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)

In Pseudomonas aeruginosa, production of exotoxin A, an ADP-ribosyltransferase, is a complex and highly regulated process. Two positively acting regulatory genes, regA and regB, have been cloned and characterized. To identify additional exotoxin A regulatory genes, we have characterized four N-methyl-N'-nitro-N-nitrosoguanidine-generated mutants of P. aeruginosa PA103 which are deficient in exotoxin A production. These mutants (PA103-8, PA103-15, PA103-16, and PA103-19) do not accumulate intracellular exotoxin A and are not complemented by the cloned toxA or regAB genes. This observation indicates that the lesion(s) in the mutants is probably in an exotoxin A regulatory gene(s) and is not in the genes for secretion of exotoxin A or in the toxA or regAB genes. To assess the effect of the putative regulatory mutations on the toxA and regAB genes, we compared the activity of the toxA and regAB promoters in the mutant and parental strains using plasmids containing the genes for beta-galactosidase or chloramphenicol acetyltransferase under the control of either the toxA or the regAB promoter. The toxA promoter-beta-galactosidase fusion plasmid could not be maintained in PA103-8. beta-Galactosidase expression driven by the toxA promoter was absent in the mutant PA103-19 and occurred at a low level, which was not repressed by iron in mutants PA103-15 and PA103-16. The regAB genes are temporally controlled by two promoters, P1 and P2. In all four mutants, regAB P1 promoter activity was reduced; however, expression under the control of the regAB P2 promoter was normal. These observations suggest the existence of one or more regulatory genes which directly affect expression of both the toxA and the regAB P1 promoters.
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PMID:Characterization of Pseudomonas aeruginosa mutants that are deficient in exotoxin A synthesis and are altered in expression of regA, a positive regulator of exotoxin A. 811 61

The Xy1R protein positively controls expression from the Pseudomonas putida TOL plasmid sigma 54-dependent Pu and Ps promoters, in response to the presence of aromatic effectors such as m-xylene, m-methylbenzyl alcohol, and p-chlorobenzaldehyde in the culture medium. Xy1R also autoregulates its own synthesis. A mutant Xy1R regulator called Xy1R7 was isolated after nitrosoguanidine mutagenesis of the wild-type gene and phenotypic selection for mutants that had acquired the ability to recognize m-nitrotoluene, a nitroarene that is not an effector for the wild-type regulator. The mutant regulator exhibited a single point mutation that resulted in a change in codon 172 (GAA-->AAA), which should result in a Glu-->Lys change in the polypeptide chain. The effector profile of the mutant regulator was determined by measuring beta-galactosidase from a fusion of the Pu promoter to a promoterless lacZ gene. The results showed that the mutant regulator had acquired the ability to recognize m-nitrotoluene, and retained the wild-type regulator's ability to recognize most of the wild-type effectors. Full transcriptional activation of the Pu promoter by Xy1R7, as with the wild-type Xy1R protein, requires its full modular structure, namely the sigma 54 recognition site, the integration host factor binding site, and the upstream activation sequences. The Xy1R7 regulator did not stimulate transcription from the Ps promoter in response to the presence of its effectors, and autoregulated its own synthesis at low levels.
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PMID:Genetic evidence for activation of the positive transcriptional regulator Xy1R, a member of the NtrC family of regulators, by effector binding. 813 29

Introduction of a Pseudomonas iron-regulated promoter lacZ fusion (SP1) and a Pseudomonas transcriptional factor into Escherichia coli allowed expression of the promoter in this heterologous host. Evaluation of this promoter in wild-type and fur mutants of E. coli, by measuring beta-galactosidase activity, indicated that E. coli Fur can regulate the Pseudomonas promoter in response to iron starvation. Gel retardation assays suggested that purified Fur protein could interact with the SP1 promoter upstream of the transcriptional start. DNase I footprinting analysis established that Fur protected a primary 58-bp region (-50 to -106 bp). These protein/DNA interactions correlate with the observed in vivo regulation of the SP1 promoter in E. coli and indicate that Fur can functionally regulate a Pseudomonas iron-regulated promoter.
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PMID:Escherichia coli ferric uptake regulator (Fur) can mediate regulation of a pseudomonad iron-regulated promoter. 820 May 8

