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

Abstract

In this study, a sporulation-specific gene (tentatively named cwlC) involved in mother cell lysis in Bacillus thuringiensis was characterized. The encoded CwlC protein consists of an N-terminal N-acetylmuramoyl-l-alanine amidase (MurNAc-LAA) domain and a C-terminal amidase02 domain. The recombinant histidine-tagged CwlC proteins purified from Escherichia coli were able to directly bind to and digest the B. 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 an N-acetylmuramoyl-l-alanine amidase. Results of transcriptional analyses indicated that cwlC is transcribed as a monocistronic unit and that its expression is dependent on sporulation sigma factor K (σ^K). Deletion of cwlC 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 for B. thuringiensis mother cell lysis during sporulation. Engineered B. thuringiensis strains targeting cwlC, 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 in Bacillus 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 in Bacillus thuringiensis CwlC of B. thuringiensis only shows 9 and 21% sequence identity with known B. subtilis mother cell hydrolases CwlB and CwlC, respectively, suggesting that mechanisms of mother cell lysis may differ between B. subtilis and B. thuringiensis The cwlC 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.

Keywords: Bacillus thuringiensis; CwlC; cell wall hydrolase; mother cell lysis.