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)

A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the beta-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the beta-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium beta-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for beta-galactosidase expression in E. coli. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and Klebsiella pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of beta-galactosidase in E. coli.
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PMID:Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli. 185 Jul 29

The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7.2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S. thermophilus ATCC 19258. Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of the beta-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of the S. thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E. coli and Klebsiella pneumoniae showed 48, 35 and 32.5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid sequence were identified when the beta-galactosidase proteins from S. thermophilus, E. coli and L. bulgaricus were compared.
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PMID:Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences. 190 4

Random Tn5 mutagenesis of the regulatory region of megaplasmid pHG1 of Alcaligenes eutrophus led to the identification of three distinct loci designated hoxA, hoxD, and hoxE. Sequencing of the hoxA locus revealed an open reading frame which could code for a polypeptide of 482 amino acids with a molecular mass of 53.5 kDa. A protein of comparable apparent molecular mass was detected in heterologous expression studies with a plasmid-borne copy of the hoxA gene. Amino acid alignments revealed striking homologies between HoxA and the transcriptional activators NifA and NtrC of Klebsiella pneumoniae and HydG of Escherichia coli. HoxA- mutants of A. eutrophus lacked both NAD-reducing soluble hydrogenase and membrane-bound hydrogenase. In HoxA- mutants, the synthesis of beta-galactosidase from a hoxS'-'lacZ operon fusion was drastically reduced, indicating that HoxA is essential for the transcription of hydrogenase genes. Mutants defective in hoxD and hoxE also lacked the catalytic activities of the two hydrogenases; however, in contrast to HoxA- mutants, they contained immunologically detectable NAD-reducing soluble hydrogenase and membrane-bound hydrogenase proteins, although at a reduced level. The low hydrogenase content in the HoxD- and HoxE- mutants correlated with a decrease in beta-galactosidase synthesized under the direction of a hoxS'-'lacZ operon fusion. Thus, hoxD and hoxE apparently intervene both in the regulation of hydrogenase synthesis and in subsequent steps leading to the formation of catalytically active enzymes.
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PMID:Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus. 200 89

Gram-negative rods were presumptively identified directly from blood cultures within 15 min as Escherichia coli, a member of the Klebsiella-Enterobacter group, or oxidase positive. Samples of artificially seeded blood cultures (193 cultures) and patient blood cultures (78 cultures) were filtered into a Dynadepth test card with the Bac-T-Screen instrument (Vitek, Inc., Hazelwood, Mo.). Triton X-100 was then filtered into the test card to lyse the blood cells but not the entrapped bacteria, and either methylumbelliferone-labeled substrates or oxidase reagent was applied to the filter surface. The oxidase test was read within 30 s, and the methylumbelliferone and indole tests were read after a 10-min incubation at room temperature. Positive beta-galactosidase, beta-glucuronidase, and indole test results predicted the identification of E. coli with a 96 to 100% sensitivity and a 99 to 100% specificity. Positive beta-xylosidase and beta-galactosidase test results and negative oxidase and beta-glucuronidase test results were 85 to 93% sensitive and 100% specific for a Klebsiella-Enterobacter organism. A positive oxidase test result and negative beta-glucuronidase, beta-xylosidase, and indole test results were highly predictive of Pseudomonas aeruginosa (sensitivity, 100%; specificity, 99%). The procedures described are rapid and simple and provide a direct presumptive identification of the gram-negative rods most commonly found in blood cultures.
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PMID:Rapid presumptive identification of gram-negative rods directly from blood cultures by simple enzymatic tests. 210 96

