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...-inducible tac promoter. The addition of MBP to the amino termini of other AraC/XylS family members has been shown to substantially enhance their solubility in vitro without perturbing their activity =-=(11, 15)-=-. We also constructed a second plasmid, pSFUM139, which expresses MBP-MxiE in cis with His 6-IpgC from tacp. The addition of a hexahistidyl epitope tag to IpgC has no effect on the activity of the pro...

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...nterchangeable with PerA (25). The DNA sequences bound by AraC-like proteins involved in the regulation of virulence factors have been characterized for only few members of the subfamily, such as Rns =-=(26)-=-, VirF of Yersinia spp. (54), UreR (47), ToxT (55), and, more recently, HilC and HilD (31). The two HTH motifs at the C terminus of these proteins seem to make contact with two adjacent major groove r...

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...in solubility was overcome by the addition of maltose binding protein (MBP) to the amino terminus of Rns, which significantly increases the solubility of the regulator without disrupting its function =-=(24)-=-. DNase I footprinting of MBP-Rns bound to the CS1 pilus promoter cooBp revealed two binding sites: one extending into the �35 hexamer and a second site between positions �93 and �129 (numbering relat...

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...vides seven rare tRNAs to supplement the rare codon usage of rns. We found that this strain produced higher yields of MBP-Rns fusion proteins than the previously described strain JM83/pEU750 produced =-=(19)-=-. KS1000/pRare2/pMBPRns1 was grown aerobically at 37°C in LB broth containing 0.2% (wt/vol) glucose, 30 �g/ml chloramphenicol, and 100 �g/ml ampicillin. After cells reached the mid-log phase, the cult...

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...s encoded on a virulence plasmid separate from the plasmid carrying the CS1 operon. Rns has two binding sites upstream of cooB, one adjacent to the -35 hexamer of CS1p and a more distal upstream site =-=[17]-=-. Mutagenesis of either binding site reduces Rns-dependent expression from CS1p in vivo. The CS1 fimbriae are more closely related, by phylogenetic analysis, to CS17 [GenBank:AY515609, GenBank:AY21649...

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... and 32P-end-labeled primer SN578 to image the noncoding strand. For the coding strand, SN577 was radiolabeled. The PCR products were purified on nondenaturing acrylamide gels as previously described =-=(34)-=-. CRP was equilibrated with radiolabeled DNA for 30 min at 37°C in 10 mM Tris-Cl (pH 7.6), 50 mM KCl, 1 mM dithiothreitol, 2 ng/l poly(dI-dC), 0.4 mM MgCl2, 0.2 mM CaCl2, 10 g/ml bovine serum albumi...

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...ate genes involved in stress response. These proteins include SoxS, Rob, and MarA, and they have been shown to function as monomers (38). A third group, including ToxT, Rns of enterotoxigenic E. coli =-=(45)-=-, BfpT (PerA) of enteropathogenic E. coli (66), and ExsA of Pseudomonas aeruginosa (29), regulate virulence gene expression. These virulence regulators may respond to physical cues, such as temperatur...

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...were used to amplify a 398 bp segment of the JRS4 chromosome including the ska promoter from 2308 to +91 bp (with respect to the start of ska transcription). The primers in the PCR were end-labelled (=-=Munson & Scott, 1999-=-) and nuclease protection assays were performed as described (Gusa & Scott, 2005). In vitro transcription assays. Transcription reactions were performed and analysed quantitatively as described by Gus...

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...ression (5). Rns binds upstream of the CSl pilin promoter, Pcoo, and the rns promoter, Prns, and at both promoters the upstream binding sites are required for full Rns-dependent transcription in vivo =-=(15, 16)-=-. Downstream of P rns there are additional Rns binding sites, one of which is also required for positive autoregulation (16; G. P. Munson, L. G. Holcomb, and J. R. Scott, unpublished data). Rns homolo...

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...RESULTS XylS specifically binds directs repeats in Pm sequence. XylS, like most members of the AraC family, aggregates when its concentration exceeds a threshold value, hindering biochemical analysis =-=(2, 28, 48, 50, 73)-=-. To overcome this problem, we devised a method to work with XylS-enriched extracts from which most DNA-binding proteins had been removed through heparin chromatography (see Materials and Methods). Br...