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

N-Acylhomoserine lactone (acyl-HSL)-mediated gene expression, also called autoinduction, is conserved among diverse gram-negative bacteria. In the paradigm Vibrio fischeri system, bioluminescence is autoinducible, and the lux operon requires the transcriptional activator LuxR and the acyl-HSL autoinducer for expression. The production of the acyl-HSL signal molecule is conferred by the luxI gene, and luxR encodes the transcriptional regulator. We show here that Erwinia stewartii, the etiological agent of Stewart's wilt of sweet corn, synthesizes an acyl-HSL. Mass spectral analysis identified the signal molecule as N-(-3-oxohexanoyl)-L-homoserine lactone, which is identical to the V. fischeri autoinducer. We have cloned and sequenced the gene that confers acyl-HSL biosynthesis, called esaI, and the linked gene, esaR, that encodes a gene regulator. The two genes are convergently transcribed and show an unusual overlap of 31 bp at their 3' ends. Sequence analysis indicates that EsaI and EsaR are homologs of LuxI and LuxR, respectively. EsaR can repress its own expression but seems not to regulate the expression of esaI. The untranslated 5' region of esaR contains an inverted repeat with similarity to the lux box-like elements located in the promoter regions of other gene systems regulated by autoinduction. However, unlike the other systems, in which the inverted repeats are located upstream of the -35 promoter elements, the esaR-associated repeat overlaps a putative -10 element. We mutagenized the esaI gene in E. stewartii by gene replacement. The mutant no longer produced detectable levels of the acyl-HSL signal, leading to a concomitant loss of extracellular polysaccharide capsule production and pathogenicity. Both phenotypes were restored by complementation with esal or by exogenous addition of the acyl-HSL.
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PMID:Capsular polysaccharide biosynthesis and pathogenicity in Erwinia stewartii require induction by an N-acylhomoserine lactone autoinducer. 766 77

The TraR and TraI proteins of Agrobacterium tumefaciens mediate cell-density-dependent expression of the Ti plasmid tra regulon. TraI synthesizes the autoinducer pheromone N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL), while TraR is an 3-oxo-C8-HSL-responsive transcriptional activator. We have compared the abilities of 3-oxo-C8-HSL and 32 related compounds to activate expression of a TraR-regulated promoter. In a strain that expresses wild-type levels of TraR, only 3-oxo-C8-HSL was strongly stimulatory, four compounds were detectably active only at high concentrations, and the remaining 28 compounds were inactive. Furthermore, many of these compounds were potent antagonists. In contrast, almost all of these compounds were stimulatory in a congenic strain that overexpresses TraR and no compound was a potent antagonist. We propose a model in which autoinducers enhance the affinity of TraR either for other TraR monomers or for DNA binding sites and that overexpression of TraR potentiates this interaction by mass action. Wild-type A. tumefaciens released a rather broad spectrum of autoinducers, including several that antagonize induction of a wild-type strain. However, under all conditions tested, 3-oxo-C8-HSL was more abundant than any other analog, indicating that other released autoinducers do not interfere with tra gene induction. We conclude that (i) in wild-type strains, only 3-oxo-C8-HSL significantly stimulates tra gene expression, while many autoinducer analogs are potent antagonists; (ii) TraR overexpression increases agonistic activity of autoinducer analogs, allowing sensitive biodetection of many autoinducers; and (iii) autoinducer stimulatory activity is potentiated by TraR overproduction, suggesting that autoinducers may shift an equilibrium between TraR monomers and dimers or oligomers. When autoinducer specificities of other quorum-sensing proteins are tested, care should be taken not to overexpress those proteins.
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PMID:Analogs of the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens. 976 71

The testicular isoform of hormone-sensitive lipase (HSLtes) is encoded by a testis-specific exon and 9 exons common to the testis and adipocyte isoforms. In mouse, HSLtes mRNA appeared during spermiogenesis in round spermatids. Two constructs containing 1.4 and 0.5 kilobase pairs (kb) of the human HSLtes gene 5'-flanking region cloned upstream of the chloramphenicol acetyltransferase gene were microinjected into mouse oocytes. Analyses of enzyme activity in male and female transgenic mice showed that 0.5 kb of the HSLtes promoter was sufficient to direct expression only in testis. Cell transfection experiments showed that CREMtau, a testis-specific transcriptional activator, does not transactivate the HSLtes promoter. Using gel retardation assays, four testis-specific binding regions (TSBR) were identified using testis and liver nuclear extracts. The testis-specific protein binding on TSBR4 was selectively competed by a probe containing a SRY/Sox protein DNA recognition site. Sox5 and Sox6 which are expressed in post-meiotic germ cells bound TSBR4. Mutation of the AACAAAG motif in TSBR4 abolished the binding. Moreover, binding of the high mobility group domain of Sox5 induced a bend within TSBR4. Together, our results showed that 0.5 kb of the human HSLtes promoter bind Sox proteins and contain cis-acting elements essential for the testis specificity of HSL.
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PMID:Testis expression of hormone-sensitive lipase is conferred by a specific promoter that contains four regions binding testicular nuclear proteins. 1009 10