A new gene whose product is required for the production of formate hydrogenlyase (FHL) has been identified in Escherichia coli. This gene, termed fhlB, maps between the frdA (94.4 min) and argI (96.6 min) genes on the E. coli chromosome and is transcribed in a clockwise direction toward argI. Biochemical analysis of an FhlB- mutant, strain SE-2011 [phi(fhlB-lacZ+)], revealed that the mutant lacks formate dehydrogenase activity associated with FHL (FDH-H) and hydrogenase activity. As a result of these defects, fermentative hydrogen production and hydrogen uptake reactions were undetectable in strain SE-2011. Fumarate reductase activity of this mutant was also reduced to about 15% of the levels of the parent (strain MC4100), and strain SE-2011 did not produce succinate as a fermentation end product. Regulation of expression of the fhlB gene, studied as production of beta-galactosidase activity by strain SE-2011, revealed that the operon is expressed at low levels under aerobic conditions. Under anaerobic growth conditions, this activity increased by two- to threefold. Addition of formate enhanced the differential rate of synthesis of the fhlB gene product to as high as 130 U of beta-galactosidase specific activity per microgram of cell protein, but only under anaerobic conditions. Formate-dependent expression of phi(fhlB-lacZ+) required the sigma 54 subunit of RNA polymerase and the fhlA gene product. The concentration of formate required for maximum expression of the fhlB gene was about 15 mM; this value decreased to about 3 mM in the presence of plasmid pSE-133, which carries the fhlA gene in a multicopy plasmid. DNA sequence analysis of the fhlA gene showed that the FhlA protein is 686 amino acids long and has an anhydrous molecular weight of 78,086. On the basis of sequence homology with other transcriptional activators such as NtrC, HydG, and Klebsiella pneumoniae NifA proteins, the FhlA protein was deduced to be a transcriptional activator controlling the production of FHL. It is proposed that formate interacts with the FhlA protein and that this active complex initiates transcription of the fhlB gene. The FhlA and FhlB proteins act as a cascade in regulating the production of FDH-H and the FHL-linked hydrogenase and ultimately the production of FHL and fermentative hydrogen.
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PMID:Genetic regulation of formate hydrogenlyase of Escherichia coli: role of the fhlA gene product as a transcriptional activator for a new regulatory gene, fhlB. 211 3

An increased resistance of laboratory animals to pulmonary infections following per os administration of a glyco-proteic complex extracted from Klebsiella pneumoniae has been reported. This was associated with an increased phagocytic capacity of alveolar macrophages (AM). In this report, the effect of treating guinea pigs with this extract on the alveolar macrophage (AM) glycosidase machinery has been studied. AM were collected by bronchoalveolar lavage, the cells were pelleted by centrifugation and AM were purified by adherence on plastic dishes. Sialidase, beta-galactosidase, beta-glucuronidase and N-acetyl-beta-D glucosaminidase activities were measured in the AM homogenate. In order to evaluate an extracellular release of these enzymes, they were also assayed in the cell free lavage fluid. Lactic dehydrogenase (LDH) activity was assayed as a control for cell lysis. In treated animals, the total number of cells as well as the number of AM increased by 25% (ns). The protein concentration was slightly reduced in the cell homogenate and unchanged in the lavage fluid. The only significant change was a decreased sialidase activity, in AM homogenate (p less than or equal to 0.01) and in lavage fluids (ns). The LDH activity was not increased in the lavage fluids.
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PMID:Glycosidase activities in alveolar macrophages from guinea pigs stimulated with a glyco-proteic complex extracted from Klebsiella pneumoniae. 212 66

Bactenecins are a class of arginine-rich antibacterial peptides of bovine neutrophil granules. Two bactenecins with approximate molecular weights of 5,000 and 7,000 designated Bac5 and Bac7, respectively, exert in vitro a potent bactericidal activity toward several gram-negative bacteria (R. Gennaro, B. Skerlavaj, and D. Romeo, Infect. Immun. 57:3142-3146, 1989). We have now found that this activity shows an inverse relationship to the ionic strength of the medium and is inhibited by divalent cations and greatly potentiated by lactoferrin. Under conditions supporting marked bactericidal activity, the two peptides cause a rapid increase in the permeability of both the outer and inner membranes of Escherichia coli, as shown by unmasking of periplasmic beta-lactamase and of cytoplasmic beta-galactosidase. In addition, the two bactenecins inhibit the respiration of E. coli and Klebsiella pneumoniae but not of Bac5- and Bac7-resistant Staphylococcus aureus. Furthermore, they induce a drop in ATP content in E. coli, K. pneumoniae, and Salmonella typhimurium and a marked decrease in the rates of transport and incorporation of [3H]leucine and [3H]uridine into E. coli protein and RNA, respectively. In general, all these effects become evident within 1 to 2 min and reach their maximal expression within about 5 min. Overall, these data strongly suggest that the decrease in bacterial viability is causally related to the increase in membrane permeability and the subsequent fall in respiration-linked proton motive force, with the attendant loss of cellular metabolites and macromolecular biosynthesis ability.
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PMID:Rapid membrane permeabilization and inhibition of vital functions of gram-negative bacteria by bactenecins. 222 43

