Transcriptional Analysis and Regulation of Expression of the Scr FI Restriction-Modification System of Lactococcus lactis subsp. cremoris UC503

Abstract

ABSTRACT Scr FI is a type II restriction-modification system from Lactococcus lactis which recognizes the nucleotide sequence 5′-CC↓ NGG-3′, cleaving at the point indicated by the arrow, and it comprises an endonuclease gene that is flanked on either side by genes encoding two 5-methylcytosine methylases. An open reading frame ( orfX ) of unknown function is located immediately upstream of these genes. In this study Northern analysis was performed, and it revealed that orfX, scrFIBM, and scrFIR are cotranscribed as a single polygenic mRNA molecule, while scrFIAM is transcribed independently. 5′ extension analysis indicated that the start site for the scrFIAM promoter was a thymine located 4 bp downstream of the −10 motif. The transcriptional start site for the orfX promoter was also found to be a thymine which is more atypically located 24 bp downstream of the −10 motif proximal to the start codon. A helix-turn-helix motif was identified at the N-terminal end of one of the methylases (M. Scr FIA). In order to determine if this motif played a role in regulation of the Scr FI locus, M. Scr FIA was purified. It was then employed in gel retardation assays using fragments containing the two promoters found on the Scr FI operon, one located upstream of orfX and the other located just upstream of scrFIAM . M. Scr FIA was found to bind to the promoter region upstream of the gene encoding it, indicating that it may have a regulatory role. In further studies the two putative promoters were introduced into a vector (pAK80) upstream of a promoterless lacZ gene, and cloned fragments of the Scr FI locus were introduced in trans with each of these promoter constructs to investigate the effect on promoter activity. These results implicated M. Scr FIA in regulation of both promoters on the Scr FI locus.