ABSTRACT
The DNA sequence of the replication module, part of the lysis module, and remnants of a lysogenic module from the lytic P335 species lactococcal bacteriophage φ31 was determined, and its regulatory elements were investigated. The identification of a characteristic genetic switch including two divergent promoters and two cognate repressor genes strongly indicates that φ31 was derived from a temperate bacteriophage. Regulation of the two early promoters was analyzed by primer extension and transcriptional promoter fusions to a lacLMreporter. The regulatory behavior of the promoter region differed significantly from the genetic responses of temperate Lactococcus lactis phages. Thecro gene homologue regulates its own production and is an efficient repressor of cI gene expression. No detectablecI gene expression could be measured in the presence ofcro. cI gene expression in the absence ofcro exerted minor influences on the regulation of the two promoters within the genetic switch. Homology comparisons revealed a replication module which is most likely expressed from the promoter located upstream of the cro gene homologue. The replication module encoded genes with strong homology to helicases and primases found in several Streptococcus thermophilus phages. Downstream of the primase homologue, an AT-rich noncoding origin region was identified. The characteristics and location of this region and its ability to reduce the efficiency of plaquing of φ31 106-fold when present at high copy number in trans provide evidence for identification of the phage origin of replication. Phage φ31 is an obligately lytic phage that was isolated from commercial dairy fermentation environments. Neither a phage attachment site nor an integrase gene, required to establish lysogeny, was identified, explaining its lytic lifestyle and suggesting its origin from a temperate phage ancestor. Several regions showing extensive DNA and protein homologies to different temperate phages ofLactococcus, Lactobacillus, andStreptococcus were also discovered, indicating the likely exchange of DNA cassettes through horizontal gene transfer in the dynamic ecological environment of dairy fermentations.
Molecular genetics in Streptococcus thermophilus: from transformation to gene expression
