Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis

In this study, a sporulation-specific gene (tentatively namedcwlC ) involved in mother cell lysis inBacillus thuringiensis was characterized. The encoded CwlC protein consists of an N-terminalN -acetylmuramoyl-l -alanine amidase (MurN Ac-LAA) domain and a C-terminal amidase02 domain. The recombinant histidine-tagged CwlC proteins purified fromEscherichia coli were able to directly bind to and digest theB. thuringiensis cell wall. The CwlC point mutations at the two conserved glutamic acid residues (Glu-24 and Glu-140) shown to be critical for the catalytic activity in homologous amidases resulted in a complete loss of cell wall lytic activity, suggesting that CwlC is anN -acetylmuramoyl-l -alanine amidase. Results of transcriptional analyses indicated thatcwlC is transcribed as a monocistronic unit and that its expression is dependent on sporulation sigma factor K (σK ). Deletion ofcwlC completely blocked mother cell lysis during sporulation without impacting the sporulation frequency, Cry1Ac protein production, and insecticidal activity. Taken together, our data suggest that CwlC is an essential cell wall hydrolase forB. thuringiensis mother cell lysis during sporulation. EngineeredB. thuringiensis strains targetingcwlC , which allows the crystal inclusion to remain encapsulated in the mother cell at the end of sporulation, may have the potential to become more effective biological control agents in agricultural applications since the crystal inclusion remains encapsulated in the mother cell at the end of sporulation.IMPORTANCE Mother cell lysis has been well studied inBacillus subtilis , which involves three distinct yet functionally complementary cell wall hydrolases. In this study, a novel cell wall hydrolase, CwlC, was investigated and found to be essential for mother cell lysis inBacillus thuringiensis . CwlC ofB. thuringiensis only shows 9 and 21% sequence identity with knownB. subtilis mother cell hydrolases CwlB and CwlC, respectively, suggesting that mechanisms of mother cell lysis may differ betweenB. subtilis andB. thuringiensis . ThecwlC gene deletion completely blocked the release of spores and crystals from the mother cell without affecting insecticidal activity. This may provide a new effective strategy for crystal encapsulation against UV light inactivation.