The luminescence (lux) operon (luxICDABEG) of the symbiotic bacterium Vibrio fischeri is regulated by the transcriptional activator LuxR and two acyl-homoserine lactone (acyl-HSL) autoinducers (the luxI-dependent 3-oxo-hexanoyl-HSL [3-oxo-C6-HSL] and the ainS-dependent octanoyl-HSL [C8-HSL]) in a population density-responsive manner called quorum sensing. To identify quorum-sensing-regulated (QSR) proteins different from those encoded by lux genes, we examined the protein patterns of V. fischeri quorum-sensing mutants defective in luxI, ainS, and luxR by two-dimensional polyacrylamide gel electrophoresis. Five non-Lux QSR proteins, QsrP, RibB, AcfA, QsrV, and QSR 7, were identified; their production occurred preferentially at high population density, required both LuxR and 3-oxo-C6-HSL, and was inhibited by C8-HSL at low population density. The genes encoding two of the QSR proteins were characterized: qsrP directs cells to synthesize an apparently novel periplasmic protein, and ribB is a homolog of the Escherichia coli gene for 3,4-dihydroxy-2-butanone 4-phosphate synthase, a key enzyme for riboflavin synthesis. The qsrP and ribB promoter regions each contained a sequence similar to the lux operon lux box, a 20-bp region of dyad symmetry necessary for LuxR/3-oxo-C6-HSL-dependent activation of lux operon transcription. V. fischeri qsrP and ribB mutants exhibited no distinct phenotype in culture. However, a qsrP mutant, in competition with its parent strain, was less successful in colonizing Euprymna scolopes, the symbiotic host of V. fischeri. The newly identified QSR genes, together with the lux operon, define a LuxR/acyl-HSL-responsive quorum-sensing regulon in V. fischeri.
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PMID:LuxR- and acyl-homoserine-lactone-controlled non-lux genes define a quorum-sensing regulon in Vibrio fischeri. 1078 50

Luminescence in Vibrio fischeri is controlled by a population density-responsive regulatory mechanism called quorum sensing. Elements of the mechanism include: LuxI, an acyl-homoserine lactone (acyl-HSL) synthase that directs synthesis of the diffusible signal molecule, 3-oxo-hexanoyl-HSL (V. fischeri autoinducer-1, VAI-1); LuxR, a transcriptional activator protein necessary for response to VAI-1; GroEL, which is necessary for production of active LuxR; and AinS, an acyl-HSL synthase that catalyzes the synthesis of octanoyl-HSL (VAI-2). The population density-dependent accumulation of VAI-1 triggers induction of lux operon (luxICDABEG; genes for luminescence enzymes and for LuxI) transcription and luminescence by binding to LuxR, forming a complex that facilitates the association of RNA polymerase with the luxoperon promoter. VAI-2, which apparently interferes with VAI-1 binding to LuxR, operates to limit premature luxoperon induction. Hierarchical control is imposed on the system by 3':5'-cyclic AMP (cAMP) and cAMP receptor protein (CRP), which are necessary for activated expression of luxR. Several non-lux genes in V. fischeri are controlled by LuxR and VAI-1. Quorum regulation in V. fischeri serves as a model for LuxI/LuxR-type quorum sensing systems in other gram-negative bacteria.
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PMID:Quorum regulation of luminescence in Vibrio fischeri. 1094 79

The Pseudomonas aeruginosa LasR protein functions in concert with N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C(12)-HSL) to coordinate the expression of target genes, including many genes that encode virulence factors, with cell density. We used a LexA-based protein interaction assay to demonstrate that LasR forms multimers only when 3O-C(12)-HSL is present. A series of LasR molecules containing internal deletions or substitutions in single, conserved amino acid residues indicated that the N-terminal portion of LasR is required for multimerization. Studies performed with these mutant versions of LasR demonstrated that the ability of LasR to multimerize correlates with its ability to function as a transcriptional activator of lasI, a gene known to be tightly regulated by the LasR-3O-C(12)-HSL regulatory system. A LasR molecule that carries a C-terminal deletion can function as a dominant-negative mutant in P. aeruginosa, as shown by its ability to decrease expression of lasB, another LasR-3O-C(12)-HSL target gene. Taken together, our data strongly support the hypothesis that LasR functions as a multimer in vivo.
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PMID:LasR, a transcriptional activator of Pseudomonas aeruginosa virulence genes, functions as a multimer. 1216 17

Aes, a 36-kDa acetylesterase from Escherichia coli, belongs to the hormone-sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon. The activity of MalT is depressed through a direct protein-protein interaction with Aes. Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive. To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E. coli and purified. At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column. A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected. By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio. The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected. The esterase activity associated with the 87-kDa complex, when assayed with p-nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa. Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the alpha-galactosidase from E. coli. The E. coli cells harboring plasmid pT7-SCII-aes and, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source. Because alpha-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E. coli.
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PMID:The Aes protein and the monomeric alpha-galactosidase from Escherichia coli form a non-covalent complex. Implications for the regulation of carbohydrate metabolism. 1237 3

