Boizet B, Lahbib-Mansais Y, Dupont L, Ritzenthaler P, Mata M
Centre de Recherche de Biochimie et de Génétique Cellulaires du CNRS, Toulouse, France.
Gene. 1990 Sep 28;94(1):61-7. doi: 10.1016/0378-1119(90)90468-7.
The lysA gene specifying an endolysin of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv1, was cloned and expressed in Escherichia coli. The 4.05-kb restriction fragment containing this gene was analysed by restriction and deletion mapping, and by subcloning. The nucleotide sequence of a 1150-bp fragment coding for an active lysin was determined. The lysA gene consists of 585 bp and codes for a protein of a deduced Mr of 21,120, which agrees with the size based on in vivo transcription/translation studies. The deduced amino acid sequence of the mv1 lysin (LysA) was compared to that of other known lytic enzymes. Significant homology was observed with the N-terminal portion of the muramidase of the fungus Chalaropsis and that of the muramidase of the Streptococcus pneumoniae phage Cp-1, suggesting that LysA might be a muramidase. In E. coli, the cloned lysA gene was able to complement the muramidase-defective bacteriophage lambda Ram5, proving that the products of these two genes are interchangeable. The lysA gene is preceded by an open reading frame with unknown function and no characteristic prokaryotic promoter sequences could be detected upstream from lysA, suggesting that this gene is part of an operon.
编码德氏乳杆菌保加利亚亚种噬菌体mv1内溶素的lysA基因在大肠杆菌中进行了克隆和表达。通过限制性酶切和缺失图谱分析以及亚克隆，对包含该基因的4.05 kb限制性片段进行了分析。测定了编码活性内溶素的1150 bp片段的核苷酸序列。lysA基因由585 bp组成，编码一个推导分子量为21120的蛋白质，这与基于体内转录/翻译研究得出的大小一致。将mv1内溶素（LysA）的推导氨基酸序列与其他已知裂解酶的序列进行了比较。观察到与拟青霉的溶菌酶N端部分以及肺炎链球菌噬菌体Cp-1的溶菌酶有显著同源性，表明LysA可能是一种溶菌酶。在大肠杆菌中，克隆的lysA基因能够互补溶菌酶缺陷型噬菌体λRam5，证明这两个基因的产物是可互换的。lysA基因之前有一个功能未知的开放阅读框，在lysA上游未检测到典型的原核启动子序列，这表明该基因是一个操纵子的一部分。
