulfment peptidoglycan hydrolase by a sporulation signature protease in Clostridioides difficile.

In the model organism Bacillus subtilis, a signaling protease produced in the forespore, SpoIVB, is essential for the activation of the sigma factor σK, which is produced in the mother cell as an inactive pro-protein, pro-σK. SpoIVB has a second function essential to sporulation, most likely during cortex synthesis. The cortex is composed of peptidoglycan (PG) and is essential for the spore's heat resistance and dormancy. Surprisingly, the genome of the intestinal pathogen Clostridioides difficile, in which σK is produced without a pro-sequence, encodes two SpoIVB paralogs, SpoIVB1 and SpoIVB2. Here, we show that spoIVB1 is dispensable for sporulation, while a spoIVB2 in-frame deletion mutant fails to produce heat-resistant spores. The spoIVB2 mutant enters sporulation, undergoes asymmetric division, and completes engulfment of the forespore by the mother cell but fails to synthesize the spore cortex. We show that SpoIIP, a PG hydrolase and part of the engulfasome, the machinery essential for engulfment, is cleaved by SpoIVB2 into an inactive form. Within the engulfasome, the SpoIIP amidase activity generates the substrates for the SpoIID lytic transglycosylase. Thus, following engulfment completion, the cleavage and inactivation of SpoIIP by SpoIVB2 curtails the engulfasome hydrolytic activity, at a time when synthesis of the spore cortex peptidoglycan begins. SpoIVB2 is also required for normal late gene expression in the forespore by a currently unknown mechanism. Together, these observations suggest a role for SpoIVB2 in coordinating late morphological and gene expression events between the forespore and the mother cell.

Disruption of Two-component System LytSR Affects Forespore Engulfment in Bacillus thuringiensis.

Two-component regulatory systems (TCSs) play pivotal roles in bacteria sensing many different stimuli from environment. Here, we investigated the role of the LytSR TCS in spore formation in Bacillus thuringiensis (Bt) subsp. kurstaki HD73. lacZ gene fusions revealed that the transcription of the downstream genes, lrgAB, encoding two putative membrane-associated proteins, is regulated by LytSR. The sporulation efficiency of a lytSR mutant was significantly lower than that of wild-type HD73. A confocal microscopic analysis demonstrated that LytSR modulates the process of forespore engulfment. Moreover, the transcription of the lytSR operon is regulated by the mother-cell transcription factor SigE, whereas the transcription of the sporulation gene spoIIP was reduced in the lytSR mutant, as demonstrated with a ß-galactosidase activity assay. These results suggest that LytSR modulates forespore engulfment by affecting the transcription of the spoIIP gene in Bt.