Molecular Characterization of a Phage-Inducible Middle Promoter and Its Transcriptional Activator from the Lactococcal Bacteriophage f31†

An inducible middle promoter from the lactococcal bacteriophage f31 was isolated previously by shotgun cloning an 888-bp fragment (P15A10) upstream of the b-galactosidase (b-Gal) gene (lacZ.st) from Streptococcus thermophilus (D. J. O’Sullivan, S. A. Walker, S. G. West, and T. R. Klaenhammer, Bio/Technology 14:82–87, 1996). The promoter showed low levels of constitutive b-Gal activity which could be induced twoto threefold over baseline levels after phage infection. During this study, the fragment was subcloned and characterized to identify a smaller, tightly regulated promoter fragment which allowed no b-Gal activity until after phage infection. This fragment, defined within nucleotides 566 to 888 (P566–888; also called fragment 566–888), contained tandem, phage-inducible transcription start sites at nucleotides 703 and 744 (703/744 start sites). Consensus 210 regions were present upstream of both start sites, but no consensus 235 regions were identified for either start site. A transcriptional activator, encoded by an open reading frame (ORF2) upstream of the 703/744 start sites, was identified for P566–888. ORF2 activated P566–888 when provided in trans in Escherichia coli. In addition, when combined with pTRK391 (P15A10::lacZ.st) in Lactococcus lactis NCK203, an antisense ORF2 construct was able to retard induction of the phage-inducible promoter as measured by b-Gal activity levels. Finally, gel shift assays showed that ORF2 was able to bind to promoter fragment 566–888. Deletion analysis of the region upstream from the tandem promoters identified a possible binding site for transcriptional activation of the phage promoters. The DNA-binding ability of ORF2 was eliminated upon deletion of part of this region, which lies centered approximately 35 bp upstream of start site 703. Deletion analysis and mutagenesis studies also elucidated a critical region downstream of the 703/744 start sites, where mutagenesis resulted in a twoto threefold increase in b-Gal activity. With these improvements, the level of expression achieved by an explosive-expression strategy was elevated from 3,000 to 11,000 b-Gal units within 120 min after induction.