Mur-LH, the broad-spectrum endolysin of Lactobacillus helveticus temperate bacteriophage φ-0303
Mur-LH, the broad-spectrum endolysin of Lactobacillus helveticus temperate bacteriophage φ-0303
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DOI：
10.1128/aem.70.1.96-103.2004
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发表时间：
2004-01-01
影响因子：
4.4
通讯作者：
Lortal, S
中科院分区：
文献类型：
--
作者：
Deutsch, SM;Guezenec, S;Lortal, S
PHI-0303是从瑞士乳杆菌CNRZ 303菌株经丝裂霉素C诱导后分离到的一株温和型噬菌体。在这项工作中，该噬菌体的裂解蛋白的编码基因被克隆到大肠杆菌DH5Alpha的Phi-0303文库。以敏感菌株L.helveticus CNRZ 892的全细胞为底物，通过其表达检测其裂解活性。溶素基因位于Phi-0303 4.1kb的DNA片段内，含有6个开放阅读框(ORF)和2个截短的ORF。在克隆的片段中没有发现与Holin基因同源的序列。在该片段中也存在一个整合酶编码基因，但它的转录方向与溶素基因相反。从噬菌体总DNA中扩增出裂解素基因，并进行亚克隆。Lys基因全长1,122个碱基，编码373个氨基酸的蛋白质(Mur-Lh)，推测其产物的相对分子质量为40,207 Da。与序列数据库中的序列比较显示与其他噬菌体的大量内溶素同源性。以瑞士乳杆菌CNRZ 892为底物，复性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定，重组蛋白的表观相对分子质量为40 kDa。该蛋白的N-末端序列证实了起始密码子。内溶酶对Helveticus CNRZ 303细胞壁的降解和残基的生化分析表明，Mur-Lh具有N-乙酰壁酰胺酶活性。最后，内溶素表现出广泛的裂解活性，因为它对不同的物种都有活性，主要是嗜热乳杆菌，但也有乳球菌、双球菌、枯草芽孢杆菌、亚麻短杆菌和粪肠球菌。
phi-0303 is a temperate bacteriophage isolated from Lactobacillus helveticus CNRZ 303 strain after mitomycin C induction. In this work, the gene coding for a lytic protein of this bacteriophage was cloned using a library of phi-0303 in Escherichia coli DH5alpha. The lytic activity was detected by its expression, using whole cells of the sensitive strain L. helveticus CNRZ 892 as the substrate. The lysin gene was within a 4.1-kb DNA fragment of phi-0303 containing six open reading frames (ORFs) and two truncated ORFs. No sequence homology with holin genes was found within the cloned fragment. An integrase-encoding gene was also present in the fragment, but it was transcribed in a direction opposite that of the lysin gene. The lysin-encoding lysin gene was verified by PCR amplification from the total phage DNA and subcloned. The lys gene is a 1,122-bp sequence encoding a protein of 373 amino acids (Mur-LH), whose product had a deduced molecular mass of 40,207 Da. Comparisons with sequences in sequence databases showed homology with numerous endolysins of other bacteriophages. Mur-LH was expressed in E. coli BL21, and by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with L. helveticus CNRZ 892 as the substrate, the recombinant protein showed an apparent molecular mass of 40 kDa. The N-terminal sequence of the protein confirmed the start codon. Hydrolysis of cell walls of L. helveticus CNRZ 303 by the endolysin and biochemical analysis of the residues produced demonstrated that Mur-LH has N-acetylmuramidase activity. Last, the endolysin exhibited a broad spectrum of lytic activity, as it was active on different species, mainly thermophilic lactobacilli but also lactococci, pediococci, Bacillus subtilis, Brevibacterium linens, and Enterococcus faecium.