Durmaz Evelyn, Madsen Søren M, Israelsen Hans, Klaenhammer Todd R
Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina 27695-7624, USA.
J Bacteriol. 2002 Dec;184(23):6532-44. doi: 10.1128/JB.184.23.6532-6543.2002.
Phages of the P335 group have recently emerged as important taxa among lactococcal phages that disrupt dairy fermentations. DNA sequencing has revealed extensive homologies between the lytic and temperate phages of this group. The P335 lytic phage phi31 encodes a genetic switch region of cI and cro homologs but lacks the phage attachment site and integrase necessary to establish lysogeny. When the putative cI repressor gene of phage phi31 was subcloned into the medium-copy-number vector pAK80, no superinfection immunity was conferred to the host, Lactococcus lactis subsp. lactis NCK203, indicating that the wild-type CI repressor was dysfunctional. Attempts to clone the full-length cI gene in Lactococcus in the high-copy-number shuttle vector pTRKH2 were unsuccessful. The single clone that was recovered harbored an ochre mutation in the cI gene after the first 128 amino acids of the predicted 180-amino-acid protein. In the presence of the truncated CI construct, pTRKH2::CI-per1, phage phi31 was inhibited to an efficiency of plaquing (EOP) of 10(-6) in NCK203. A pTRKH2 subclone which lacked the DNA downstream of the ochre mutation, pTRKH2::CI-per2, confirmed the phenotype and further reduced the phi31 EOP to <10(-7). Phage phi31 mutants, partially resistant to CI-per, were isolated and showed changes in two of three putative operator sites for CI and Cro binding. Both the wild-type and truncated CI proteins bound the two wild-type operators in gel mobility shift experiments, but the mutated operators were not bound by the truncated CI. Twelve of 16 lytic P335 group phages failed to form plaques on L. lactis harboring pTRKH2::CI-per2, while 4 phages formed plaques at normal efficiencies. Comparisons of amino acid and DNA level homologies with other lactococcal temperate phage repressors suggest that evolutionary events may have led to inactivation of the phi31 CI repressor. This study demonstrated that a number of different P335 phages, lytic for L. lactis NCK203, have a common operator region which can be targeted by a truncated derivative of a dysfunctional CI repressor.
P335 组噬菌体最近已成为破坏乳制品发酵的乳球菌噬菌体中的重要分类群。DNA 测序揭示了该组裂解性噬菌体和温和性噬菌体之间存在广泛的同源性。P335 裂解性噬菌体 phi31 编码 cI 和 cro 同源物的遗传开关区域，但缺乏建立溶原性所需的噬菌体附着位点和整合酶。当噬菌体 phi31 的推定 cI 阻遏基因被亚克隆到中拷贝数载体 pAK80 中时，宿主乳酸乳球菌乳亚种 NCK203 未获得超感染免疫，这表明野生型 CI 阻遏蛋白功能失调。尝试在高拷贝数穿梭载体 pTRKH2 中将全长 cI 基因克隆到乳球菌中未成功。回收的单个克隆在预测的 180 个氨基酸的蛋白质的前 128 个氨基酸之后的 cI 基因中存在赭石突变。在截短的 CI 构建体 pTRKH2::CI-per1 存在的情况下，噬菌体 phi31 在 NCK203 中的噬菌斑形成效率（EOP）被抑制到 10^(-6)。一个缺少赭石突变下游 DNA 的 pTRKH2 亚克隆 pTRKH2::CI-per2，证实了该表型并进一步将 phi31 的 EOP 降低到<10^(-7)。分离出对 CI-per 部分抗性的噬菌体 phi31 突变体，其在 CI 和 Cro 结合的三个推定操纵位点中的两个位点发生了变化。在凝胶迁移率变动实验中，野生型和截短的 CI 蛋白都能结合两个野生型操纵子，但截短的 CI 不结合突变的操纵子。16 种裂解性 P335 组噬菌体中有 12 种未能在携带 pTRKH2::CI-per2 的乳酸乳球菌上形成噬菌斑，而 4 种噬菌体以正常效率形成噬菌斑。与其他乳球菌温和性噬菌体阻遏蛋白的氨基酸和 DNA 水平同源性比较表明，进化事件可能导致了 phi31 CI 阻遏蛋白的失活。这项研究表明，许多对乳酸乳球菌 NCK203 具有裂解性的不同 P335 噬菌体具有一个共同的操纵子区域，该区域可被功能失调的 CI 阻遏蛋白的截短衍生物靶向。
