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首页> 外文期刊>Journal of bacteriology >Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation.
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Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation.

机译：编码乳酸乳球菌的主要肽聚糖水解酶的基因的分子克隆和核苷酸序列，乳酸菌是细胞分离所需的村酰胺酶。

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A gene of Lactococcus lactis subsp. cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells. In cell extracts of L. lactis MG1363 and several halo-producing E. coli transformants, lytic bands of similar sizes were identified by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing L. lactis or M. lysodeikticus cell walls. Of these clearing bands, corresponding to the presence of lytic enzymes with sizes of 46 and 41 kDa, the 41-kDa band was also present in the supernatant of an L. lactis culture. Deletion analysis of one of the recombinant plasmids showed that the information specifying lytic activity was contained within a 2,428-bp EcoRV-Sau3A fragment. Sequencing of part of this fragment revealed a gene (acmA) that could encode a polypeptide of 437 amino acid residues. The calculated molecular mass of AcmA (46,564 Da) corresponded to that of one of the lytic activities detected. Presumably, the enzyme is synthesized as a precursor protein which is processed by cleavage after the Ala at position 57, thus producing a mature protein with a size of 40,264 Da, which would correspond to the size of the enzyme whose lytic activity was present in culture supernatants of L. lactis. The N-terminal region of the mature protein showed 60% identity with the N-terminal region of the mature muramidase-2 of Enterococcus hirae and the autolysin of Streptococcus faecalis. Like the latter two enzymes, AcmA contains C-terminal repeated regions. In AcmA, these three repeats are separated by nonhomologous intervening sequences highly enriched in serine, threonine, and asparagine. Genes specifying identical activities were detected in various strains of L. lactis subsp. lactis and L. lactis subsp. cremoris by the SDS-polyacrylamide gel electrophoresis detection assay and PCR experiments. By replacement recombination, an acmA deletion mutant which grew as long chains was constructed, indicating that AcmA is required for cell separation.
机译：乳酸乳球菌亚种的基因。通过在含有高压灭菌的冻干微球菌lysodeikticus细胞的培养基中筛选大肠杆菌转化株的细胞壁裂解活性，在pUC19中的菌株基因组文库中鉴定出编码肽聚糖水解酶的cremoris MG1363。在乳酸乳球菌MG1363和几种产生卤素的大肠杆菌转化子的细胞提取物中，通过变性十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶变性乳酸乳球菌或溶乳支原体，鉴定出大小相似的裂解带。在这些清除带中，对应于大小分别为46和41 kDa的裂解酶的存在，乳酸乳球菌培养物的上清液中也存在41 kDa的带。重组质粒之一的缺失分析表明，指定裂解活性的信息包含在2,428-bp的EcoRV-Sau3A片段内。该片段的部分测序揭示了一个基因（acmA），该基因可以编码437个氨基酸残基的多肽。计算得出的AcmA分子质量（46,564 Da）与检测到的一种裂解活性相对应。据推测，该酶是作为前体蛋白合成的，通过在57位的Ala裂解后进行加工，从而生成大小为40,264 Da的成熟蛋白，这与培养物中存在裂解活性的酶的大小相对应。乳酸乳杆菌的上清液。成熟蛋白的N末端区域与平肠肠球菌的成熟muramidase-2的N末端区域和粪链球菌的自溶素显示60％的同一性。与后两种酶一样，AcmA包含C末端重复区域。在AcmA中，这三个重复序列被高度富含丝氨酸，苏氨酸和天冬酰胺的非同源插入序列隔开。在各种乳酸乳球菌亚种中检测到指定相同活性的基因。乳酸和乳酸乳亚种。通过SDS-聚丙烯酰胺凝胶电泳检测assay蒲和PCR实验。通过替换重组，构建了随着长链而增长的acmA缺失突变体，表明AcmA是细胞分离所必需的。

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