The Lactobacillus bulgaricus beta-galactosidase gene was cloned on a ca. 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter. The nucleotide sequence of the gene and approximately 400 bases of 3'- and 5'-flanking sequences was determined. The amino acid sequence of the beta-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca. 114 kilodaltons, slightly smaller than the E. coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E. coli evolved (ebgA) beta-galactosidase. The cloned beta-galactosidase was found to be indistinguishable from the native enzyme by several criteria. From amino acid sequence alignments, the L. bulgaricus beta-galactosidase has a 30 to 34% similarity to the E. coli lacZ, E. coli ebgA, and K. pneumoniae lacZ enzymes. There are seven regions of high similarity common to all four of these beta-galactosidases. Also, the putative active-site residues (Glu-461 and Tyr-503 in the E. coli lacZ beta-galactosidase) are conserved in the L. bulgaricus enzyme as well as in the other two beta-galactosidases mentioned above. The conservation of active-site amino acids and the large regions of similarity suggest that all four of these beta-galactosidases evolved from a common ancestral gene. However, these enzymes are quite different from the thermophilic beta-galactosidase encoded by the Bacillus stearothermophilus bgaB gene.
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PMID:Expression and nucleotide sequence of the Lactobacillus bulgaricus beta-galactosidase gene cloned in Escherichia coli. 249 11

232 isolates of gram-negative, oxidase-negative bacteria, isolated from various samples of different animal species, were tested with help of RAPIDEC coli for the production of beta-glucuronidase, beta-galactosidase and indole. The test correctly identified 164 of 175 E. coli strains (sensitivity 93.7%) and correctly indicated that 57 of 57 isolates of the family Enterobacteriaceae (7 Citrobacter sp., 18 Enterobacter sp., 16 Klebsiella sp., 10 Proteus sp., 2 Providencia sp. and 4 Salmonella sp.) were not E. coli (specificity 100%).
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PMID:[The rapid identification of E. coli by the detection of 3 enzymes]. 249 26

The ebg system has been used as a model to study the artificial selection of new catalytic functions of enzymes and of inducer specificities of repressors. A series of mutant enzymes with altered catalytic specificities were previously characterized biochemically as were the changes in inducer specificities of mutant, but fully functional, repressors. The wild type ebg operon has been sequenced, and the sequence differences of the mutant enzymes and repressors have been determined. We now report that, contrary to our previous understanding, ebg enzyme contains 180-kD alpha-subunits and 20-kD beta-subunits, both of which are required for full activity. Mutations that dramatically affect substrate specificity and catalytic efficiency lie in two distinct regions, both well outside of the active site region. Mutations that affect inducer specificity of the ebg repressor lie within predicted sugar binding domains. Comparisons of the ebg beta-galactosidase and repressor with homologous proteins of the Escherichia coli and Klebsiella pneumoniae lac operons, and with the galactose operon repressor, suggest that the ebg and lac operons diverged prior to the divergence of E. coli from Klebsiella. One case of a triple substitution as the consequence of a single event is reported, and the implications of that observation for mechanisms of spontaneous mutagenesis are discussed.
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PMID:DNA sequence analysis of artificially evolved ebg enzyme and ebg repressor genes. 251 8


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