Binding site recognition by Rns, a virulence regulator in the AraC family. (1/134)

The expression of CS1 pili by enterotoxigenic strains of Escherichia coli is regulated at the transcriptional level and requires the virulence regulator Rns, a member of the AraC family of regulatory proteins. Rns binds at two separate sites upstream of Pcoo (the promoter of CS1 pilin genes), which were identified in vitro with an MBP::Rns fusion protein in gel mobility and DNase I footprinting assays. At each site, Rns recognizes asymmetric nucleotide sequences in two regions of the major groove and binds along one face of the DNA helix. Both binding sites are required for activation of Pcoo in vivo, because mutagenesis of either site significantly reduced the level of expression from this promoter. Thus, Rns regulates the expression of CS1 pilin genes directly, not via a regulatory cascade. Analysis of Rns-nucleotide interactions at each site suggests that binding sites for Rns and related virulence regulators are not easily identified because they do not bind palindromic or repeated sequences. A strategy to identify asymmetric binding sites is presented and applied to locate potential binding sites upstream of other genes that Rns can activate, including those encoding the CS2 and CFA/I pili of enterotoxigenic E. coli and the global regulator virB of Shigella flexneri.  (+info)

InvF is required for expression of genes encoding proteins secreted by the SPI1 type III secretion apparatus in Salmonella typhimurium. (2/134)

The expression of genes encoding proteins secreted by the SPI1 (Salmonella pathogenicity island) type III secretion apparatus is known to require the transcriptional activators SirA and HilA. However, neither SirA nor HilA is believed to directly activate the promoters of these genes. invF, the first gene of the inv-spa gene cluster, is predicted to encode an AraC-type transcriptional activator and is required for invasion into cultured epithelial cells. However, the genes which are regulated by InvF have not been identified. In this work, an in-frame deletion in invF was constructed and tested for the expression of Phi(sigD-lacZYA), sipC::Tn5lacZY, and a plasmid-encoded Phi(sicA-lacZYA). SigD (Salmonella invasion gene) is a secreted protein required for the efficient invasion of Salmonella typhimurium into cultured eucaryotic cells. sicA (Salmonella invasion chaperone) is the first gene of a putative operon encoding the Sip/Ssp (Salmonella invasion/Salmonella secreted proteins) invasion proteins secreted by the SPI1 type III export apparatus. invF was required for the expression of the sigD, sicA, and sipC fusions. This is the first demonstration that there is a functional promoter in the intergenic sequence between spaS and sicA. In addition, several proteins were either absent from or found in reduced amounts in the culture supernatants of the invF mutant. Therefore, invF is required for the optimal expression of several genes encoding SPI1-secreted proteins. Genetic evidence is also presented suggesting there is HilA-dependent readthrough transcription from the invF promoter at least through sipC.  (+info)

Amino acid-DNA contacts by RhaS: an AraC family transcription activator. (3/134)

RhaS, an AraC family protein, activates rhaBAD transcription by binding to rhaI, a site consisting of two 17-bp inverted repeat half-sites. In this work, amino acids in RhaS that make base-specific contacts with rhaI were identified. Sequence similarity with AraC suggested that the first contacting motif of RhaS was a helix-turn-helix. Assays of rhaB-lacZ activation by alanine mutants within this potential motif indicated that residues 201, 202, 205, and 206 might contact rhaI. The second motif was identified based on the hypothesis that a region of especially high amino acid similarity between RhaS and RhaR (another AraC family member) might contact the nearly identical DNA sequences in one major groove of their half-sites. We first made targeted, random mutations and then made alanine substitutions within this region of RhaS. Our analysis identified residues 247, 248, 250, 252, 253, and 254 as potentially important for DNA binding. A genetic loss-of-contact approach was used to identify whether any of the RhaS amino acids in the first or second contacting motif make base-specific DNA contacts. In motif 1, we found that Arg202 and Arg206 both make specific contacts with bp -65 and -67 in rhaI1, and that Arg202 contacts -46 and Arg206 contacts -48 in rhaI2. In motif 2, we found that Asp250 and Asn252 both contact the bp -79 in rhaI1. Alignment with the recently crystallized MarA protein suggest that both RhaS motifs are likely helix-turn-helix DNA-binding motifs.  (+info)

The 17-gene ethanolamine (eut) operon of Salmonella typhimurium encodes five homologues of carboxysome shell proteins. (4/134)

