State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
Department of Biology, Northeastern University, Boston, Massachusetts, USA.
Appl Environ Microbiol. 2018 Mar 19;84(7). doi: 10.1128/AEM.02640-17. Print 2018 Apr 1.
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.
在这项研究中，我们对 中参与母细胞裂解的孢子特异性基因（暂命名为 ）进行了研究。编码的 CwlC 蛋白由 N 端乙酰胞壁酰-L-丙氨酸酰胺酶（MurAc-LAA）结构域和 C 端酰胺酶 02 结构域组成。从 中纯化的重组组氨酸标记的 CwlC 蛋白能够直接结合并消化细胞壁。两个保守的谷氨酸残基（Glu-24 和 Glu-140）的 CwlC 点突变对同源酰胺酶的催化活性至关重要，导致细胞壁裂解活性完全丧失，表明 CwlC 是一种乙酰胞壁酰-L-丙氨酸酰胺酶。转录分析结果表明， 作为单顺反子转录单位转录，其表达依赖于孢子形成 σ 因子 K（σ）。 缺失完全阻断了孢子形成过程中的母细胞裂解，而不影响孢子形成频率、Cry1Ac 蛋白的产生和杀虫活性。综上所述，我们的数据表明 CwlC 是 孢子形成过程中母细胞裂解所必需的细胞壁水解酶。针对 的工程菌株可能具有成为更有效的农业应用生物防治剂的潜力，因为在孢子形成结束时，晶体包含物仍被包裹在母细胞中。 在 中，母细胞裂解已得到充分研究，涉及三种不同但功能互补的细胞壁水解酶。在这项研究中，研究了一种新型细胞壁水解酶 CwlC，发现它是 中母细胞裂解所必需的。 的 CwlC 与已知的 母细胞水解酶 CwlB 和 CwlC 分别只有 9%和 21%的序列同一性，表明母细胞裂解的机制可能在 和 之间有所不同。 基因缺失完全阻止了孢子和晶体从母细胞中释放，而不影响杀虫活性。这可能为防止晶体被紫外线灭活提供了一种新的有效晶体包裹策略。
