Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Expression of the Vibrio fischeri luminescence genes (luxR and luxICDABEG) in Escherichia coli requires autoinducer (N-3-oxohexanoyl homoserine lactone) and LuxR protein, which activate transcription of luxICDABEG (genes for autoinducer synthase and the luminescence enzymes), and cyclic AMP (cAMP) and cAMP receptor protein (CRP), which activate transcription of the divergently expressed luxR gene. In E. coli and in V. fischeri, the autoinducer-LuxR protein-dependent induction of luxICDABEG transcription (called autoinduction) is delayed by glucose, whereas it is promoted by iron restriction, but the mechanisms for these effects are not clear. To examine in V. fischeri control of lux gene expression by autoinducer, cAMP, glucose, and iron, lux::Mu dI(lacZ) and lux deletion mutants of V. fischeri were constructed by conjugation and gene replacement procedures. beta-Galactosidase synthesis in a luxC::lacZ mutant exhibited autoinduction. In a luxR::lacZ mutant, complementation by the luxR gene was necessary for luminescence, and addition of cAMP increased beta-galactosidase activity four- to sixfold. Furthermore, a luxI::lacZ mutant produced no detectable autoinducer but responded to its addition with induced synthesis of beta-galactosidase. These results confirm in V. fischeri key features of lux gene regulation derived from studies with E. coli. However, beta-galactosidase specific activity in the luxI::lacZ mutant, without added autoinducer, exhibited an eight- to tenfold decrease and rise back during growth, as did beta-galactosidase and luciferase specific activities in the luxR::lacZ mutant and luciferase specific activity in a delta(luxR luxICD) mutant. The presence of glucose delayed the rise back in beta-galactosidase and luciferase specific activities in these strains, whereas iron restriction promoted it. Thus, in addition to transcriptional control by autoinducer and LuxR protein, the V. fischeri lux system exhibits a cell density-dependent modulation of expression that does not require autoinducer, LuxR protein, or known lux regulatory sites. The response of autoinducer-LuxR protein-independent modulation to glucose and iron may account for how these environmental factors control lux gene expressions.
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PMID:Cell density-dependent modulation of the Vibrio fischeri luminescence system in the absence of autoinducer and LuxR protein. 131 12

The gene coding for the Neurospora crassa copper metallothionein (MT) was synthesized and inserted in the lacZ' gene of pUC18 plasmid to give the same translational reading frame as the latter gene. The MT-beta-galactosidase fused gene was expressed in Escherichia coli to produce a fused protein in which the amino and carboxy termini of MT are linked to the beta-galactosidase through methionine residues. An MT derivative containing an extra homoserine residue at the carboxy terminus was prepared by cyanogen bromide cleavage of the fused protein followed by a reverse-phase HPLC separation. The spectral features of the MT derivative and its copper complex were similar to those of the corresponding native MTs.
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PMID:Chemical synthesis and expression of copper metallothionein gene of Neurospora crassa. 297 86

A series of structural analogs of the Pseudomonas aeruginosa autoinducer [PAI, N-3-oxo-dodecanoyl homoserine lactone] were obtained and tested for their ability to act as autoinducers in stimulating the expression of the gene for elastase (lasB) by measuring beta-galactosidase production from a lasB-lacZ gene fusion in the presence of the transcriptional activator LasR. The data suggest that the length of the acyl side chain of the autoinducer molecule is the most critical factor for activity. Replacement of the ring O by S in the homoserine lactone moiety can be tolerated. Tritium-labelled PAI ([3H]PAI) was synthesized and used to demonstrate the association of [3H]PAI with cells overexpressing LasR. The PAI analogs were also tested for their ability to compete with [3H]PAI for binding of LasR. Results from the competition assays suggest that once again the length of the acyl side chain appears to be crucial for antagonist activity. The presence of the 3-oxo moiety also plays a significant role in binding since analogs which lacked this moiety were much less effective in blocking binding of [3H]PAI. All analogs demonstrating competition with PAI in binding to LasR also exhibited the ability to activate lasB expression, suggesting that they are functional analogs of PAI.
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PMID:Functional analysis of the Pseudomonas aeruginosa autoinducer PAI. 883 Jun 97

