Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P51532 (transcriptional activator)
 6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

AmpR, the transcriptional regulator for the Citrobacter freundii ampC beta-lactamase gene, was purified. The purified AmpR had DNA-binding activity, the same molecular mass (32 kDa) on sodium dodecyl sulphate/polyacrylamide gel electrophoresis as previously described, and N-terminal sequencing of the first 15 amino acids was in agreement with that predicted from the nucleotide sequence. Two mutants were isolated that abolish DNA-binding and beta-lactamase induction and which map in the amino- and carboxyl-terminal ends of AmpR, respectively. The mutation in the amino terminus (S35F) was located in a helix-turn-helix region showing high homology to other members of the LysR regulator family. Therefore this mutation may directly abolish the contact between AmpR and its operator sequence. It is suggested that the C-terminal mutation (Y264N) affects subunit interactions in AmpR. One constitutive mutant was isolated which mapped in the centre of the ampR gene. This G102E mutant leads to constitutive beta-lactamase expression in the absence of both beta-lactam inducer and ampG, a gene essential for induction in wild-type enterobacteria. Another mutant protein, D135Y, showed wild-type properties in an ampG+ and an ampG::kan background, but could, unlike wild-type AmpR, activate the ampC gene in an ampG1 mutant background. It is thought that ampG1 is a missense mutant. These two types of ampR mutants suggest that activation of ampC transcription is dependent on the conversion of AmpR into a transcriptional activator and that this activation may normally involve interactions with AmpG.
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PMID:Purification and mutant analysis of Citrobacter freundii AmpR, the regulator for chromosomal AmpC beta-lactamase. 194 5

Citrobacter freundii encodes an inducible chromosomal beta-lactamase. Induction requires the product of the ampR gene, which is transcribed in the opposite orientation from the ampC beta-lactamase gene. We show here that the AmpR protein acts as a transcriptional activator by binding to a DNA region immediately upstream of the ampC promoter. The DNase I footprint pattern was not affected by growth in the presence of beta-lactam inducer or by the use of extracts prepared from cells carrying the ampD2 allele leading to semiconstitutive production of beta-lactamase. It is suggested that activation of AmpR facilitates binding or open complex formation for RNA polymerase at the ampC promoter. The AmpR-binding site overlaps the ampR promoter, and beta-galactosidase activity was decreased from an ampR-lacZ transcriptional fusion when AmpR was expressed from a coresident plasmid, suggesting that ampR is autogenously controlled. The AmpR protein belongs to a family of highly homologous transcriptional activators that includes LysR, which regulates the E. coli lysine synthetase gene, and the NodD protein, which regulates expression of a number of genes involved in nodulation in Rhizobium. The lack of sequence homology to any known beta-lactam-binding protein suggests that AmpR does not bind directly to the beta-lactam inducer but interacts with a second messenger of unknown nature.
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PMID:Binding of the Citrobacter freundii AmpR regulator to a single DNA site provides both autoregulation and activation of the inducible ampC beta-lactamase gene. 278 68

OmpR and EnvZ, the protein products of the ompB locus, are regulatory components required for osmoexpression of outer membrane porin proteins, OmpF and OmpC, in Escherichia coli. EnvZ is considered to be an osmosensor which transmits signals across the membrane to OmpR, a transcriptional activator for ompF and ompC. We inserted the envZ gene into a high expression vector, pIN-III. Following cellular fractionation, EnvZ was found to be localized in the inner membrane. Sequence analysis revealed that the signal peptide-like N-terminal sequence was not removed from the purified EnvZ. A genetic approach using EnvZ/beta-lactamase fusion proteins was taken to determine the topology of EnvZ in the inner membrane. When beta-lactamase was fused after the N-terminal signal peptide-like sequence, ampicillin resistance, conferred by the beta-lactamase moiety of the fusion protein, was expressed. However, when beta-lactamase was fused after the second downstream apolar sequence, the cells showed very poor ampicillin resistance indicating that the enzyme was localized on the cytoplasmic side of the inner membrane. The results of this approach reveal that the hydrophilic region of EnvZ between the two apolar sequences is periplasmically localized and that the hydrophilic region downstream of the second apolar sequence is cytoplasmically directed. These results were confirmed by partial proteolysis of the fusion proteins in intact cells.
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PMID:Localization and membrane topology of EnvZ, a protein involved in osmoregulation of OmpF and OmpC in Escherichia coli. 282 92

