Affiliation:
1. National Food Biotechnology Centre and Department of Microbiology, University College Cork, Cork, Ireland
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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
24 articles.
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