Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes.
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PMID:Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology. 283 40

The scr genes located on plasmid pUR400 and responsible for sucrose (Scr) metabolism of Escherichia coli K12 and other enteric bacteria have been cloned on a 9.3 kb DNA fragment. The different genes were mapped by transposon insertion mutagenesis, by restriction endonuclease and deletion mapping, and the corresponding gene products were identified. Besides the known structural genes scrA, coding for an EnzymeII(Scr) (45 kD) of the phosphoenolypyruvate-dependent phosphotransferase system (PTS), and scrB, coding for a sucrose 6-phosphate hydrolase (invertase) (55 kD), two new structural genes were discovered. Gene scrK apparently codes for an intracellular and ATP-dependent fructokinase (39 kD), while scrY seems to code for a sucrose porin (58 kD) in the outer cell membrane. No genes for an Enzyme III(Scr) of the PTS or for (a) glycosyltransferase(s) were detected. The four genes form an scr operon (gene order, scrK scrY scrA scrB, transcription from K to B), regulated by a repressor (gene scrR, 37 kD) and inducible by sucrose, fructose and fructose-containing oligosaccharides.
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PMID:Plasmid-mediated sucrose metabolism in Escherichia coli K12: mapping of the scr genes of pUR400. 283 84

The 25-kDa peptidoglycan-associated outer-membrane protein and most likely porin of Vibrio cholerae is a major immunogenic species. It has been purified by ion-exchange elution on hydroxyapatite followed by gel filtration on Bio-Gel P150 both in the presence of sodium dodecyl sulfate. This protein, of greater than 90% purity as judged by Western blotting, has been used to raise antibodies in rabbits. The antisera were then used to screen V. cholerae gene banks, constructed in Escherichia coli K12, and this has enabled us to isolate several colonies harbouring the cloned gene. The plasmids in these colonies have been designated pPM451, pPM455 and pPM472. These plasmids have a 5.3 X 10(3)-base BamHI fragment of V. cholerae DNA in common. Restriction endonuclease mapping of these plasmids has been performed and the protein identified both by Western blot analysis and in E. coli K12 minicells. The protein is not efficiently expressed in E. coli K12. It is proposed to use the name ompV to describe the structural gene, present in the cloned DNA, for this V. cholerae outer membrane protein.
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PMID:Purification of the 25-kDa Vibrio cholerae major outer-membrane protein and the molecular cloning of its gene: ompV. 398 95

Nitric oxide is a free radical (NO) formed biologically through the oxidation of L-arginine by nitric oxide synthases. NO is produced transiently in mammalian cells for intercellular signaling and in copious quantities to cause cytostasis and cytotoxicity. In the latter situation, NO is a deliberate cytotoxic product of activated macrophages, along with other reactive oxygen species such as hydrogen peroxide (H2O2) and superoxide (O2-). Escherichia coli has a complex set of responses to H2O2 and O2- that involves approximately 80 inducible proteins; we wondered whether these bacteria might induce analogous defenses against nitric oxide. We show here that a multigene system controlled by the redox-sensitive transcriptional regulator SoxR is activated by NO in vivo. This induction confers bacterial resistance to activated murine macrophages with kinetics that parallel the production of NO by these cells. Elimination of specific SoxR-regulated genes diminishes the resistance of these bacteria to the cytotoxic macrophages. The required functions include manganese-containing superoxide dismutase, endonuclease IV (a DNA-repair enzyme for oxidative damage), and micF, an antisense regulator of the outer membrane porin OmpF. These results demonstrate that SoxR is a sensor for cellular exposure to NO, and that the soxRS response system may contribute to bacterial virulence.
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PMID:Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages. 823 47

The major outer-membrane protein. FomA, of Fusobacterium nucleatum has been associated with porin activity, interbacterial adherence and stimulation of host immune cells. Until now, molecular analysis of FomA has not been possible because previous attempts to clone the fomA gene were not successful. The inability to clone F. nucleatum genes led to speculation that Escherichia coli may not be a suitable host. This report concerns the amplification of the fomA gene of F. nucleatum T18 using oligonucleotide primers containing restriction endonuclease sites that allow cloning of fomA into the E. coli expression vector pMMB67. The resultant plasmid, pXWI, was transformed into E. coli DH5 alpha, providing high-level expression of recombinant FomA (rFomA). Amino acid sequencing of rFomA demonstrated that the FomA signal peptide was correctly processed by E. coli signal peptidase I. rFomA was correctly localized to the outer membrane by the E. coli export pathway. The rFomA protein also displayed the heat-modifiable oligomeric and conformational properties of native FomA (nFomA). This demonstration of rFomA expression, processing, export, and secondary and tertiary structure in E. coli provides support for the feasibility of molecular analysis of the structure and function of FomA and other F. nucleatum proteins using recombinant techniques.
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PMID:Cloning and expression of FomA, the major outer-membrane protein gene from Fusobacterium nucleatum T18. 913 12