Escherichia coli JRG582 is an ampD ampE deletion derivative of strain HfrH and accordingly it is derepressed for expression of the cloned inducible beta-lactamase gene of Citrobacter freundii, carried on plasmid pNU305. Following chemical mutagenesis of JRG582(pNU305) a cefotaxime sensitive mutant was isolated, CS51(pNU305), which produced low levels of beta-lactamase due to a mutation in the host chromosome. Two recombinant plasmids containing genomic DNA from E. coli HfrH, namely pUB5608 and pUB5611, were isolated as a consequence of their ability to restore the beta-lactam resistant phenotype to CS51(pNU305). This ability was due to direct transcriptional activation of the beta-lactamase gene, ampC, rather than complementation of the CS51 mutation. Transposon mutagenesis and subcloning showed that restoration of ampicillin resistance to CS51(pNU305) was the function of a single gene, which maps at 60.3 min on the E. coli chromosome. The gene encodes a 33 kDa protein with significant homology to members of the LysR family of bacterial activator proteins, in particular the AmpR protein from C. freundii. Homology is especially strong over the N-terminal region which includes the helix-turn-helix DNA-binding motif. This gene was shown to complement the gcvA1 mutation at 60.3 min on the E. coli chromosome, and the DNA sequence agrees exactly with the published sequence of gcvA which encodes the transcriptional activator of the inducible glycine cleavage enzyme system. It is suggested that GcvA can activate transcription of ampC by binding to the AmpR binding region upstream of ampC so as to mimic the activated state of AmpR and hence provides an example of cross-talk between DNA-binding proteins of different inducible enzyme systems.
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PMID:GcvA, a LysR-type transcriptional regulator protein, activates expression of the cloned Citrobacter freundii ampC beta-lactamase gene in Escherichia coli: cross-talk between DNA-binding proteins. 770 73

The AmpR transcriptional activator for the chromosomal ampC beta-lactamase gene of Citrobacter freundii was found to interact with an operator sequence located in the 5' half of the 38 bp region protected by AmpR in DNase I footprinting experiments. AmpR binding was associated with significant DNA bending of target DNA. A glycine to glutamic acid alteration at position 102 in AmpR converts AmpR into a transcriptional activator even in the absence of beta-lactam inducer. AmpRG102E interacted with the operator binding sequence and induced DNA bending. A glycine to lysine alteration at residue 102 completely abolished the ability of AmpR to transcriptionally affect the ampC promoter, i.e. to repress in the absence of beta-lactam inducer and induce in the presence of beta-lactam. Nevertheless, AmpRG102K could repress the oppositely orientated ampR promoter. AmpRG102K could also specifically interact with the operator but the resulting complex migrated faster in gel retardation experiments and no significant DNA bending was observed.
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PMID:Interactions of wild-type and mutant AmpR of Citrobacter freundii with target DNA. 796 33

OmpT and OmpU are pore-forming proteins of the outer membrane of Vibrio cholerae, a pathogen that colonizes the intestine and produces cholera. Expression of the ompU and ompT genes is under the regulation of ToxR, a transmembrane transcriptional activator that also controls expression of virulence factors. It was recently shown that bile stimulates the ToxR-mediated transcription of ompU and that ompU-expressing strains are more resistant to bile and anionic detergents than ompT-expressing cells. In order to further understand the role of the OmpT and OmpU porins in the ability of V. cholerae to survive and colonize the host intestine, we examined the outer membrane permeability of cells expressing only ompU or only ompT or both genes in the absence and in the presence of bile. By comparing various strains in terms of the rate of degradation of the beta-lactam antibiotic cephaloridine by the periplasmic beta-lactamase, we found that the permeation of the antibiotic through the outer membrane of OmpU-containing cells was slower than the permeation in OmpT-containing cells. In addition, the OmpU-mediated outer membrane permeability was not affected by external bile, while the OmpT-mediated antibiotic flux was reduced by bile in a concentration-dependent manner. Our results confirm that OmpT and OmpU provide a passageway for hydrophilic solutes through the outer membrane and demonstrate that bile might interfere with this traffic in OmpT-producing cells by functionally inhibiting the OmpT pore. The insensitivity of OmpU to bile may be due to its small pore size and may provide an explanation for the resistance of OmpU-producing cells to bile in vivo.
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PMID:Vibrio cholerae OmpU and OmpT porins are differentially affected by bile. 1174 72

