Díaz E, López R, García J L
Unidad de Genética Bacteriana, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
J Bacteriol. 1992 Sep;174(17):5516-25. doi: 10.1128/jb.174.17.5516-5525.1992.
The first temperate bacteriophage (EJ-1) of Streptococcus pneumoniae with Myoviridae morphotype A1 isolated from a clinical atypical strain has been purified and characterized. This phage has a double-stranded linear genome about 42 kb long, but in contrast to the other pneumococcal temperate phages that have been characterized so far, EJ-1 does not contain any protein covalently linked to it. We have sequenced a fragment of EJ-1 DNA containing the ejl gene, encoding a cell wall lytic enzyme (EJL amidase). This gene has been cloned and expressed in Escherichia coli, and the EJL enzyme was purified and biochemically characterized as an N-acetylmuramyl-L-alanine amidase that shares many similarities with the major pneumococcal autolysin. The EJL amidase is a choline-dependent enzyme that needs the process of conversion to achieve full enzymatic activity, but in contrast to the wild-type pneumococcal LYTA amidase, this process was found to be reversible. Comparisons of the primary structure of this new lytic enzyme with that of the other cell wall lytic enzymes of S. pneumoniae and its bacteriophages characterized so far provided new insights as to the evolutionary relationships between phages and bacteria. The nucleotide sequences of the attachment site (attP) on the phage genome and one of the junctions created by the insertion of the prophage were determined. Interestingly, the attP site was located near the ejl gene, as previously observed for the pneumococcal temperate bacteriophage HB-3 (A. Romero, R. López, and P. García, J. Virol. 66:2860-2864, 1992). A stem-and-loop structure, some adjacent direct and inverted repeats, and two putative integration host factor-binding sites were found in the att sites.
从临床非典型菌株中分离出的第一株具有肌尾噬菌体科A1形态型的肺炎链球菌温和噬菌体（EJ-1）已被纯化和鉴定。该噬菌体具有约42 kb长的双链线性基因组，但与迄今已鉴定的其他肺炎链球菌温和噬菌体不同，EJ-1不含有任何与之共价连接的蛋白质。我们对包含ejl基因的EJ-1 DNA片段进行了测序，该基因编码一种细胞壁裂解酶（EJL酰胺酶）。该基因已在大肠杆菌中克隆并表达，EJL酶被纯化，并经生化鉴定为一种N-乙酰胞壁酰-L-丙氨酸酰胺酶，与主要的肺炎链球菌自溶素具有许多相似之处。EJL酰胺酶是一种胆碱依赖性酶，需要转化过程才能实现完全酶活性，但与野生型肺炎链球菌LYTA酰胺酶不同，该过程被发现是可逆的。将这种新的裂解酶的一级结构与迄今已鉴定的肺炎链球菌及其噬菌体的其他细胞壁裂解酶的一级结构进行比较，为噬菌体与细菌之间的进化关系提供了新的见解。确定了噬菌体基因组上附着位点（attP）和前噬菌体插入产生的一个连接点的核苷酸序列。有趣的是，attP位点位于ejl基因附近，如先前对肺炎链球菌温和噬菌体HB-3所观察到的那样（A. Romero、R. López和P. García，《病毒学杂志》66:2860 - 2864，1992年）。在att位点发现了一个茎环结构、一些相邻的正向和反向重复序列以及两个假定的整合宿主因子结合位点。
