Ramadurai Lakshmi, Lockwood Katherine J, Lockwood J, Nadakavukaren Mathew J, Jayaswal Radheshyam K
Microbiology (Reading). 1999 Apr;145 ( Pt 4):801-808. doi: 10.1099/13500872-145-4-801.
The authors previously reported the cloning of a lytic-enzyme-encoding gene, lytM, from an autolysis-defective mutant of Staphylococcus aureus. In the present work, the lytM gene was overexpressed in Escherichia coli and the product was purified to homogeneity by affinity chromatography and HPLC. Biochemical analysis of LytM-cleaved peptidoglycan fragments indicated that LytM is a glycylglycine endopeptidase. Immunoelectron microscopic studies with anti-LytM rabbit IgG showed that LytM is expressed during the early exponential phase and is overexpressed in an autolysis-defective mutant compared with the parent strain. Also, a uniform distribution of gold particles on the surface of actively growing bacterial cells indicates that LytM plays a role in cell growth. Northern blot analyses of lytM expression in two global regulatory mutants, agr and sar, showed that expression of lytM is increased about twofold in these mutants as compared with the parents. Protein homology searches revealed that LytM could be a member of the zinc protease family, as it contained a homologous 38-amino-acid motif, Tyr-X-His-X11-Val-X12/20-Gly-X5-6-His. Atomic absorption spectrometric analysis of LytM revealed the presence of 0.9 mol zinc (mol LytM)(-1).
作者之前报道了从金黄色葡萄球菌的自溶缺陷突变体中克隆出一个编码裂解酶的基因lytM。在本研究中，lytM基因在大肠杆菌中过表达，其产物通过亲和层析和高效液相色谱法纯化至均一。对LytM切割的肽聚糖片段进行生化分析表明，LytM是一种甘氨酰甘氨酸内肽酶。用抗LytM兔IgG进行的免疫电子显微镜研究表明，LytM在指数生长期早期表达，与亲本菌株相比，在自溶缺陷突变体中过表达。此外，在活跃生长的细菌细胞表面金颗粒的均匀分布表明LytM在细胞生长中起作用。对lytM在两个全局调控突变体agr和sar中的表达进行Northern印迹分析，结果显示与亲本相比，这些突变体中lytM的表达增加了约两倍。蛋白质同源性搜索显示，LytM可能是锌蛋白酶家族的一员，因为它含有一个同源的38个氨基酸的基序，即Tyr-X-His-X11-Val-X12/20-Gly-X5-6-His。对LytM进行原子吸收光谱分析发现，每摩尔LytM含有0.9摩尔锌。