beta-Lactam resistance in enteric bacteria is frequently caused by mutations in ampD encoding a cytosolic N-acetylmuramyl- l-alanine amidase. Such mutants are blocked in murein (peptidoglycan) recycling and accumulate cytoplasmic muropeptides that interact with the transcriptional activator ampR, which de-represses beta-lactamase expression. Salmonella enterica serovar Typhimurium, an extensively studied enteric pathogen, was used to show that mutations in ampD decreased the ability of S. typhimurium to enter a macrophage derived cell line and made the bacteria more potent as inducers of inducible nitric oxide synthase (iNOS), as compared with the wild-type. ampG mutants, defective in the transport of recycled muropeptides across the cytoplasmic membrane, behaved essentially as the wild-type in invasion assays and in activation of iNOS. As ampD mutants also have reduced in vivo fitness in a murine model, we suggest that the cytoplasmic accumulation of muropeptides affects the virulence of the ampD mutants.
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PMID:Components of the peptidoglycan-recycling pathway modulate invasion and intracellular survival of Salmonella enterica serovar Typhimurium. 1561 30

Little is known about the significance of insects as environmental reservoirs of antibiotic-resistant bacteria. We characterized the antibiotic resistome of the microbial community in gypsy moth larval midguts by applying functional metagenomics to cultured isolates. The minimum inhibitory concentrations of 12 antibiotics were determined for 44 cultured isolates, and antibiotic resistance genes were selected from metagenomic libraries derived from DNA extracted from a pool of the isolates. Six unique clones were identified. Two were highly resistant to penicillin-type beta-lactams, two were moderately resistant to erythromycin, and two were moderately resistant to a range of antibiotics, including erythromycin, carbenicillin, and chloramphenicol. Sequence analysis predicted that the active genes encoded efflux pumps, a transcriptional activator of efflux pump protein expression, and an extended-spectrum class A beta-lactamase. Insect guts are a reservoir of antibiotic resistance genes with the potential for dissemination.
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PMID:Resident microbiota of the gypsy moth midgut harbors antibiotic resistance determinants. 1926 80

This study describes the screening of a plant extract library for inhibitors of signal transduction pathways mediated by the cholecystokinin receptor, CCK1. CCK1 receptors are coupled to Galpha(q/11)-proteins, localized mainly in the gastrointestinal tract, and implicated in the regulation of various digestive functions. A primary screen was performed using a cell-based assay that used the beta-lactamase gene reporter controlled by the transcriptional activator NFAT. The assay was validated with the CCK1 receptor antagonist, lorglumide, and automated by the use of a liquid-handling robot MultiProbe II. Off-target hits were triaged by counterscreening against gene reporter cells activated by a combination of thapsigargin and phorbol ester. Purification of active compounds was guided by the beta-lactamase gene reporter and Ca2+ mobilization assays. Pure compounds were characterized by Ca2+ mobilization, radioligand binding, inositol-1 phosphate formation, and Eu-GTP binding assays. The selectivity of inhibition was tested against a panel of Galpha(q/11), Galpha(s), and Galpha(i/0)-coupled receptors. These studies led to the identification of a novel Galpha(q/11)-selective inhibitor.
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PMID:Screening a plant extract library for inhibitors of cholecystokinin receptor CCK1 pathways. 2046 Feb 49