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

Abstract

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

Construction of a Conditionally Asporogenous Bacillus thuringiensis Recombinant Strain Overproducing Cry Protein by Deletion of the leuB Gene

“…As shown in Figures 3A,B , comparing to Bt 4.0718, except that cry1Ac was increased, cry2Aa and cwlC in Bt Δ leuB were all significantly down-regulated at the transcriptional level in both culture conditions. CwlC is a cell wall hydrolase newly characterized, which is essential for Bt mother cell lysis ( Chen et al, 2018 ). Interestingly, consistent with the blocked mother cell lysis of Bt Δ leuB in LB medium, the relative mRNA level of cwlC was also decreased to an extreme low degree ( Figure 3B ).…”

“…The number of spores was determined by counting the heat-resistant (65°C for 40 min) CFU on plates (the unlysed cells of Bt Δ leuB group needed sonication disruption to release the spores prior to heat treatment). Sporulation efficiency was calculated as described previously ( Chen et al, 2018 ). To perform the relative heat-resistance assay quantitatively, 30 μl samples of each group were heated at 90°C for 15 min, and then serially diluted for CFU determination.…”

“…However, a drawback of the spo0A deletion recombinant Bt strains is that the expression of sporulation-dependent cry genes was seriously inhibited and must be located under the control of the sporulation-independent cry3A gene promoter. Recently, another study showed that deletion of the cell wall hydrolase gene cwlC in Bt strain completely blocked the mother cell lysis without impacting sporulation or ICPs production (Chen et al, 2018). These aforementioned studies indicate that the genetic engineering approach provides a promising strategy for developing new recombinant Bt insecticides with better environmental stability.…”

Evolution, purification, and characterization of RC0497: a peptidoglycan amidase from the prototypical spotted fever species Rickettsia conorii

“…It is likely that both these amidases are independently expressed and are subject to differential regulation depending on the phases of rickettsial growth, colonization, and survival in mammalian hosts or arthropod vectors. The Bacillus PG amidases CwlB, CwlC, and CwlD, although involved in the cleavage of the same amide bond, are expressed at different physiological conditions as demonstrated by the expression of CwlB, CwlC, and CwlD primarily during vegetative growth, mother cell lysis, and sporulation, respectively (Shida et al, 2001;Chen et al, 2018).…”

2‐Methylcitrate cycle: a well‐regulated controller of Bacillus sporulation

“…To determine the efficiency of sporulation, 1 ml of GYS culture samples was heated at 80°C for 10 min at indicated time points to test for heat‐resistant spore formation. The sporulation efficiency was defined as the ratio of the heat‐resistant spores at indicated time points to the total number of cells at early stationary phase (at 10 h in GYS medium) (Chen et al ., ).…”