Characterization of CwlC, an autolysin, and its role in mother cell lysis of Bacillus thuringiensis subsp. israelensis

Bacillus thuringiensis subsp. israelensis (Bti) has been proven to efficiently control mosquitoes, of which many species are important vectors of human disease. The larvicidal action is attributed to the parasporal crystals formed in the sporulating cells and released upon cell autolysis. In this study, a sporulation-specific cwlC gene that encodes an N-acetylmuramoyl-L-alanine amidase was characterized in Bti strain Bt-59. CwlC was the only cell wall hydrolase in Bti found to contain both MurNAc-LAA and Amidase02_C domains. A recombinant CwlC-His protein was able to digest the Bacillus cell wall. Deletion of the cwlC gene delayed Bti mother cell lysis without impacting vegetative growth or insecticidal efficacy. Transcriptional analyses indicated that cwlC was expressed at the late sporulation stage and was controlled by SigK. Two other cell wall hydrolase genes, cwlB and cwlE, with high expression levels at T14 in Bt-59, were also identified. Like cwlC, cwlB expression was controlled by SigK; in contrast, cwlE was found not to be under the control of this sigma factor and unlike the other two, its gene was found to be plasmid encoded

Characterization of a novel cell wall hydrolase CwlE involved in Bacillus thuringiensis subsp. israelensis mother cell lysis

Cell wall hydrolases are ubiquitous among spore-form bacteria and essential for mother cell lysis. In this study, a novel cell wall hydrolase gene cwlE involved in mother cell lysis was characterized from Bacillus thuringiensis subsp. israelensis (Bti) strain Bt-59. cwlE was specifically expressed in Bti and located in the large plasmid carrying the insecticidal genes. The encoded CwlE protein consists of a MurNAc-LAA domain and two highly conserved catalytic residues (E26 and E151). The recombinant CwlE-His protein was able to digest the cell wall of Bti, indicating that CwlE is an N-acetylmuramoyl-L-alanine amidase. Transcriptional analysis indicated that cwlE began to express at the early stage of stationary phase and was controlled by SigE. Single mutation of cwlE gene delayed Bti mother cell lysis, while double mutation of cwlE and sigK completely blocked Bti mother cell lysis. After exposure to UV light to deactivate the crystal proteins, the level of decrease of insecticidal activity against mosquito larvae of Bt-59 (ΔcwlE-sigK) was less than that observed for Bt-59. This study elucidates the mechanism of Bti mother cell lysis and provides an effective strategy for mosquito control using Bt products with increased persistence.</p

Characterization of a novel cell wall hydrolase CwlE involved in Bacillus thuringiensis subsp. israelensis mother cell lysis

Cell wall hydrolases are ubiquitous among spore-form bacteria and essential for mother cell lysis. In this study, a novel cell wall hydrolase gene cwlE involved in mother cell lysis was characterized from Bacillus thuringiensis subsp. israelensis (Bti) strain Bt-59. cwlE was specifically expressed in Bti and located in the large plasmid carrying the insecticidal genes. The encoded CwlE protein consists of a MurNAc-LAA domain and two highly conserved catalytic residues (E26 and E151). The recombinant CwlE-His protein was able to digest the cell wall of Bti, indicating that CwlE is an N-acetylmuramoyl-L-alanine amidase. Transcriptional analysis indicated that cwlE began to express at the early stage of stationary phase and was controlled by SigE. Single mutation of cwlE gene delayed Bti mother cell lysis, while double mutation of cwlE and sigK completely blocked Bti mother cell lysis. After exposure to UV light to deactivate the crystal proteins, the level of decrease of insecticidal activity against mosquito larvae of Bt-59 (ΔcwlE-sigK) was less than that observed for Bt-59. This study elucidates the mechanism of Bti mother cell lysis and provides an effective strategy for mosquito control using Bt products with increased persistence.</p