Type II restriction-modification (R-M) systems are composed of linked restriction endonuclease and modification methyltransferase genes and serve as barriers to horizontal gene transfer even though they are mobile in themselves. Their products kill host bacterial cells that have lost the R-M genes, a process that helps to maintain the frequency of the R-M systems in the viable cell population. Their establishment and maintenance in a bacterial host are expected to involve fine regulation of their gene expression. In the present study, we analyzed transcription of the modification gene and its regulation within the EcoRIR-M system. Northern blotting revealed that the downstream ecoRIM gene is transcribed as a monocistronic mRNA and as part of a larger bicistronic mRNA together with the upstream ecoRIR gene. Primer extension, RNase protection, and mutational analysis using lacZ gene fusions identified two overlapping promoters for ecoRIM gene transcription within the ecoRIR gene. Further mutational analysis revealed that two upstream AT-rich elements within the ecoRIR gene, "AATAAA" and "ATTATAAATATA," function as negative regulators of these promoters. Simultaneous substitution of these two elements resulted in a fourfold increase in beta-galactosidase activity and a five-fold increase in transcript levels as measured by RNase protection assay. RNA measurements of the ecoRIM transcript suggested that these elements decreased ecoRIM expression by interfering with transcription initiation of the ecoRIM promoters. Possible roles for these ecoRIM promoters and their negative regulators in the EcoRIR-M system are discussed. (c) 2007 Elsevier B.V. All rights reserved.