A new lacZ transcriptional fusion vector, pHRP309, based on the IncQ plasmid RSF1010, was constructed and shown to be easily mobilized into a variety of Gram- eubacteria. We also developed a two-step cloning procedure to facilitate the cloning of small promoter fragments into the fusion vector. A set of 'cohort' vectors was constructed which allowed directed cloning of fragments downstream from an omega streptomycin/spectinomycin-resistance cassette while maintaining multiple flanking restriction sites. The omega cassette provides a selectable antibiotic-resistance marker for cloning promoters into the fusion vector and makes mapping to determine fragment orientation unnecessary. The presence of the omega cassette also decreases background beta-galactosidase activity by decreasing readthrough transcription from plasmid sequences. The fusion vector carries a gentamicin-resistance-encoding gene as the selectable marker and can therefore be used in Tn5 (kanamycin-resistant) and Tn10 (tetracycline-resistant) mutant strains. Since pHRP309 is a member of the IncQ incompatibility group, it is compatible with IncP cloning vectors and can be used in strains carrying cloned regulatory genes. Using this system, we cloned the positively regulated Pseudomonas putida pcaI promoter and studied its regulation.
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PMID:Construction and use of a new broad-host-range lacZ transcriptional fusion vector, pHRP309, for gram- bacteria. 822 91

The regulatory gene camR on the CAM plasmid of Pseudomonas putida (ATCC 17453) negatively controls expression of the cytochrome P-450cam hydroxylase operon (camDCAB) for the camphor degradation pathway and is oriented in a direction opposite to that of the camDCAB operon. In this study, we examined expression of the camR gene by monitoring the beta-galactosidase activity of camR-lacZ translational fusions in P. putida camR and camR+ strains. We found that the camR gene was autogenously regulated by its own product, CamR. To search for an operator site of the camR gene, a cam repressor (CamR)-overproducing plasmid, pHAOV1, was constructed by placing the camR gene under the control of a pL promoter. The translational initiation codon of CamR was changed by site-directed mutagenesis from GTG to ATG to improve translation efficiency. Judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the CamR protein was expressed up to about 10% of the soluble protein of CamR-overproducing Escherichia coli JM83/pHAOV1 cells. Results of DNase I footprinting assays using the cell lysate indicated that the CamR repressor covered a single region between the camR gene and the camDCAB operon. Our findings also suggest that the camR gene autogenously regulates its own expression by binding of the gene product, CamR, to the operator, which also serves as an operator of the camDCAB operon.
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PMID:Evidence for autoregulation of camR, which encodes a repressor for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid. 825 71

The Tat protein of human immunodeficiency virus 1 (HIV-1) can enter cells efficiently when added exogenously in tissue culture. To assess if Tat can carry other molecules into cells, we chemically cross-linked Tat peptides (residues 1-72 or 37-72) to beta-galactosidase, horseradish peroxidase, RNase A, and domain III of Pseudomonas exotoxin A (PE) and monitored uptake colorimetrically or by cytotoxicity. The Tat chimeras were effective on all cell types tested, with staining showing uptake into all cells in each experiment. In mice, treatment with Tat-beta-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in kidney and brain. The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages. Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells.
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PMID:Tat-mediated delivery of heterologous proteins into cells. 829 May 79

This work investigates the ability of laser-based, time-resolved fluorometry to detect the lactose operon genetic marker in microorganisms and to study protein-DNA interactions. In the first study, rapid detection of the Escherichia coli lacZY operon inserted in two strains of Pseudomonas proposed as fungal biological control organisms was achieved. Optimization of incubation time, immobilization apparatus size, and reagent volumes, along with the laser-based instrumentation, yielded an assay capable of detecting 10(4) immobilized lac+ Pseudomonas fluorescens cells within a 30-min incubation time. In the second study, the synthesis of E. coli beta-galactosidase was monitored in "real-time" with observable enzymatic activity beginning 4 to 5 min after induction with isopropylthiogalactoside.
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PMID:Fast and sensitive laser-based enzymatic detection of the lactose operon in microorganisms. 839 62

