DNA methylation at the CfrBI site is involved in expression control in the CfrBI restriction-modification system

Abstract

We have previously found that genes of the CFR:BI restriction-modification (R-M) system from Citrobacter freundii are oriented divergently and that their promoter regions overlap. The overlapping promoters suggest regulation of gene expression at the transcriptional level. In this study the transcription regulation of CFR:BI R-M genes was analyzed in vivo and in vitro in Escherichia coli. It was shown that in the presence of CFR:BI methyltransferase (M.CFR:BI), cell galactokinase activity decreases 10-fold when the galactokinase gene (galK) is under the control of the cfrBIM promoter and increases 20-fold when galK is under the control of the cfrBIR promoter. The CFR:BI site, proven to be unique for the entire CFR:BI R-M gene sequence, is located in the -35 cfrBIM promoter region and is in close vicinity of the -10 cfrBIR promoter region. A comparison of the cfrBIM and the cfrBIR promoter activities in the in vitro transcription system using methylated and unmethylated DNA fragments as templates demonstrated that the efficiency of CFR:BI R-M gene transcription is regulated by enzymatic modification at the N-4-position of cytosine bases of the CFR:BI site by M.CFR:BI. From the results of the in vivo and in vitro experiments we suggest a new model of gene expression regulation in type II R-M systems.

Figure 1

In vitro transcription from the cfrBIM and cfrBIR gene promoters. (A) The 7% PAGE autoradiogram of mRNAs produced by in vitro transcription from a 135 bp DNA fragment. In vitro transcription was performed as described (21) using E.coli RNA polymerase. The transcripts from the DNA fragment containing the cfrBIR gene promoter are presented in lanes 1 and 2 and those from the cfrBIM gene promoter in lanes 3 and 4. Transcription products in the absence of m⁴C DNA modification at the CfrBI site (lanes marked by a minus) and in the presence of MTase (lanes marked by a plus) were analyzed. The mRNA fragments resulting from transcription initiated by the cfrBIR and cfrBIM promoters were 80 nt (lane 2) and 79 nt (lane 3), respectively. Lane 5 represents DNA fragments of known length resulting from the sequencing reaction. (B) Schematic representation of the DNA fragment utilized for studies on in vitro transcription from the CfrBI R-M gene promoters. The 135 bp EcoRI–SnaBI DNA fragment was derived from plasmids pMet4 and pRes8. The EcoRI–BamHI (19 bp) and HindIII–SnaBI (76 bp) DNA fragments were from plasmid pFD51; the shaded BamHI–HindIII (40 bp) DNA fragment containing either the cfrBIM or cfrBIR promoter was constructed from pMet4 or pRes8, respectively.

Figure 2

A putative cruciform structure of the CfrBI-involving DNA sequence. The CfrBI site is shown in bold. *, cytosine undergoing m⁴-modification; shaded boxes, the –35 cfrBIM and –10 cfrBIR promoter regions. The PC/Gene (IntelliGenetics, Mountain View, CA) package of programs was used to analyze the nucleotide sequences.