The outer membrane (OM) vitamin B(12) receptor, BtuB, is the primary receptor for E group colicin adsorption to Escherichia coli. Cell death by this family of toxins requires the OM porin OmpF but its role remains elusive. We show that OmpF enhances the ability of purified BtuB to protect bacteria against the endonuclease colicin E9, demonstrating either that the two OM proteins form the functional receptor or that OmpF is recruited for subsequent translocation of the bacteriocin. While stable binary colicin E9-BtuB complexes could be readily shown in vitro, OmpF-containing complexes could not be detected, implying that OmpF association with the BtuB-colicin complex, while necessary, must be weak and/or transient in nature.
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PMID:OmpF enhances the ability of BtuB to protect susceptible Escherichia coli cells from colicin E9 cytotoxicity. 1280 62

Binding of enzymatic E colicins to the vitamin B12 receptor, BtuB, is the first stage in a cascade of events that culminate in the translocation of the cytotoxic nuclease into the Escherichia coli cytoplasm and release of its tightly bound immunity protein. A dogma of colicin biology is that the toxin coiled-coil connecting its functional domains must unfold or unfurl to span the periplasm, with recent reports claiming this reaction is initiated by receptor binding. We report isothermal titration calorimetry data of BtuB binding the endonuclease toxin ColE9 and a disulfide form (ColE9S-S) where unfolding of the coiled-coil is prevented and, as a consequence, the toxin is biologically inactive. Contrary to expectation, the thermodynamics of receptor binding, characterized by large negative values for TDeltaS, are identical for the two colicins, arguing against any form of BtuB-induced unfolding. We go on to delineate key features of the "colicin translocon" that assembles at the cell surface after BtuB binding by using a complex of histidine-tagged Im9 bound to ColE9S-S. First, we show that the porin OmpF is recruited directly to the BtuB.colicin complex to form the translocon. Second, recruitment is through the natively unfolded region of the colicin translocation domain, with this domain likely having two contact points for OmpF. Finally, the immunity protein is not released during its assembly. Our study demonstrates that although colicin unfolding is undoubtedly a prerequisite for E. coli cell death, it must occur after assembly of the translocon.
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PMID:Cell entry mechanism of enzymatic bacterial colicins: porin recruitment and the thermodynamics of receptor binding. 1616 65

A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E. coli DE3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a(+) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E. coli DE3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28% of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1:4000 titer determined by indirect EISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.
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PMID:Construtcion of Neisseria gonorrhoeae porin B plasmid recombinant and its expression in E. coli. 1620 Dec 62

Bacteria producing endonuclease colicins are protected against the cytotoxic activity by a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. This complex is released into the extracellular medium, and the immunity protein is jettisoned upon binding of the complex to susceptible cells. However, it is not known how and at what stage during infection the immunity protein release occurs. Here, we constructed a hybrid immunity protein composed of the enhanced green fluorescent protein (EGFP) fused to the colicin E2 immunity protein (Im2) to enhance its detection. The EGFP-Im2 protein binds the free colicin E2 with a 1:1 stoichiometry and specifically inhibits its DNase activity. The addition of this hybrid complex to susceptible cells reveals that the release of the hybrid immunity protein is a time-dependent process. This process is achieved 20 min after the addition of the complex to the cells. We showed that complex dissociation requires a functional translocon formed by the BtuB protein and one porin (either OmpF or OmpC) and a functional import machinery formed by the Tol proteins. Cell fractionation and protease susceptibility experiments indicate that the immunity protein does not cross the cell envelope during colicin import. These observations suggest that dissociation of the immunity protein occurs at the outer membrane surface and requires full translocation of the colicin E2 N-terminal domain.
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PMID:Release of immunity protein requires functional endonuclease colicin import machinery. 1701 83