Specific induction of the copper resistance operon (cop) promoter from Pseudomonas syringae was measured by beta-galactosidase production from a cop promoter-lacZ fusion. Induction of the cop promoter in P. syringae pv. syringae required trans-acting factors from copper resistance plasmid pPT23D, from which cop was originally cloned. Tn5 mutagenesis of pPT23D was used to localize two complementation groups immediately downstream from copABCD. Cloning and sequencing of the DNA in this region revealed two genes, copR and copS, expressed in the same orientation as the cop operon but from a separate constitutive promoter. The amino acid sequence deduced from these genes showed distinct similarities to known two-component regulatory systems, including PhoB-PhoR and OmpR-EnvZ. In addition, CopR showed strong similarity to copper resistance activator protein PcoR from Escherichia coli. Functional chromosomal homologs to copRS activated the cop promoter, in a copper-inducible manner, in copper-resistant or -sensitive strains of P. syringae pv. tomato and other Pseudomonas species. This implies that copper-inducible gene regulation is associated with a common chromosomally encoded function, as well as plasmid-borne copper resistance, in Pseudomonas spp.
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PMID:A two-component regulatory system required for copper-inducible expression of the copper resistance operon of Pseudomonas syringae. 844 73

The lasR gene of Pseudomonas aeruginosa is required for transcription of the genes for elastase (lasB) and LasA protease (lasA), two proteases associated with virulence. We report here that the alkaline protease gene (apr) also requires the lasR gene for transcription. Alkaline protease mRNA was absent in the lasR mutant PAO-R1 and present when an intact lasR gene was supplied in trans as determined by Northern (RNA) analysis. The lasR gene also enhances exotoxin A production. Exotoxin A activity in supernatants of PAO-R1 were 30% less than in supernatants of the parental strain, PAO-SR. Multiple copies of lasR in trans in PAO-R1 in increased toxin A activity to twice the parental levels. Analysis of PAO-R1 containing the toxA promoter fused to beta-galactosidase suggests that LasR acts at the toxA promoter or at upstream toxA mRNA sequences. beta-Galactosidase activity was approximately 40% lower in PAO-R1 than in the parental strain, PAO-SR. Furthermore, the effect of LasR on the toxA promoter is not due to the stimulation of transcription of regA, a transcriptional activator of toxA. No difference in chloramphenicol acetyltransferase (CAT) activity was noted between PAO-SR and PAO-R1 containing transcriptional regA promoter-CAT gene fusions. These results broaden the regulatory dominion of lasR and suggest that the lasR gene plays a global role in P. aeruginosa pathogenesis.
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PMID:LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression. 845 22

Reporter gene technology was employed to detect the activity of an alginate promoter of Pseudomonas aeruginosa when the organism was grown as a biofilm on a Teflon mesh substratum and as planktonic cells in liquid medium. Alginate biosynthetic activity was determined with a mucoid cell line derived from a cystic fibrosis isolate and containing an alginate algC promoter fused to a lacZ reporter gene. Reporter activity was demonstrated with chromogenic and fluorogenic substrates for beta-galactosidase. Expression of algC was shown to be upregulated in biofilm cells compared with planktonic cells in liquid medium. Gene up-expression correlated with alginate biosynthesis as measured by Fourier transform infrared spectroscopy, uronic acid accumulation, and alginate-specific enzyme-linked immunosorbent assay. The algC promoter was shown to have maximum activity in planktonic cultures during the late lag and early log phases of the cell growth cycle. During a time course experiment, biofilm algC activity exceeded planktonic activity except during the period immediately following inoculation into fresh medium. In continuous-culture experiments, conversion of lacZ substrate was demonstrated microscopically in individual cells by epifluorescence microscopy.
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PMID:Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa. 847 92


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