The malate dehydrogenase of Ralstonia eutropha and functionality of the C(3)/C(4) metabolism in a Tn5-induced mdh mutant.

The Tn5-induced mutant VG12 of Ralstonia eutropha HF39, which was isolated in this study, revealed an interesting phenotype: it grew on fructose and pyruvate as well as autotrophically like the wild-type, whereas growth on tricarboxylic acid intermediates and glyoxylic acid was reduced, and no growth occurred if acetate, propionate or levulinate were provided as carbon source. Tn5 was mapped in a gene encoding an NAD(H)-dependent malate dehydrogenase (MDH), and MDH activity was strongly diminished in VG12. Furthermore, the mdh gene was cloned, sequenced and heterologously expressed in Escherichia coli, conferring significantly higher specific MDH activity to the recombinant strain. The phenotype of VG12 sheds light on the C(3)/C(4) metabolism of R. eutropha, which mediates between the Entner-Doudoroff pathway and the tricarboxylic acid cycle (TCC), demonstrating that enzymes catalyzing the conversion of C(3) and C(4) metabolites can circumvent the metabolic disruption of the TCC in VG12 and that the phosphoenolpyruvate carboxykinase serves a dual and important function.

[1]  J. Weissenbach,et al.  Genome sequence of the plant pathogen Ralstonia solanacearum , 2002, Nature.

[2]  A. Steinbüchel,et al.  The methylcitric acid pathway in Ralstonia eutropha: new genes identified involved in propionate metabolism. , 2001, Microbiology.

[3]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[4]  Yunje Cho,et al.  Structural Basis for Cold Adaptation , 1999, The Journal of Biological Chemistry.

[5]  R. Fleischmann,et al.  Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. , 1995, Science.

[6]  M. Nishiyama,et al.  Determinants of protein thermostability observed in the 1.9-A crystal structure of malate dehydrogenase from the thermophilic bacterium Thermus flavus. , 1993, Biochemistry.

[7]  A. Abdelal,et al.  Complete sequence of the Salmonella typhimurium gene encoding malate dehydrogenase. , 1993, Gene.

[8]  M. Hall,et al.  Crystal structure of Escherichia coli malate dehydrogenase. A complex of the apoenzyme and citrate at 1.87 A resolution. , 1992, Journal of molecular biology.

[9]  A. Steinbüchel,et al.  Isolation of prokaryotic RNA and detection of specific mRNA with biotinylated probes , 1990 .

[10]  L. Banaszak,et al.  Refined crystal structure of cytoplasmic malate dehydrogenase at 2.5-A resolution. , 1989, Biochemistry.

[11]  L. Hood,et al.  Specific-primer-directed DNA sequencing. , 1986, Analytical biochemistry.

[12]  B. Bowien,et al.  Unusual C3 and C4 metabolism in the chemoautotroph Alcaligenes eutrophus , 1984, Journal of bacteriology.

[13]  D. Hanahan.  Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[14]  S. Srivastava,et al.  Mutagenesis of Alcaligenes eutrophus by insertion of the drug-resistance transposon Tn5 , 1982, Archives of Microbiology.

[15]  B. Friedrich,et al.  Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus , 1981, Journal of bacteriology.

[16]  B. Hohn,et al.  A small cosmid for efficient cloning of large DNA fragments. , 1980, Gene.

[17]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.

[18]  F. Bolivar.  Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules. , 1978, Gene.

[19]  B. Hohn,et al.  Packaging recombinant DNA molecules into bacteriophage particles in vitro. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[20]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[21]  W. Tanner.  Methodon der enzymatischen Analyse, Hans Ulrich Bergmeyer (Ed.). Verl. Chemie, Weinheim (1974) , 1975 .

[22]  D. Hogness,et al.  Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[23]  H. Schlegel,et al.  Zur Synthese von C4-Dicarbonsäuren aus Pyruvat durch Hydrogenomonas eutropha Stamm H 16 , 1971, Archiv für Mikrobiologie.

[24]  J. Marmur.  A procedure for the isolation of deoxyribonucleic acid from micro-organisms , 1961 .

[25]  H. Schlegel,et al.  Synthese der Enzyme des Tricarbonsäure-Cyclus in Hydrogenomonas eutropha Stamm H 16 , 2004, Archiv für Mikrobiologie.

[26]  H. Schlegel,et al.  Ein Submersverfahren zur Kultur wasserstoffoxydierender Bakterien: Wachstumsphysiologische Untersuchungen , 2004, Archiv für Mikrobiologie.

[27]  R. Simon.  High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon , 2004, Molecular and General Genetics MGG.

[28]  V. Pirrotta,et al.  Microdissection and cloning of DNA from a specific region of Drosophila melanogaster polytene chromosomes , 2004, Chromosoma.

[29]  K. Entian,et al.  Cloning and sequence of the mdh structural gene of Escherichia coli coding for malate dehydrogenase , 2004, Archives of Microbiology.

[30]  Amos Bairoch,et al.  The PROSITE database, its status in 2002 , 2002, Nucleic Acids Res..

[31]  Christian O. Bra,et al.  The methylcitric acid pathway in Ralstonia eutropha: new genes identified involved in propionate metabolism , 2001 .

[32]  Gapped BLAST and PSI-BLAST: A new , 1997 .

[33]  A. Bairoch,et al.  The PROSITE database, its status in 1997 , 1997, Nucleic Acids Res..

[34]  W. Bullock.  XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. , 1987 .

[35]  A. Pühler,et al.  A Broad Host Range Mobilization System for In Vivo Genetic Engineering: Transposon Mutagenesis in Gram Negative Bacteria , 1983, Bio/Technology.

[36]  W. Frings,et al.  [Synthesis of C 4 -dicarboxylic acids from pyruvate by Hydrogenomonas eutropha strain H16]. , 1971, Archiv fur Mikrobiologie.

[37]  H. Bergmeyer.  Methoden der enzymatischen Analyse , 1962 .