Sekiguchi J, Akeo K, Yamamoto H, Khasanov F K, Alonso J C, Kuroda A
Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Nagano, Japan.
J Bacteriol. 1995 Oct;177(19):5582-9. doi: 10.1128/jb.177.19.5582-5589.1995.
DNA sequencing of a region upstream of the mms223 gene of Bacillus subtilis showed the presence of two open reading frames, orf1 and orf2, which may encode 18- and 27-kDa polypeptides, respectively. The predicted amino acid sequence of the latter shows high similarity to a major autolysin of B. subtilis, CwlB, with 35% identity over 191 residues, as well as to other autolysins (CwlC, CwlM, and AmiB). The gene was tentatively named cwlD. Bright spores produced by a B. subtilis mutant with an insertionally inactivated cwlD gene were committed to germination by the addition of L-alanine, and spore darkening, a slow and partial decrease in A580, and 72% dipicolinic acid release compared with that of the wild-type strain were observed. However, degradation of the cortex was completely blocked. Spore germination of the cwlD mutant measured by colony formation after heat treatment was less than 3.7 x 10(-8). The germination deficiency of the cwlD mutant was only partially removed when the spores were treated with lysozyme. Analysis of the chromosomal transcription of cwlD demonstrated that a transcript (RNA2) appearing 3 h after initiation of sporulation may have originated from an internal sigma E-dependent promoter of the cwlD operon, and a longer transcript (RNA1) appearing 4.5 h after sporulation may have originated from a sigma G-dependent promoter upstream of the orf1 gene. The cwlD mutant harboring a B. subtilis vector plasmid containing the intact cwlD gene recovered germination at a frequency 26% of the wild-type level.
对枯草芽孢杆菌mms223基因上游区域进行DNA测序，结果显示存在两个开放阅读框，即orf1和orf2，它们可能分别编码18 kDa和27 kDa的多肽。后者的预测氨基酸序列与枯草芽孢杆菌的一种主要自溶素CwlB高度相似，在191个残基上有35%的同一性，同时也与其他自溶素（CwlC、CwlM和AmiB）相似。该基因被暂命名为cwlD。一个cwlD基因插入失活的枯草芽孢杆菌突变体产生的明亮孢子，通过添加L-丙氨酸可启动萌发，与野生型菌株相比，观察到孢子变暗、A580缓慢且部分降低以及72%的吡啶二羧酸释放。然而，皮层的降解被完全阻断。通过热处理后菌落形成测定的cwlD突变体的孢子萌发率低于3.7×10⁻⁸。当用溶菌酶处理孢子时，cwlD突变体的萌发缺陷仅部分消除。对cwlD基因的染色体转录分析表明，在芽孢形成开始后3小时出现的转录本（RNA2）可能起源于cwlD操纵子的一个内部σE依赖性启动子，而在芽孢形成后4.5小时出现的较长转录本（RNA1）可能起源于orf1基因上游的一个σG依赖性启动子。携带含有完整cwlD基因的枯草芽孢杆菌载体质粒的cwlD突变体以野生型水平26%的频率恢复萌发。
