Walker S A, Dombroski C S, Klaenhammer T R
Department of Food Science, North Carolina State University, Raleigh 27695-7624, USA.
Appl Environ Microbiol. 1998 Mar;64(3):1147-52. doi: 10.1128/AEM.64.3.1147-1152.1998.
A phage-inducible middle promoter (P15A10) from the lytic, lactococcal bacteriophage phi 31, a member of the P335 species, is located in an 888-base pair fragment near the right cohesive end. Sequence analysis revealed extensive homology (> 95%) to the right cohesive ends of two temperate phages of the P335 species, phi r1t and phi LC3. Sequencing upstream and downstream of P15A10 showed that the high degree of homology between phi 31 and phi r1t continued beyond the phage promoter. With the exception of one extra open reading frame in phi 31, the sequences were highly homologous (95 to 98%) between nucleotides 13,448 and 16,320 of the published phi r1t sequence. By use of a beta-galactosidase (beta-Gal) gene under the control of a smaller, more tightly regulated region within the P15A10 promoter, P566-888, it was established that mitomycin C induction of a lactococcal strain harboring the prophage phi r1t induced the P566-888 promoter, as determined from an increase in beta-Gal activity. Hybridization of nine other lactococcal strains with 32P-labeled P566-888 showed that the Lactococcus lactis strains C10, ML8, and NCK203 harbored sequences homologous to that of the phage-inducible promoter. Mitomycin C induced the resident prophages in all these strains and concurrently induced the P566-888 promoter, as determined from an increase in beta-Gal activity. DNA restriction analysis revealed that the prophages in C10, ML8, and NCK203 had identical restriction patterns which were different from that of phi r1t. In addition, DNA sequencing showed that the promoter elements in the three phages were identical to each other and to P566-888 from the lytic phage phi 31. These results point to a conserved mechanism in the regulation of gene expression between the lytic phage phi 31 and at least two temperate bacteriophages and provide further evidence for a link in the evolution of certain temperate phages and lytic phages.
来自裂解性乳酸菌噬菌体phi 31（P335物种的成员）的噬菌体诱导性中间启动子（P15A10）位于右侧粘性末端附近一个888碱基对的片段中。序列分析显示，它与P335物种的两个温和噬菌体phi r1t和phi LC3的右侧粘性末端具有广泛的同源性（> 95%）。对P15A10上下游进行测序表明，phi 31和phi r1t之间的高度同源性在噬菌体启动子之外仍持续存在。除了phi 31中有一个额外的开放阅读框外，已发表的phi r1t序列中13448至16320核苷酸之间的序列高度同源（95%至98%）。通过使用在P15A10启动子内一个较小、调控更严格的区域P566 - 888控制下的β - 半乳糖苷酶（β - Gal）基因，证实了携带原噬菌体phi r1t的乳酸菌株经丝裂霉素C诱导后，P566 - 888启动子被诱导，这是通过β - Gal活性的增加来确定的。用32P标记的P566 - 888与其他九个乳酸菌株杂交表明，乳酸乳球菌菌株C10、ML8和NCK203含有与噬菌体诱导性启动子同源的序列。丝裂霉素C诱导了所有这些菌株中的原噬菌体，并同时诱导了P566 - 888启动子，这是通过β - Gal活性的增加来确定的。DNA限制性分析表明，C10、ML8和NCK203中的原噬菌体具有相同的限制性图谱，与phi r1t的不同。此外，DNA测序表明，这三种噬菌体中的启动子元件彼此相同，且与裂解性噬菌体phi 31的P566 - 888相同。这些结果表明，在裂解性噬菌体phi 31和至少两种温和噬菌体之间的基因表达调控存在保守机制，并为某些温和噬菌体和裂解性噬菌体的进化联系提供了进一步的证据。
