Genomic Sequence of Bacteriophage ATCC 8074-B1 and Activity of Its Endolysin and Engineered Variants against Clostridium sporogenes
Genomic Sequence of Bacteriophage ATCC 8074-B1 and Activity of Its Endolysin and Engineered Variants against Clostridium sporogenes
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DOI：
10.1128/aem.07884-11
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发表时间：
2012-05-01
影响因子：
4.4
通讯作者：
Narbad, Arjan
中科院分区：
文献类型：
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作者：
Mayer, Melinda J.;Gasson, Michael J.;Narbad, Arjan
溶菌噬菌体ATCC 8074-B1在其宿主生孢梭菌上产生大噬斑。对47，595-bp基因组的测序允许鉴定82个推定的开放阅读框，包括编码头部和尾部形态发生和裂解的蛋白质的那些。然而，通常与溶原性相关的序列不存在。ORF 22编码内溶素CS 74 L，其显示与N-乙酰胞壁酰-L-丙氨酸酰胺酶的同源性，并且当在大肠杆菌中表达时，该蛋白质引起C.当外部添加时，产生孢子的细胞。CS 74 L对酪丁酸梭菌和丙酮丁醇梭菌也有活性。单独表达的催化结构域（CS 74 L（1-177））表现出与全长内溶素相似的活性和相同的宿主范围。产生了由CS 74 L催化结构域融合至内溶素CD 27 L的C末端结构域组成的嵌合内溶素，所述内溶素CD 27 L衍生自艰难梭菌噬菌体Phi CD 27。该嵌合体（CSCD）裂解C.具有与单独的催化结构域的活性相当的活性。相反，当针对C.酪丁酸菌添加CD 27 L C-末端结构域不能使溶素靶向艰难梭菌或其他CD 27 L敏感性细菌。
Lytic bacteriophage ATCC 8074-B1 produces large plaques on its host Clostridium sporogenes. Sequencing of the 47,595-bp genome allowed the identification of 82 putative open reading frames, including those encoding proteins for head and tail morphogenesis and lysis. However, sequences commonly associated with lysogeny were absent. ORF 22 encodes an endolysin, CS74L, that shows homology to N-acetylmuramoyl-L-alanine amidases, and when expressed in Escherichia coli, the protein causes effective lysis of C. sporogenes cells when added externally. CS74L was also active on Clostridium tyrobutyricum and Clostridium acetobutylicum. The catalytic domain expressed alone (CS74L(1-177)) exhibited a similar activity and the same host range as the full-length endolysin. A chimeric endolysin consisting of the CS74L catalytic domain fused to the C-terminal domain of endolysin CD27L, derived from Clostridium difficile bacteriophage Phi CD27, was produced. This chimera (CSCD) lysed C. sporogenes cells with an activity equivalent to that of the catalytic domain alone. In contrast, the CD27L C-terminal domain reduced the efficacy of the CS74L catalytic domain when tested against C. tyrobutyricum. The addition of the CD27L C-terminal domain did not enable the lysin to target C difficile or other CD27L-sensitive bacteria.