The eut operon of Salmonella typhimurium encodes proteins involved in the cobalamin-dependent degradation of ethanolamine. Previous genetic analysis revealed six eut genes that are needed for aerobic use of ethanolamine; one (eutR), encodes a positive regulator which mediates induction of the operon by vitamin B12 plus ethanolamine. The DNA sequence of the eut operon included 17 genes, suggesting a more complex pathway than that revealed genetically. We have correlated an open reading frame in the sequence with each of the previously identified genes. Nonpolar insertion and deletion mutations made with the Tn10-derived transposable element T-POP showed that at least 10 of the 11 previously undetected eut genes have no Eut phenotype under the conditions tested. Of the dispensable eut genes, five encode apparent homologues of proteins that serve (in other organisms) as shell proteins of the carboxysome. This bacterial organelle, found in photosynthetic and sulfur-oxidizing bacteria, may contribute to CO2 fixation by concentrating CO2 and excluding oxygen. The presence of these homologues in the eut operon of Salmonella suggests that CO2 fixation may be a feature of ethanolamine catabolism in Salmonella.  (+info)

Functional domains of the TOL plasmid transcription factor XylS. (5/134)

The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.  (+info)

Cooperative action of the catabolite activator protein and AraC in vitro at the araFGH promoter. (6/134)

Full activation of transcription of the araFGH promoter, p(FGH), requires both the catabolite activator protein (CAP) and AraC protein. At p(FGH), the binding site for CAP is centered at position -41.5, an essential binding site for AraC is centered at position -79.5, and a second, nonessential binding site is centered at position -154.5. In this work, we used the minimal promoter region required for in vivo activation of p(FGH) to examine the roles of CAP and AraC in stimulating formation of open complexes at p(FGH). Migration retardation assays of open complexes showed that RNA polymerase binds exceptionally tightly to the AraC-CAP-p(FGH) complex and that the order of addition of proteins to the initiating complex is important. Similar assays with RNA polymerase containing truncated alpha subunits suggest that AraC interacts with the C-terminal domain of the alpha subunit. Finally, AraC protein also acts to prevent the improper binding of RNA polymerase at a pseudo promoter near the true p(FGH) promoter.  (+info)

Mutational analysis of the highly conserved C-terminal residues of the XylS protein, a member of the AraC family of transcriptional regulators. (7/134)

The XylS protein of the TOL plasmid of Pseudomonas putida belongs to the so-called AraC/XylS family of regulators, that includes more than 100 different bacterial proteins. A conserved stretch of about 100 amino acids is present at the C-terminal end. This conserved region is believed to contain seven alpha-helices, including two helix-turn-helix (HTH) DNA binding motifs (alpha(2)-T-alpha(3) and alpha(5)-Talpha-(6)), connected by a linker alpha-helix (alpha(4)), and two flanking alpha-helices (alpha(1) and alpha(7)). The second HTH motif is the region with the highest homology in the proteins of the family, with certain residues showing almost 90% identity. We have constructed XylS single mutants in the most conserved residues and have analysed their ability to stimulate transcription from its cognate promoter, Pm, fused to 'lacZ. The analysis revealed that mutations in the alpha(5)-helix conserved residues had little effect on the XylS transcriptional activity, whereas the distribution of polarity in the alpha(6)-helix was important for the activity. The strongest effect of the mutations was observed in conserved residues located outside the DNA binding domain, namely, Gly-290 in the turn between the two helices, Pro-309 located downstream of alpha(6), and Leu-313, in the small last helix alpha(7), that seems to play an important role in the activation of RNA-polymerase. Our analysis shows that conservation of amino acids in the family reflects structural requirements rather than functionality in specific DNA interactions.  (+info)

Recognition of overlapping nucleotides by AraC and the sigma subunit of RNA polymerase. (8/134)

The Escherichia coli promoter p(BAD), under the control of the AraC protein, drives the expression of mRNA encoding the AraB, AraA, and AraD gene products of the arabinose operon. The binding site of AraC at p(BAD) overlaps the RNA polymerase -35 recognition region by 4 bases, leaving 2 bases of the region not contacted by AraC. This overlap raises the question of whether AraC substitutes for the sigma subunit of RNA polymerase in recognition of the -35 region or whether both AraC and sigma make important contacts with the DNA in the -35 region. If sigma does not contact DNA near the -35 region, p(BAD) activity should be independent of the identity of the bases in the hexamer region that are not contacted by AraC. We have examined this issue in the p(BAD) promoter and in a second promoter where the AraC binding site overlaps the -35 region by only 2 bases. In both cases promoter activity is sensitive to changes in bases not contacted by AraC, showing that despite the overlap, sigma does read DNA in the -35 region. Since sigma and AraC are thus closely positioned at p(BAD), it is possible that AraC and sigma contact one another during transcription initiation. DNA migration retardation assays, however, showed that there exists only a slight degree of DNA binding cooperativity between AraC and sigma, thus suggesting either that the normal interactions between AraC and sigma are weak or that the presence of the entire RNA polymerase is necessary for significant interaction.  (+info)

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