Conjugal transfer of the Ti plasmid pTiC58 is regulated by a quorum-sensing system involving the transcriptional activator TraR and the acyl homoserine lactone autoinducer N-(3-oxo-octanoyl)-L-homoserine lactone (AAI). Activation of tra gene expression by TraR and AAI is inhibited by TraM, an 11 kDa protein also coded for by the Ti plasmid. Previous studies suggested that TraM interferes with TraR activity by directly interacting with the activator protein. Using the yeast two-hybrid system, constructs of Saccharomyces cerevisiae containing a fusion of traR to the B42 domain of the prey plasmid pJG4.5 and a fusion of traM to the lexA gene of the bait plasmid pEG202 produced beta-galactosidase and grew on medium lacking leucine, both phenotypes indicative of an interaction between the two proteins. Early termination mutants and substitution mutants mapping to the C-terminus of TraM were isolated by screening for alleles unable to interfere with TraR activity in Agrobacterium tumefaciens. These mutants all failed to interact with the TraR fusion in the two-hybrid system. An N-terminal deletion mutant of TraM lacking the first 27 residues weakly interacted with TraR in the two-hybrid system whereas deletions of 48 amino acids or more abolished the interaction. As assessed by Western blot analysis, the mutant fusion proteins were produced at levels indistinguishable from that of the wild-type TraM in the yeast tester strain. Mutants of TraR that were not inhibited by TraM in A. tumefaciens were isolated and fell into two classes. In the first, the mutation resulted in increased expression of wild-type TraR. In the second, a proline residue at position 176 was changed to serine (P176 --> S) or to leucine (P176 --> L). The P176 --> S mutant interacted with wild-type TraM, but at a detectably lower level, in the two-hybrid assay. Mutants of TraR with N-terminal deletions as large as 105 amino acids interfered with the ability of TraM to inhibit wild-type TraR in A. tumefaciens. Two-hybrid assays indicated that these mutants, as well as a C-terminal 49 residue fragment of TraR, can interact with TraM. We conclude that TraM and TraR interact in vivo and that this interaction is responsible for inhibition of TraR-mediated activation. We also conclude that the two proteins interact with each other through domains located at their respective C-termini.
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PMID:Modulating quorum sensing by antiactivation: TraM interacts with TraR to inhibit activation of Ti plasmid conjugal transfer genes. 1056 72

Most bacterial proteins are stable, with half-lives considerably longer than the generation time. In Escherichia coli, the few exceptions are unstable regulatory proteins. The results presented here indicate that the first enzyme in methionine biosynthesis - homoserine trans-succinylase (HTS) - is unstable and subject to energy-dependent proteolysis. The enzyme is stable in triple mutants defective in Lon-, HslVU- and ClpP-dependent proteases. The instability of the protein is determined by the amino-terminal part of the protein, and its removal or substitution by the N-terminal part of beta-galactosidase confers stability. The effect of the amino-terminal segment is not caused by the N-end rule, as substitution of the first amino acid does not affect the stability of the protein. HTS is the first biosynthetic E. coli enzyme shown to have a short half-life and may represent a group of biosynthetic enzymes whose expression is controlled by proteolysis. Alternatively, the proteolytic processing of HTS may be unique to this enzyme and could reflect its central role in regulating bacterial growth, especially at elevated temperatures.
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PMID:Control of methionine biosynthesis in Escherichia coli by proteolysis. 1099 74

The CepR-CepI quorum-sensing system has been shown to regulate production of the siderophore ornibactin, extracellular proteases, and N-octanoyl-homoserine-L-lactone (OHL) in Burkholderia cepacia strain K56-2. To examine the effect of cepIR on production of other siderophores, cepR mutants were constructed in strains that produce pyochelin in addition to salicylic acid and ornibactins. Pc715j-R1 (cepR::tp) hyperproduced ornibactin but produced parental levels of pyochelin and salicylic acid, suggesting that CepR is a negative regulator of ornibactin synthesis but not pyochelin or salicylic acid. Pc715j-R1 was also protease deficient and OHL negative. The effects of cepR on ornibactin biosynthetic genes were examined by constructing cepR pvdA-lacZ and cepR pvdD-lacZ mutants and monitoring beta-galactosidase activity. There was an increase in expression of pvdA in the cepR mutant compared to the level in its parent strain in both low- and high-iron media during stationary phase. When the outer membrane protein profiles of a cepR mutant and the wild-type strain were compared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there did not appear to be any difference in levels of expression of the ornibactin receptor. Experiments with cepI-lacZ and cepR-lacZ transcriptional fusions indicated that cepI was not expressed in the cepR mutant and that cepR acts as a negative regulator of its own expression. By a thin-layer chromatography assay for N-acyl homoserine lactones, OHL and N-hexanoyl-L-homoserine lactone (HHL) were detectable in K56-2 and Pc715j, both wild-type strains. OHL was not detectable and HHL was only weakly detectable in the cepI and cepR mutants. These results suggest that CepR is both a positive and negative transcriptional regulator and that CepR may influence the expression of ornibactin biosynthetic genes in addition to the expression of the cepIR quorum-sensing system.
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PMID:Regulation of ornibactin biosynthesis and N-acyl-L-homoserine lactone production by CepR in Burkholderia cepacia. 1124 59

