Restriction-modification (R-M) system Ecl18kI is representative of R-M systems whose coordinated transcription is achieved through a separate DNA-binding domain of the methyltransferase. M.Ecl18kI recognizes an operator sequence located in the noncoding region that separates the divergently transcribed R and M genes. Here we show that, contrary to previous predictions, the two ecl18kI promoters are not divergent, but actually face one another. The binding of M.Ecl18kI to its operator prevents RNA polymerase (RNAP) binding to the M promoter by steric exclusion, but has no direct effect on RNAP interaction with the R promoter. The start point for R transcription is located outside of the intergenic region, opposite the initiation codon of the M gene. Regulated transcription of the potentially toxic ecl18kI R gene is accomplished (i) at the stage of promoter complex formation, through direct competition from complexes formed at the M promoter, and (ii) at the stage of promoter clearance, since R promoter-bound RNAP escapes the promoter more slowly than RNAP bound to the M promoter.

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The capacity of pathogens to respond to environmental signals, such as iron concentration, is key to bacterial survival and establishment of a successful infection. Bacillus cereus is a widely distributed bacterium with distinct pathogenic properties. Hemolysin II (HlyII) is one of its pore-forming cytotoxins and has been shown to be involved in bacterial pathogenicity in a number of cell and animal models. Unlike many other B. cereus pathogenicity factors, HlyII is not regulated by pleiotropic transcriptional regulator PlcR but is controlled by its own regulator, HlyIIR. Using a combination of in vivo and in vitro techniques, we show that hlyII expression is also negatively regulated by iron by the global regulator Fur via direct interaction with the hlyII promoter. DNase I footprinting and in vitro transcription experiments indicate that Fur prevents RNA polymerase binding to the hlyII promoter. HlyII expression profiles demonstrate that both HlyIIR and Fur regulate HlyII expression in a concerted fashion, with the effect of Fur being maximal in the early stages of bacterial growth. In sum, these results show that Fur serves as a transcriptional repressor for hlyII expression.

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The interaction of DNA-methyltransferase Ecl18kI (M.Ecl18kI) with a fragment of promoter region of restriction-modification system SsoII was studied. It is shown that dissociation constants of M.Ecl18kI and M.SsoII complexes with DNA ligand carrying a regulatory site previously characterized for M.SsoII have comparable values. A deletion derivative of M.Ecl18kI, Delta(72-379)Ecl18kI, representing the N-terminal protein region responsible for regulation, was obtained. It is shown that such polypeptide fragment has virtually no interaction with the regulatory site. Therefore, the existence of a region responsible for methylation is necessary for maintaining M.Ecl18kI regulatory function. The properties of methyltransferase NlaX, which is actually a natural deletion derivative of M.Ecl18kI and M.SsoII lacking the first 70 amino acid residues and not being able to regulate gene expression of the SsoII restriction-modification system, were studied. The ability of mutant forms of M.Ecl18kI incorporating single substitutions in regions responsible for regulation and methylation to interact with both sites of DNA recognition was characterized. The data show a correlation between DNA-binding activity of two M.Ecl18kI regions-regulatory and methylating.