Genetic modifications to temperate Enterococcus faecalis phage Ef11 that abolish the establishment of lysogeny and sensitivity to repressor, and increase host range and productivity of lytic infection.
Genetic modifications to temperate Enterococcus faecalis phage Ef11 that abolish the establishment of lysogeny and sensitivity to repressor, and increase host range and productivity of lytic infection.
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DOI：
10.1099/mic.0.067116-0
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发表时间：
2013-06
期刊：
影响因子：
1.5
通讯作者：
Hongming Zhang;D. Fouts;Jessica DePew;R. Stevens
中科院分区：
文献类型：
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作者：
Hongming Zhang;D. Fouts;Jessica DePew;R. Stevens
Ef11是一种温带噬菌体，最初是从从感染的根管中回收的溶原性粪肠球菌菌株中通过诱导分离出来的，Ef11前体噬菌体在粪肠球菌菌株中广泛传播。由于粪肠球菌已经成为一种重要的机会人类病原体，我们有兴趣研究Ef11建立/维持溶原性的关键基因和调控序列，作为构建毒力强、高度溶解的噬菌体基因组的第一步，该噬菌体可用于治疗严重的粪肠球菌感染。Ef11在粪肠球菌JH2-2中的传代产生了一种变异体，在指示细胞的草坪上产生了大的、广泛传播的斑块，并提高了肉汤培养物中的噬菌体滴度。对产生大斑块的克隆病毒的遗传分析表明，该变异体是Ef11与粪肠球菌JH2-2染色体上有缺陷的Fl1C样噬菌体之间的重组。该重组体具有5个有缺陷的Fl1C样噬菌体ORF，而不是Ef11基因组的6个ORF。重组噬菌体基因组中假定的溶原性基因模块(ORF31-36)的缺失和可能的cro启动子被Nisin诱导的启动子替换，不会导致病毒感染性的丧失。融合了上述所有Ef11基因组修饰的遗传结构导致了一种不能溶原性和对阻遏物不敏感的变体的产生，使其具有毒力和高度裂解能力，并具有显著扩大的宿主范围。
Ef11 is a temperate bacteriophage originally isolated by induction from a lysogenic Enterococcus faecalis strain recovered from an infected root canal, and the Ef11 prophage is widely disseminated among strains of E. faecalis. Because E. faecalis has emerged as a significant opportunistic human pathogen, we were interested in examining the genes and regulatory sequences predicted to be critical in the establishment/maintenance of lysogeny by Ef11 as a first step in the construction of the genome of a virulent, highly lytic phage that could be used in treating serious E. faecalis infections. Passage of Ef11 in E. faecalis JH2-2 yielded a variant that produced large, extensively spreading plaques in lawns of indicator cells, and elevated phage titres in broth cultures. Genetic analysis of the cloned virus producing the large plaques revealed that the variant was a recombinant between Ef11 and a defective FL1C-like prophage located in the E. faecalis JH2-2 chromosome. The recombinant possessed five ORFs of the defective FL1C-like prophage in place of six ORFs of the Ef11 genome. Deletion of the putative lysogeny gene module (ORFs 31-36) and replacement of the putative cro promoter from the recombinant phage genome with a nisin-inducible promoter resulted in no loss of virus infectivity. The genetic construct incorporating all the aforementioned Ef11 genomic modifications resulted in the generation of a variant that was incapable of lysogeny and insensitive to repressor, rendering it virulent and highly lytic, with a notably extended host range.