, 2010), CpxR and OmpR (Jubelin et al., 2005), CpxR and H-NS (Ogasawara et al., 2010), and RstA and IHF (Ogasawara et al., 2010). As an extension, here we focused on the regulatory role of an as yet uncharacterized transcription factor, MlrA (MerR-like regulator; renamed from YehV), which was identified as a novel positive regulator for the synthesis of curli fimbriae and extracellular matrix in E. coli and Salmonella typhimurium (Brown et

al., 2001), although the mode of its action remains largely unidentified. For transcription initiation of the mlrA gene, not www.selleckchem.com/products/Roscovitine.html only is RpoD involved but so too is RpoS, and thus expression of the mlrA gene is induced in the stationary phase (Brown et al., 2001). MlrA contains a conserved N-terminal DNA-binding domain present in members of the MerR family, implying that MlrA is a DNA-binding regulatory protein. Selleck Ganetespib However, its C-terminal domain exhibits no similarity to any of the hitherto characterized MerR family members. In the present study, we found that MlrA is indeed a DNA-binding transcription factor, and is involved in activation of the csgD promoter by binding between the promoter-distal IHF-binding site in hot-spot I and the OmpR-binding site in hot-spot II for interaction with transcription factors. Interplay between three activators, MlrA, OmpR and IHF, was also analysed with respect to control of this complex promoter. Escherichia coli K-12 wild-type K-12 BW25113 and

mutant strains lacking the genes for transcription factors were the products of the Keio Collection, and were obtained from the E. coli stock centre of the National Institute of Genetics (see Supporting Information, Table S1). Escherichia coli BWcsgD and BWmlrA were KmS derivatives of JW1023 and JW2115, respectively, that were constructed using pCP20. Escherichia coli BL21(DE3) F-ompT hsd (rB− mB−) dcm galλ(DE3) was used for expression and purification of the transcription factors used in this study. Escherichia coli MC4100 was used for construction of the csgD–lacZ, csgB-lacZ and mlrA-lacZ reporter vectors. For construction of plasmids (pMlrA and pOmpR) for expression

of His-tagged MlrA and OmpR, DNA fragments corresponding to the coding sequences of their respective transcription factors were amplified by PCR using E. coli W3110 genome DNA as a template and pairs of primers, which were designed so (-)-p-Bromotetramisole Oxalate as to hybridize upstream or downstream of each coding sequence (Table S2). After digestion with NdeI and NotI (note that the restriction enzyme sites were included within the primer sequences), PCR products were cloned into pET21a(+) (Novagen) between NdeI and NotI sites. The plasmid constructs were confirmed by DNA sequencing. For protein expression, the plasmids were transformed into E. coli BL21 (DE3), and the transcription factors were overexpressed and purified as His-tagged forms (Igarashi & Ishihama, 1991; Yamamoto et al., 2005). In brief, E.