Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae.
Bottom Line: Like MarA, RamA binds the promoter region, implying that it might be subject to autoregulation.Intriguingly, levels of romA and ramA expression were not uniformly affected by changes within the ramR gene, thereby supporting the dual promoter finding.Furthermore, a subset of strains sustained no changes within the ramR gene but which still overexpressed the romA-ramA genes, strongly suggesting that a secondary regulator may control ramA expression.
Affiliation: Centre for Infection and Immunity, 4th Floor, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK.Show MeSH
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Mentions: Bioinformatic analyses indicate the presence of a putative but conserved palindromic binding site for RamR within the pI region. However, no such site could be observed for the pII promoter. We also wondered whether RamA, like other similar regulators (i.e. MarA), autoregulates itself by binding to its own promoter region. The intergenic regions, designated pI and pII, bound both purified RamR and RamA proteins (Fig. 3A). The interactions of the pI and pII promoter regions with the RamA and RamR proteins were found to be specific, as no shifts were observed with BSA alone (Fig. 3A) and addition of cold promoter was able to reduce binding of the proteins to the labelled probe (data not shown). Of note, the fragment of DNA (pII*) that contained no transcriptional signals was found not to bind either RamA or RamR (Fig. 3B). As indicated previously, the pI promoter has a conserved RamR binding site that is required for RamR-mediated control. However, the lack of a similar or identical RamR binding site (by bioinformatic analyses) indicates that RamR-mediated regulation of the pII promoter may be mediated via a more degenerate palindromic binding site. Accordingly, bioinformatic analyses using the EMBOSS Pairwise Alignment Tool (http://www.ebi.ac.uk/Tools/emboss/align/) of the pII promoter region implies that there is a putative but degenerate palindromic site upstream of the ramA gene (Fig. 1A).
Affiliation: Centre for Infection and Immunity, 4th Floor, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK.