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 transformation assay was used to assay the amount of DNA present in the extracellular medium of a growing culture of Acinetobacter calcoaceticus. It was observed that small amounts of DNA were liberated during the entire exponential growth phase in a batch culture. Release of DNA could be fully accounted for by lysis of cells. Lysis was quantified via simultaneous measurement of beta-galactosidase activity of cells and supernatant, with a strain that contained a plasmid (pAPA100) with lacZ under control of a constitutive beta-lactamase promoter. In conclusion, no evidence could be obtained indicating that Acinetobacter calcoaceticus actively excretes DNA, to be used for DNA exchange.
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PMID:Acinetobacter calcoaceticus liberates chromosomal DNA during induction of competence by cell lysis. 776 85

In Escherichia coli with group II capsules, the synthesis and cellular expression of capsular polysaccharide are encoded by the kps gene cluster. This gene cluster is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of the K5 polysaccharide, which is a group II capsular polysaccharide, have been cloned and sequenced. Region 1 contains the kpsE, -D, -U, -C, and -S genes. In this communication we describe the KpsE protein, the product of the kpsE gene. A truncated kpsE gene was fused with a truncated beta-galactosidase gene to generate a fusion protein containing the first 375 amino acids of beta-galactosidase and amino acids 67 to 382 of KpsE (KpsE'). This fusion protein was isolated and cleaved with factor Xa, and the purified KpsE' was used to immunize rabbits. Intact KpsE was extracted from the membranes of a KpsE-overexpressing recombinant strain with octyl-beta-glucoside. It was purified by affinity chromatography with immobilized anti-KpsE antibodies. Cytofluorometric analysis using the anti-KpsE antibodies with whole cells and spheroplasts, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of proteins from spheroplasts and membranes before and after treatment with proteinase K, indicated that the KpsE protein is associated with the cytoplasmic membrane and has an exposed periplasmic domain. By TnphoA mutagenesis and by constructing beta-lactamase fusions to the KpseE protein, it was possible to determine the topology of the KpsE protein within the cytoplasmic membrane.
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PMID:Characterization and localization of the KpsE protein of Escherichia coli K5, which is involved in polysaccharide export. 786 84

The periplasmic enzyme beta-lactamase was selectively released from Escherichia coli K12 by the amphiphilic quaternary ammonium compound tetradecyl betainate at certain concentration intervals. At low concentrations little enzyme was released, and at high concentrations enzyme inactivation occurred. Greater effects of tetradecyl betainate were seen both with respect to release and inactivation at higher pH. At intermediate concentrations of tetradecyl betainate high yields of beta-lactamase were obtained with no detectable contribution of the cytoplasmic marker beta-galactosidase. The highest yields of beta-lactamase activity were obtained when high concentrations of salt were added 1 min after permeation of the bacteria with tetradecyl betainate.
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PMID:Selective release of the periplasmic enzyme beta-lactamase from Escherichia coli with tetradecyl betainate. 803 73

The topology of Escherichia coli diacylglycerol kinase (DAGK) within the cytoplasmic membrane was elucidated by a combined approach involving both multiple aligned sequence analysis and fusion protein experiments. Hydropathy plots of the five prokaryotic DAGK sequences available were uniform in their prediction of three transmembrane segments. The hydropathy predictions were experimentally tested genetically by fusing C-terminal deletion derivatives of DAGK to beta-lactamase and beta-galactosidase. Following expression, the enzymatic activities of the chimeric proteins were measured and used to determine the cellular location of the fusion junction. These studies confirmed the hydropathy predictions for DAGK with respect to the number and approximate sequence locations of the transmembrane segments. Further analysis of the aligned DAGK sequences detected probable alpha-helical N-terminal capping motifs and two amphipathic alpha-helices within the enzyme. The combined fusion and sequence data indicate that DAGK is a polytopic integral membrane protein with three transmembrane segments with the N terminus of the protein in the cytoplasm, the C terminus in the periplasmic space, and two amphipathic helices near the cytoplasmic surface.
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PMID:Membrane topology of Escherichia coli diacylglycerol kinase. 807 Dec 24

A novel expression system based on the Bacillus subtilis bacteriophage phi 105 has been developed to permit the high-level synthesis and secretion of beta-lactamase I (BlaI) from Bacillus cereus. Shotgun insertion of a promoterless lacZ gene into the phage genome permitted the identification of a clone producing large amounts of beta-galactosidase (beta Gal), indicating the transcription of the reporter gene from a strong phage promoter. The insertion also blocked lysis of the host cell. Although the insertion in the original prophage was complex, plasmid vectors and prophage derivatives have been developed to facilitate the replacement of lacZ with other genes for expression. The new prophages contain two additional mutations: an ind mutation, which greatly enhances the normally poor transformability of phi 105 lysogens, and a cts mutation, which allows thermo-induction of phage development and protein production. Induction of a derivative prophage containing the blaI gene from B. cereus resulted in the production of up to 500 micrograms of secreted BlaI per ml of culture supernatant.
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PMID:An efficient expression and secretion system based on Bacillus subtilis phage phi 105 and its use for the production of B. cereus beta-lactamase I. 822 93

P-type ATPases are a family of cation transport enzymes present in all species from bacteria to mammals whose members mediate membrane flux of all common biologically relevant cations. More than 50 members of this family of transporters have been sequenced; extensive structural data are available, and several members have been analyzed by site-directed mutagenesis. Nonetheless, there is no current consensus regarding their membrane topology. In this work, the Salmonella typhimurium Mg2+ transporting P-type ATPase encoded by the MgtB locus has been used as a model for P-type ATPases. Unlike other prokaryotic P-type ATPases, the MgtB protein is similar in length, amino acid sequence, and hydropathy profile to known eukaryotic P-type ATPases. The membrane topology of MgtB was analyzed by several epitope insertions in MgtB and from the activity of 35 protein fusions between MgtB and the reporter enzymes BlaM (beta-lactamase) and LacZ (beta-galactosidase). The epitope insertions within MgtB all retained function as assessed by cation uptake assays and were regulated normally by the level of Mg2+ within the growth medium. The epitope insertion and fusion protein data are completely incompatible with the numerous previously proposed models for P-type ATPases predicting 7, 8, 9, or 12 transmembrane segments. Rather, they indicate that MgtB contains 10 transmembrane segments with both amino and carboxyl termini residing within the cytosol. By extension, we suggest that all eukaryotic P-type ATPases contain 10 transmembrane segments with both termini within the cytosol.
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PMID:Membrane topology of a P-type ATPase. The MgtB magnesium transport protein of Salmonella typhimurium. 822 55

TolR is a 142-amino-acid protein required for the import of colicins and bacteriophage and for maintenance of cell envelope integrity. The topology of TolR in the inner membrane was analyzed by two methods. First, bacteria expressing a series of TolR-beta-galactosidase, TolR-alkaline phosphatase, and TolR-beta-lactamase fusions were assayed for the appropriate enzymatic activity. Second, the accessibility of TolR to proteinase K was determined in permeabilized cells and everted vesicles with an antibody elicited against the carboxyl-terminal 70% of TolR. The results are consistent with TolR spanning the inner membrane once via residues 23 to 43 and with the carboxyl-terminal moiety being exposed to the periplasm. Quantitative studies with the anti-TolR antibody indicated the presence of 2 x 10(3) to 3 x 10(3) TolR molecules per cell.
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PMID:Membrane topology of the Escherichia coli TolR protein required for cell envelope integrity. 837 53

The sensitivity of selected bacteria to a range of antibiotics was tested under hyperbaric conditions used in saturation diving. The effect of hyperbaric helium and oxygen (heliox) on antibiotic stability and on induction of beta-lactamase was also determined. Increased resistance to penicillin (up to 23%) was shown by Staphylococcus aureus and to gentamicin (up to 46%) and rifampicin (up to 18%) by Escherichia coli and Salmonella typhimurium at 36 and 71 bar pressure. Exposure to 71 bar heliox did not affect antibiotic activity but increased the production of beta-lactamase in inducible S. aureus and Bacillus subtilis and production of beta-galactosidase in inducible E. coli. Increased resistance to antibiotics in saturation diving conditions can be attributed in some cases to the influence of hyperbaric pressure on induction mechanisms in bacteria. The experimental system devised for this work is suitable for more detailed examination of the influence of hyperbaric stress on antibiotic resistance and of its effect on induction mechanisms in general.
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PMID:The effect of antibiotics on bacteria under hyperbaric conditions. 870 35

The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the Escherichia coli beta-galactosidase (lacZ) gene. Two processes affected the accumulation of pGP1 variants with deletions in the penP-lacZ region. First, divergent transcription from genes upstream of penP-lacZ increased pGP1 deletion frequencies up to about 10-fold. Second, the removal of the PenP signal peptide resulted in completely stable plasmids, indicating that the entry of the PenP fragment into the protein export pathway is an important factor in the instability of pGP1. On the basis of these results, we propose a model in which the temporary anchoring of the plasmid to the membrane through the cotranscriptional and cotranslational entry of PenP into the protein export pathway creates domains of local hypersupercoiling, which we assume to be targets for deletion formation.
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PMID:The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis. 875 43

We have developed plasmid and phage vectors for the display of foreign proteins on the surface of bacteriophage lambda capsid by modifying the D gene which encodes the major head protein gpD. The vectors have multiple cloning sites, and permit colour selection and conditional chain termination for recombinants. Displayed proteins can be fused to either the N or C terminus of gpD by a peptide linker. The conditional chain termination scheme, via a host Escherichia coli suppressor activity, allows the fusion and assembly of homomultimeric proteins as well as control of the number of fusion proteins per phage particle. We have successfully displayed beta-lactamase, IgG-binding domains of the Staphylococcus aureus protein A, and beta-galactosidase by cloning the genes into the vector. The constructs express functionally active proteins fused to gpD that assemble into phage particles. These results suggest that gpD may be fused to many other peptides and proteins at their N or C terminus and the fusion products may be accessible on the surface of bacteriophage lambda particles.
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PMID:Surface display of proteins on bacteriophage lambda heads. 880 76


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