Bacteria adapt their pattern of gene expression in response to a variety of external cues, including fluctuations in population density. This type of bacterial cell-to-cell communication is referred to as quorum-sensing. Quorum-sensing systems are present in many bacterial species and constitute a large collection of ligands and cognate receptors. The availability of such diversity offers interesting opportunities for biotechnological exploitation. We describe here the transformation of the quorum-sensing system of Agrobacterium tumefaciens into a transcription regulatory system that works in mammalian cells. The A. tumefaciens TraR protein was fused to the eukaryotic activation domain of NF-kappaB p65, generating a novel chimaeric transcriptional activator that stimulates gene transcription in different human cell lines from a minimal promoter containing the TraR DNA recognition sequence in the presence of the Agrobacterium quorum-sensing signal molecule N-(3-oxo-octanoyl)homoserine lactone (3-oxo-C(8)-HSL). The basal level of transcription was low in the absence of 3-oxo-C(8)-HSL, and gene expression was stimulated up to 1,000-fold at a saturating concentration of 3-oxo-C(8)-HSL.
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PMID:A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription factor TraR. 1261 5

Conjugal transfer of Ti plasmids of Agrobacterium tumefaciens is controlled by a quorum-sensing system composed of the transcriptional activator TraR and its acyl-homoserine lactone quormone N-(3-oxo-octanoyl)-L-homoserine lactone (3-oxo-C8-HSL). The population density dependence of quorum-sensing systems can often be circumvented by addition of the quormone to cultures at low cell numbers. However, the quorum-dependent activation of Ti plasmid conjugal transfer exhibited a lag of almost 8 h when the quormone was added to donor cells at low population densities (Piper and Farrand, J. Bacteriol. 182:1080-1088, 2000). As measured by activation of a TraR-dependent traG::lacZ reporter fusion, TraR in cells exposed to the cognate signal for 5 min showed detectable activity, while exposure for 15 min resulted in full activity. Thus, the lag in activation is not due to some intrinsic property of TraR. Cells exposed to the agonistic analog N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) exhibited similar induction kinetics. However, activation of the reporter in cells exposed to the poorly effective alkanoyl acyl-HSL N-hexanoyl-L-homoserine lactone (C6-HSL) required the continued presence of the signal. As measured by an in vivo repressor assay, TraR activated by 3-oxo-C6-HSL or by 3-oxo-C8-HSL remained active for as long as 8 h after removal of exogenous signal. However, TraR activated by the alkanoyl quormone C6-HSL rapidly lost activity following removal of the signal. In quormone retention assays, which measure signal binding by TraR, cells grown with either of the two 3-oxo-acyl-HSL quormones retained the ligand after washing, while cells grown with C6-HSL lost the alkanoyl-HSL concomitant with the rapid loss of TraR activity. We conclude that TraR rapidly binds its quormone and that, once bound, the cognate signal and its close homologs are tightly retained. Moreover, in the absence of other regulatory factors, activated TraR remains functional after removal of the signal. On the other hand, poorly active signals are not tightly bound, and their removal by washing leads to rapid loss of TraR activity.
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PMID:In situ activation of the quorum-sensing transcription factor TraR by cognate and noncognate acyl-homoserine lactone ligands: kinetics and consequences. 1312 37

The RhlR transcriptional regulator of Pseudomonas aeruginosa, along with its cognate autoinducer, N-butyryl homoserine lactone (C(4)-HSL), regulates gene expression in response to cell density. With an Escherichia coli LexA-based protein interaction system, we demonstrated that RhlR multimerized and that the degree of multimerization was dependent on the C(4)-HSL concentration. Studies with an E. coli lasB::lacZ lysogen demonstrated that RhlR multimerization was necessary for it to function as a transcriptional activator. Deletion analysis of RhlR indicated that the N-terminal domain of the protein is necessary for C(4)-HSL binding. Single amino acid substitutions in the C-terminal domain of RhlR generated mutant RhlR proteins that had the ability to bind C(4)-HSL and multimerize but were unable to activate lasB expression, demonstrating that the C-terminal domain is important for target gene activation. Single amino acid substitutions in both the N-terminal and C-terminal domains of RhlR demonstrated that both domains possess residues involved in multimerization. RhlR with a C-terminal deletion and an RhlR site-specific mutant form that possessed multimerization but not transcriptional activation capabilities were able to inhibit the ability of wild-type RhlR to activate rhlA expression in P. aeruginosa. We conclude that C(4)-HSL binding is necessary for RhlR multimerization and that RhlR functions as a multimer in P. aeruginosa.
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PMID:Functional domains of the RhlR transcriptional regulator of Pseudomonas aeruginosa. 1464 72


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