Durmaz E, Klaenhammer T R
Department of Food Science, Southeast Dairy Foods Research Center, College of Agriculture and Life Sciences, North Carolina State University, Raleigh, North Carolina 27695, USA.
Appl Environ Microbiol. 2000 Mar;66(3):895-903. doi: 10.1128/AEM.66.3.895-903.2000.
Recombinant phages are generated when Lactococcus lactis subsp. lactis harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species. These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations. They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of L. lactis chromosomal DNA. The objective of this study was to characterize four recombinant variants that arose from infection of L. lactis NCK203 (Abi(+)) with phage phi31. HindIII restriction maps of the variants (phi31.1, phi31.2, phi31.7, and phi31.8) were generated, and these maps revealed the regions containing recombinant DNA. The recombinant region of phage phi31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, phi31. This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and Escherichia coli Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes. A sequence analysis and phenotypic tests revealed a new origin of replication in the phi31.1 DNA, which replaced the phi31 origin. Three separate HindIII fragments, accounting for most of the recombinant region of phi31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203. Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages. The chromosomal insertions did not affect an inducible prophage present in NCK203. Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses. Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions.
当携带编码噬菌体抗性机制流产型（Abi）质粒的乳酸乳球菌乳亚种被属于P335种的小等轴噬菌体感染时，就会产生重组噬菌体。这些噬菌体变体很可能是乳制品发酵中出现的有毒新噬菌体的重要来源。它们与它们的祖先的区别在于对Abi防御的抗性以及基因组组织的改变，包括乳酸乳球菌染色体DNA区域。本研究的目的是表征由噬菌体phi31感染乳酸乳球菌NCK203（Abi(+)）产生的四种重组变体。生成了变体（phi31.1、phi31.2、phi31.7和phi31.8）的HindIII限制性图谱，这些图谱揭示了包含重组DNA的区域。对最常出现的变体噬菌体phi31.1的重组区域进行了测序，与亲本噬菌体phi31相比，发现了7.8 kb的新DNA。该区域包含与各种乳球菌温和噬菌体的大量同源实例，以及λ重组蛋白BET和大肠杆菌霍利迪连接点解离酶Rus的同源物，这些因子可能有助于高效的重组过程。序列分析和表型测试揭示了phi31.1 DNA中有一个新的复制起点，它取代了phi31的起点。占phi31.1重组区域大部分的三个独立的HindIII片段分别克隆到革兰氏阳性自杀载体pTRK333中，并转化到NCK203中。每个质粒的染色体插入阻止了不同组合的重组噬菌体的出现。染色体插入不影响NCK203中存在的可诱导原噬菌体。我们的结果表明，重组噬菌体可以从染色体的不同区域获取DNA盒，以克服Abi防御。通过插入破坏这些区域可以改变噬菌体-宿主相互作用期间出现的新噬菌体的类型和多样性。
