Transcriptional analysis and regulation of expression of the ScrFI restriction-modification system of Lactococcus lactis subsp. cremoris UC503

Abstract

ScrFI is a type II restriction-modification system from Lactococcus lactis which recognizes the nucleotide sequence 5'-CC downward arrow 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.ScrFIA). In order to determine if this motif played a role in regulation of the ScrFI locus, M.ScrFIA was purified. It was then employed in gel retardation assays using fragments containing the two promoters found on the ScrFI operon, one located upstream of orfX and the other located just upstream of scrFIAM. M.ScrFIA 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 ScrFI locus were introduced in trans with each of these promoter constructs to investigate the effect on promoter activity. These results implicated M.ScrFIA in regulation of both promoters on the ScrFI locus.

FIG. 1

Schematic representation of the molecular organization of the ScrFI R/M locus, indicating the position of the restriction endonuclease gene (scrFIR) flanked by two 5-methylcytosine methylase genes. An ORF of unknown function (orfX) and two ribosomal protein genes (rpmF and rpmG) are situated upstream of scrFIBM. Promoter sequences are indicated by P. (Modified from reference .)

FIG. 2

Northern analysis of the ScrFI locus using RNA from L. lactis UC503. The probes used were orfX (lane orfX), scrFIBM (lane BM), scrFIR (lane R), and scrFIAM (lane AM). The sizes of the transcripts are indicated on the left and right.

FIG. 3

(a) 5′ extension analysis of the promoter region upstream of scrFIAM of the ScrFI locus of L. lactis subsp. cremoris UC503. The arrow indicates the position of the extension product. (b) Sequence of an extended region between scrFIR and scrFIAM, showing the regulatory signals. The transcriptional start site is indicated by a solid triangle. RBS, ribosome-binding site.

FIG. 4

(a) 5′ extension analysis of the orfX promoter region of the ScrFI locus of L. lactis subsp. cremoris UC503. The arrow indicates the position of the extension product. A deviation from the correct sequence published by Twomey et al. (28) is indicated by the underlined base. (b) Sequence of an extended region upstream of orfX, showing the regulatory signals. The transitional start site is indicated by a solid triangle. A direct repeat upstream of the −35 hexamer is indicated by boldface type. RBS, ribosome-binding site.

FIG. 5

Gel retardation assay results, showing binding between the M.ScrFIA protein and the scrFIAM promoter region. A 0.3-ng portion of ³²P-labeled fragment harboring scrFIAM promoter DNA (300-bp region) was used as the probe. Lanes 1 to 7, 0, 0.1, 0.2, 0.5, 1.0, 2.0, and 4.0 pmol of M.ScrFIA, respectively; lane 8, negative control containing a ³²P-labeled 300-bp lactococcal DNA fragment from the origin of replication region of pCI372 plus 4 pmol of M.ScrFIA.

FIG. 6

Schematic diagram of the scrFIAM promoter region. The transcriptional signals of the promoter are indicated. A to G represent the ³²P-labeled PCR-generated fragments used in the gel retardation assays. The positions of these fragments as found in the GenBank sequence are indicated.