HETEROGENEOUS ENDOLYSINS IN LISTERIA-MONOCYTOGENES BACTERIOPHAGES - A NEW CLASS OF ENZYMES AND EVIDENCE FOR CONSERVED HOLIN GENES WITHIN THE SIPHOVIRAL LYSIS CASSETTES
HETEROGENEOUS ENDOLYSINS IN LISTERIA-MONOCYTOGENES BACTERIOPHAGES - A NEW CLASS OF ENZYMES AND EVIDENCE FOR CONSERVED HOLIN GENES WITHIN THE SIPHOVIRAL LYSIS CASSETTES
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DOI：
10.1111/j.1365-2958.1995.tb02345.x
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发表时间：
1995-06-01
影响因子：
3.6
通讯作者：
SCHERER, S
中科院分区：
文献类型：
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作者：
LOESSNER, MJ;WENDLINGER, G;SCHERER, S
单核细胞增生李斯特菌噬菌体A118、A500和A511是具有特征宿主范围的三个不同噬菌体群的成员。它们的内溶素（ply）基因被克隆并在大肠杆菌中表达，如当重组细胞的菌落与李斯特菌细胞的菌苔覆盖时所赋予的裂解表型所证明的。测定克隆的DNA片段的核苷酸序列，并且显示各个酶（PLY 118，30.8 kDa; PLY 500，33.4 kDa; PLY 511，36.5 kDa）在其N-末端或C-末端结构域内具有不同程度的同源性。转录分析显示，它们是“晚期”基因，在感染后15-20分钟开始转录。酶被过表达和部分纯化，并检查其个体特异性。当外源施用时，溶素诱导所有物种的李斯特菌菌株的快速裂解，但通常不影响其他细菌。使用纯化的胞壁的水解，PLY 511被表征为N-乙酰胞壁酰-L-丙氨酸酰胺酶（EC 3.5.1.28），并且在其N-末端结构域中显示出与该类型的其它酶的同源性。相比之下，PLY 118和PLY 500被证明代表一类新的细胞壁裂解酶，其在β-肽聚糖的L-丙氨酸和D-谷氨酸残基之间裂解;这些被命名为L-丙氨酰-D-谷氨酸肽酶。这两种酶在N-末端结构域中具有同源性，我们建议这决定了水解特异性。高度保守的holin（hol）基因序列存在于p118和p1500的上游。它们编码与噬菌体λ基因S的产物结构相似的蛋白质，并且被预测为形成孔以允许溶素接近其肽聚糖底物的膜蛋白。在管蚜病毒裂解盒中保守的洞蛋白基因与下游溶素基因的这种排列解释了为什么单独的细胞质内溶素不是致命的，因为它们需要跨细胞膜的特异性运输功能。
Listeria monocytogenes bacteriophages A118, A500 and A511 are members of three distinct phage groups with characteristic host ranges. Their endolysin (ply) genes were cloned and expressed in Escherichia coli as demonstrated by the conferred lytic phenotype when colonies of recombinant cells were overlaid with a lawn of Listeria cells. The nucleotide sequences of the cloned DNA fragments were determined and the individual enzymes (PLY118, 30.8 kDa; PLY500, 33.4 kDa; PLY511, 36.5 kDa) were shown to have varying degrees of homology within their N-terminal or C-terminal domains. Transcriptional analysis revealed them to be 'late' genes with transcription beginning 15-20 min post-infection. The enzymes were overexpressed and partially purified and their individual specificities examined. When applied exogenously, the lysins induced rapid lysis of Listeria strains from all species but generally did not affect other bacteria. Using hydrolysis of purified listerial cell walls, PLY511 was characterized as an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) and shows homology in its N-terminal domain to other enzymes of this type. In contrast, PLY118 and PLY500 were shown to represent a new class of cell wall lytic enzymes which cleave between the L-alanine and D-glutamate residues of listerial peptidoglycan; these were designated as L-alanoyl-D-glutamate peptidases. These two enzymes share homology in the N-terminal domain which we propose determines hydrolytic specificity. Highly conserved holin (hol) gene sequences are present upstream of ply118 and ply500. They encode proteins of structural similarity to the product of phage lambda gene S, and are predicted to be membrane proteins which form pores to allow access of the lysins to their peptidoglycan substrates. This arrangement of conserved holin genes with downstream lysin genes among the siphoviral lysis cassettes explains why the cytoplasmic endolysins alone are not lethal, since they require a specific transport function across the cell membrane.