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Query: UMLS:C0519030 (Klebsiella)
 21,988 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pseudomonas syringae pv. syringae 61 contains a 25-kb cluster of hrp genes that are required for elicitation of the hypersensitive response (HR) in tobacco. TnphoA mutagenesis of cosmid pHIR11, which contains the hrp cluster, revealed two genes encoding exported or inner-membrane-spanning proteins (H.-C. Huang, S. W. Hutcheson, and A. Collmer, Mol. Plant-Microbe Interact. 4:469-476, 1991). The gene in complementation group X, designated hrpH, was subcloned on a 3.1-kb SalI fragment into pCPP30, a broad-host-range, mobilizable vector. The subclone restored the ability of hrpH mutant P. syringae pv. syringae 61-2089 to elicit the HR in tobacco. DNA sequence analysis of the 3.1-kb SalI fragment revealed a single open reading frame encoding an 81,956-Da preprotein with a typical amino-terminal signal peptide and no likely inner-membrane-spanning hydrophobic regions. hrpH was expressed in the presence of [35S]methionine by using the T7 RNA polymerase-promoter system and vector pT7-3 in Escherichia coli and was shown to encode a protein with an apparent molecular weight of 83,000 on sodium dodecyl sulfate-polyacrylamide gels. The HrpH protein in E. coli was located in the membrane fraction and was absent from the periplasm and cytoplasm. The HrpH protein possessed similarity with several outer membrane proteins that are known to be involved in protein or phage secretion, including the Klebsiella oxytoca PulD protein, the Yersinia enterocolitica YscC protein, and the pIV protein of filamentous coliphages. All of these proteins possess a possible secretion motif, GG(X)12VP(L/F)LXXIPXIGXL(F/L), near the carboxyl terminus, and they lack a carboxyl-terminal phenylalanine, in contrast to other outer membrane proteins with no known secretion function. These results suggest that the P. syringae pv. syringae HrpH protein is involved in the secretion of a proteinaceous HR elicitor.
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PMID:The Pseudomonas syringae pv. syringae 61 hrpH product, an envelope protein required for elicitation of the hypersensitive response in plants. 140 Feb 38

We describe the cloning of an ntrC gene of Agrobacterium tumefaciens C58 by interspecific complementation of an Escherichia coli ntrC mutant. Restriction mapping and Southern blot analysis of the complementing clone identified a 1.7-kb EcoRI-PvuII DNA fragment whose sequence was determined. Analysis of this sequence revealed coding regions corresponding to a complete ntrC gene and the C-terminal region of an ntrB gene. Amino acid sequence comparisons of A. tumefaciens NTRC protein with NTRC sequences from Rhizobium meliloti, Bradyrhizobium sp. (Parasponia), Klebsiella pneumoniae, E. coli, and Salmonella typhimurium show strong sequence conservation supporting DNA hybridization data, demonstrating strong evolutionary homology among ntrC genes of Rhizobiaceae. The C58 NTRC protein has been identified, by 35S-labeling, in a T7 RNA polymerase (pT7-7) expression vector system.
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PMID:Identification, cloning, and sequence analysis of the nitrogen regulation gene ntrC of Agrobacterium tumefaciens C58. 252 Aug 24

The oadGAB genes encoding the gamma, alpha and beta-subunits of the oxaloacetate decarboxylase Na+ pump in Klebsiella pneumoniae have been cloned on plasmid pSK-GAB and expressed in Escherichia coli. The membranes of the recombinant E. coli clone contained about three times as much catalytically active oxaloacetate decarboxylase (3 mg protein/2 g wet cells) as those of the K. pneumoniae strain from which the genes were derived. The enzyme was solubilised from the membranes with Triton X-100 and purified. Its Na+ transport function was demonstrated after reconstitution into proteoliposomes. Proteoliposomes containing only the membrane-bound subunits beta and gamma (not the peripheral alpha-subunit) were unable to catalyse Na+ translocation in response to a transmembrane Na+ (delta pNa+) or electrical gradient (delta psi). Individual subunits of oxaloacetate decarboxylase and combinations of two subunits were expressed from appropriate derivatives of plasmid pSK-GAB. The hydrophobic subunits beta and beta gamma were membrane-bound as expected. Interestingly, the alpha-subunit was located in the cytoplasm if expressed separately or together with beta, but became membrane-bound if expressed together with gamma. A gamma alpha complex was isolated from such membranes by avidin-Sepharose affinity chromatography. Interactions of the gamma-subunit with the water-soluble alpha-subunit and with the membrane-bound beta-subunit are therefore required to form the oxaloacetate decarboxylase complex. The combinations of separately expressed subunits gamma alpha + beta and beta gamma+alpha were shown to yield the catalytically active enzyme. The alpha or the beta-subunit and the combinations of these subunits with the gamma-subunit were therefore expressed in E. coli in a catalytically competent state. Functional expression of the separate gamma-subunit, however, could not be demonstrated. The alpha-subunit was strongly overexpressed from a pT7-7 derived plasmid, but was only partially biotinylated under these conditions. On coexpression of the birA gene encoding biotin ligase the major part (80-100%) of the overexpressed alpha-subunit was biotinylated. Highly purified alpha-subunit was obtained by fractionated precipitation of the soluble cell fraction with ammonium sulfate. Incubation of the alpha-subunit with oxaloacetate led to a CO2 transfer to its prosthetic biotin group with the formation of stoichiometric amounts of pyruvate. The velocity of the CO2 transfer to the biotin on the alpha-subunit was about three orders of magnitude too low to account for the rate of the overall reaction. The carboxyltransfer reaction was significantly accelerated if the gamma-subunit was additionally present.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Synthesis of the oxaloacetate decarboxylase Na+ pump and its individual subunits in Escherichia coli and analysis of their function. 764 79

The production of biofuels by recombinant Escherichia coli is restricted by the toxicity of the products. 2,3-Butanediol (2,3-BD), a platform and fuel bio-chemical with low toxicity to microbes, could be a promising alternative for biofuel production. However, the yield and productivity of 2,3-BD produced by recombinant E. coli strains are not sufficient for industrial scale fermentation. In this work, the production of 2,3-BD by recombinant E. coli strains was optimized by applying a systematic approach. 2,3-BD biosynthesis gene clusters were cloned from several native 2,3-BD producers, including Bacillus subtilis, Bacillus licheniformis, Klebsiella pneumoniae, Serratia marcescens, and Enterobacter cloacae, inserted into the expression vector pET28a, and compared for 2,3-BD synthesis. The recombinant strain E. coli BL21/pETPT7-EcABC, carrying the 2,3-BD pathway gene cluster from Enterobacter cloacae, showed the best ability to synthesize 2,3-BD. Thereafter, expression of the most efficient gene cluster was optimized by using different promoters, including PT7, Ptac, Pc, and Pabc. E. coli BL21/pET-RABC with Pabc as promoter was superior in 2,3-BD synthesis. On the basis of the results of biomass and extracellular metabolite profiling analyses, fermentation conditions, including pH, agitation speed, and aeration rate, were optimized for the efficient production of 2,3-BD. After fed-batch fermentation under the optimized conditions, 73.8g/L of 2,3-BD was produced by using E. coli BL21/pET-RABC within 62h. The values of both yield and productivity of 2,3-BD obtained with the optimized biological system are the highest ever achieved with an engineered E. coli strain. In addition to the 2,3-BD production, the systematic approach might also be used in the production of other important chemicals through recombinant E. coli strains.
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PMID:Systematic metabolic engineering of Escherichia coli for high-yield production of fuel bio-chemical 2,3-butanediol. 2452 31