A method for introduction of unmarked mutations in the genome of Paracoccus denitrificans: construction of strains with multiple mutations in the genes encoding periplasmic cytochromes c550, c551i, and c553i

A new suicide vector, pRVS1, was constructed to facilitate the site-directed introduction of unmarked mutations in the chromosome of Paracoccus denitrificans. The vector was derived from suicide vector pGRPd1, which was equipped with the lacZ gene encoding beta-galactosidase. The reporter gene was found to be a successful screening marker for the discrimination between plasmid integrant strains and mutant strains which had lost the plasmid after homologous recombination. Suicide vectors pGRPd1 and pRVS1 were used in gene replacement techniques for the construction of mutant strains with multiple mutations in the cycA, moxG, and cycB genes encoding the periplasmic cytochromes c550, c551i, and c553i, respectively. Southern analyses of the DNA and protein analyses of the resultant single, double, and triple mutant strains confirmed the correctness of the mutations. The wild type and mutant strains were all able to grow on succinate and choline chloride. In addition, all strains grew on methylamine and displayed wild-type levels of methylamine dehydrogenase activities. cycA mutant strains, however, showed a decreased maximum specific growth rate on the methylamine substrate. The wild-type strain, cycA and cycB mutant strains, and the cycA cycB double mutant strain were able to grow on methanol and showed wild-type levels of methanol dehydrogenase activities. moxG mutant strains failed to grow on methanol and had low levels of methanol dehydrogenase activities. The maximum specific growth rate of the cycA mutant strain on methanol was comparable with that of the wild-type strain. The data indicate the involvement of the soluble cytochromes c in clearly defined electron transport routes.

[1]  R. V. van Spanning,et al.  Mutagenesis of the gene encoding amicyanin of Paracoccus denitrificans and the resultant effect on methylamine oxidation , 1990, FEBS letters.

[2]  H. V. van Verseveld,et al.  The respiratory chain and energy conservation in the mitochondrion-like bacterium Paracoccus denitrificans. , 1987, Microbiological sciences.

[3]  B. Ludwig,et al.  Paracoccus denitrificans cytochrome c1 gene replacement mutants , 1990, Journal of bacteriology.

[4]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[5]  J. Vieira,et al.  The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. , 1982, Gene.

[6]  C. W. Jones,et al.  Bacterial respiration. , 1977, Bacteriological reviews.

[7]  C. M. Joyce,et al.  Method for determining whether a gene of Escherichia coli is essential: application to the polA gene , 1984, Journal of bacteriology.

[8]  A. H. Stouthamer,et al.  Isolation and characterization of ubiquinol oxidase complexes from Paracoccus denitrificans cells cultured under various limiting growth conditions in the chemostat. , 1987, European journal of biochemistry.

[9]  J Messing,et al.  A system for shotgun DNA sequencing. , 1981, Nucleic acids research.

[10]  P. Thomas,et al.  An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. , 1976, Analytical biochemistry.

[11]  S. Falkow,et al.  The construction of a cloning vector designed for gene replacement in Bordetella pertussis. , 1986, Gene.

[12]  R. V. van Spanning,et al.  Mutagenesis of the gene encoding cytochrome c550 of Paracoccus denitrificans and analysis of the resultant physiological effects , 1990, Journal of bacteriology.

[13]  G. C. Barr,et al.  A method for transformation of Paracoccus denitrificans , 1981 .

[14]  V. Davidson,et al.  An inducible periplasmic blue copper protein from Paracoccus denitrificans. Purification, properties, and physiological role. , 1985, The Journal of biological chemistry.

[15]  W. Hagen,et al.  A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy. , 1980, Biochimica et biophysica acta.

[16]  V. Davidson,et al.  Characterization of two inducible periplasmic c-type cytochromes from Paracoccus denitrificans. , 1986, The Journal of biological chemistry.

[17]  A. H. Stouthamer,et al.  Subfractionation and characterization of soluble c-type cytochromes from Paracoccus denitrificans cultured under various limiting conditions in the chemostat. , 1987, European journal of biochemistry.

[18]  J. G. Morris,et al.  Studies on the utilization of nitrate by Micrococcus denitrificans. , 1962, Journal of general microbiology.

[19]  A. Pühler,et al.  Vector Plasmids for in-Vivo and in-Vitro Manipulations of Gram-Negative Bacteria , 1983 .

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

[21]  A. Puustinen,et al.  Cytochrome o (bo) is a proton pump in Paracoccus denitrificans and Escherichia coli , 1989, FEBS letters.

[22]  V. Davidson,et al.  Cytochrome c‐550 mediates electron transfer from inducible periplasmic c‐type cytochromes to the cytoplasmic membrane of Paracoccus denitrificans , 1989, FEBS letters.

[23]  V. Davidson,et al.  Purification and properties of methylamine dehydrogenase from Paracoccus denitrificans , 1987, Journal of bacteriology.

[24]  C. Anthony,et al.  Characterization of the periplasmic cytochromes c of Paracoccus denitrificans: identification of the electron acceptor for methanol dehydrogenase, and description of a novel cytochrome c heterodimer , 1991 .

[25]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[26]  L. Smith,et al.  Monoclonal antibodies to cytochrome c from Paracoccus denitrificans: effects on electron transport reactions. , 1985, Biochimica et biophysica acta.

[27]  D. Kell,et al.  The Electron Transport System and Hydrogenase of Paracoccus denitrificans , 1981 .

[28]  S. Cohen,et al.  Molecular and Genetic Studies of an R Factor System Consisting of Independent Transfer and Drug Resistance Plasmids , 1973, Journal of bacteriology.

[29]  Philip John,et al.  Paracoccus denitrificans and the evolutionary origin of the mitochondrion , 1975, Nature.

[30]  I. Wilson,et al.  The cellular location and specificity of bacterial cytochrome c peroxidases. , 1990, The Biochemical journal.

[31]  M. Saraste,et al.  Are there isoenzymes of cytochrome c oxidase in Paracoccus denitrificans? , 1990, FEBS letters.

[32]  C. Anthony,et al.  The Biochemistry of Methylotrophs , 1982 .

[33]  A. H. Stouthamer,et al.  Isolation and nucleotide sequence of the methanol dehydrogenase structural gene from Paracoccus denitrificans , 1987, Journal of bacteriology.

[34]  E. Southern Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[35]  M. Finel,et al.  Deletion of the gene for subunit III leads to defective assembly of bacterial cytochrome oxidase. , 1989, The EMBO journal.

[36]  B. Trumpower,et al.  Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes. , 1985, The Journal of biological chemistry.