RsmA (for regulator of secondary metabolism), RsmC, and rsmB RNA, the components of a posttranscriptional regulatory system, control extracellular protein production and pathogenicity in Erwinia carotovora subsp. carotovora. RsmA, an RNA binding protein, acts as a negative regulator by promoting message decay. rsmB RNA, on the other hand, acts as a positive regulator by neutralizing the effect of RsmA. RsmC modulates the levels of RsmA and rsmB RNA by positively regulating rsmA and negatively controlling rsmB. The level of rsmB RNA is substantially higher in RsmA(+) bacteria than in RsmA(-) mutants. We show that rsmB RNA is more stable in the presence of RsmA than in its absence. RsmA does not stimulate the expression of an rsmB-lacZ transcriptional fusion; in fact, the beta-galactosidase level is somewhat higher in RsmA(-) bacteria than in RsmA(+) bacteria. We also investigated the basis for increased levels of rsmA and rsmB RNAs in the absence of the quorum-sensing signal, N-[3-oxohexanoyl]-L-homoserine lactone (OHL). The absence of OHL activates transcription of rsmA but not of rsmB. Instead, increased stability of rsmB RNA in the presence of RsmA accounts for the elevated levels of the rsmB RNA in OHL(-) bacteria. Mutant studies disclosed that while RsmA, OHL, and RsmC control the levels of rsmB RNA, high levels of rsmB RNA occur in the absence of RsmC or OHL only in RsmA(+) bacteria, indicating a critical role for RsmA in modulating the levels of rsmB RNA. The findings reported here firmly establish that the quorum-sensing signal is channeled in E. carotovora subsp. carotovora via the rsmA-rsmB posttranscriptional regulatory system.
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PMID:RsmA and the quorum-sensing signal, N-[3-oxohexanoyl]-L-homoserine lactone, control the levels of rsmB RNA in Erwinia carotovora subsp. carotovora by affecting its stability. 1210 25

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis patients and is a major source of nosocomial infections. This bacterium controls many virulence factors by using two quorum-sensing systems, las and rhl. The las system is composed of the LasR regulator protein and its cell-to-cell signal, N-(3-oxododecanoyl) homoserine lactone, and the rhl system is composed of RhlR and the signal N-butyryl homoserine lactone. A third intercellular signal, the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4-quinolone), also regulates numerous virulence factors. PQS synthesis requires the expression of multiple operons, one of which is pqsABCDE. Previous experiments showed that the transcription of this operon, and therefore PQS production, is negatively regulated by the rhl quorum-sensing system and positively regulated by the las quorum-sensing system and PqsR (also known as MvfR), a LysR-type transcriptional regulator protein. With the use of DNA mobility shift assays and beta-galactosidase reporter fusions, we have studied the regulation of pqsR and its relationship to pqsA, lasR, and rhlR. We show that PqsR binds the promoter of pqsA and that this binding increases dramatically in the presence of PQS, implying that PQS acts as a coinducer for PqsR. We have also mapped the transcriptional start site for pqsR and found that the transcription of pqsR is positively regulated by lasR and negatively regulated by rhlR. These results suggest that a regulatory chain occurs where pqsR is under the control of LasR and RhlR and where PqsR in turn controls pqsABCDE, which is required for the production of PQS.
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PMID:Regulation of Pseudomonas quinolone signal synthesis in Pseudomonas aeruginosa. 1596 46

A simple, rapid, sensitive microtiter plate method detecting N-acyl homoserine lactone (HSL)-related compounds was established using an Agrobacterium tumefaciens strain harboring a traG::lacZ/traR reporter gene responsive to HSLs. This strain did not produce its own HSL, but the traG::lacZ reporter gene was induced only when its transcription activator TraR detected a cognate exogenous HSL. Therefore, the assay was expected to be highly specific for HSL-related compounds. Induction of the reporter gene, leading to production of beta-galactosidase enzyme, was measured by using two different beta-galactosidase substrates, X-gal and Galacton-Star, for colorimetric and chemiluminometric detection, respectively. The screen was validated in both the 96-well and 384-well plate formats, and extracts derived from 696 different microbial isolates, mostly unidentified actinomycetes isolated from diverse locations, were tested. Crude extracts of 81 (11.64%) cultures tested positive for HSL-related compounds, and an additional 34 (4.8%) crude extracts showed a moderate to weak signal for HSLs. Data from the fractionated samples, however, suggested a much higher prevalence of HSL signals in these extracts. Of 144 crude extracts fractionated into 10 individual samples at a 10x concentration, 72 (50%) cultures tested positive for HSLs. Six cultures were active only in the crude extract, 18 were active both in crude and one or more of their fractions, and an additional 48 were active in just one or more of their fractions. This finding may be the first to suggest such a high prevalence of HSL-signals found in nature, and a large number of actinomycetes in our collection appeared to produce HSL-related compounds.
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PMID:A simple, rapid, sensitive method detecting homoserine lactone (HSL)-related compounds in microbial extracts. 1601 90

One hundred and forty-two Bradyrhizobium strains were screened for their ability to produce N-acyl homoserine lactone-like molecules (AHLs) by using an Agrobacterium tumefaciens biosensor strain containing a traI-lacZ fusion. Approximately 22% (31 of 142) of the tested strains produced AHLs that induced moderate to elevated beta-galactosidase activity levels in the biosensor strain. Bradyrhizobium japonicum and Bradyrhizobium elkanii strains were both shown to produce AHLs. Age of culture, and media composition were each shown to influence production of AHL(s), with greater production occurring in 2 day-old cultures grown in rich media. Reverse-phase high-performance liquid chromatography and thin-layer chromatography analyses indicated that the B. japonicum strain USDA 290 produced at least two types of AHLs. Our results indicate that the production AHL-like autoinducers is widespread among both B. japonicum and B. elkanii strains.
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PMID:Detection of homoserine lactone-like quorum sensing molecules in bradyrhizobium strains. 1613 57


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