Construction of insertion and deletion mxa mutants of Methylobacterium extorquens AM1 by electroporation.

Methylobacterium extorquens AM1 is a pink-pigmented facultative methylotroph which is widely used for analyzing pathways of C1 metabolism with biochemical and molecular biological techniques. To facilitate this approach, we have applied a new method to construct insertion or disruption mutants with drug resistance genes by electroporation. By using this method, mutants were obtained in four genes present in the mxa methylotrophy gene cluster for which the functions were unknown, mxaR, mxaS, mxaC and mxaD. These mutants were unable to grow on methanol except the mutant of mxaD, which showed reduced growth on methanol.

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

[2]  R. Fleischmann,et al.  The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus , 1997, Nature.

[3]  C. Anthony,et al.  Erratum to "The methanol oxidation genes mxaFJGIR (S) ACKLD in Methylobacterium extorquens" [FEMS Microbiol. Lett. 146 (1997) 31-38]. , 1997, FEMS microbiology letters.

[4]  C. Gliesche.  Transformation of methylotrophic bacteria by electroporation. , 1997, Canadian journal of microbiology.

[5]  S. Porwollik,et al.  The 52'-55' segment of the Bacillus subtilis chromosome: a region devoted to purine uptake and metabolism, and containing the genes cotA, gabP and guaA and the pur gene cluster within a 34960 bp nucleotide sequence , 1996 .

[6]  M. Lidstrom,et al.  Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate in the isocitrate-lyase-negative methylotroph Methylobacterium extorquens AM1. , 1996, Microbiology.

[7]  M. Lidstrom,et al.  Identification and nucleotide sequences of mxaA, mxaC, mxaK, mxaL, and mxaD genes from Methylobacterium extorquens AM1 , 1995, Journal of bacteriology.

[8]  M. Lidstrom,et al.  Genetics of the serine cycle in Methylobacterium extorquens AM1: identification, sequence, and mutation of three new genes involved in C1 assimilation, orf4, mtkA, and mtkB , 1994, Journal of bacteriology.

[9]  M. Lidstrom,et al.  Genetic organization of the mau gene cluster in Methylobacterium extorquens AM1: complete nucleotide sequence and generation and characteristics of mau mutants , 1994, Journal of bacteriology.

[10]  M. Lidstrom,et al.  Genetics of the serine cycle in Methylobacterium extorquens AM1: identification of sgaA and mtdA and sequences of sgaA, hprA, and mtdA , 1994, Journal of bacteriology.

[11]  K. Takahashi,et al.  A novel quinoprotein methanol dehydrogenase containing an additional 32-kilodalton peptide purified from Acetobacter methanolicus: identification of the peptide as a MoxJ product. , 1993, Biochemistry.

[12]  M. Lidstrom,et al.  Genetics of serine pathway enzymes in Methylobacterium extorquens AM1: phosphoenolpyruvate carboxylase and malyl coenzyme A lyase , 1993, Journal of bacteriology.

[13]  C. Anthony,et al.  Characterization of mutant forms of the quinoprotein methanol dehydrogenase lacking an essential calcium ion. , 1992, The Biochemical journal.

[14]  T. Yamane,et al.  Transformation of a Methylotrophic Bacterium, Methylobacterium extorquens, with a Broad-Host-Range Plasmid by Electroporation , 1991, Annals of the New York Academy of Sciences.

[15]  M. Lidstrom,et al.  Isolation and complementation analysis of 10 methanol oxidation mutant classes and identification of the methanol dehydrogenase structural gene of Methylobacterium sp. strain AM1 , 1986, Journal of bacteriology.

[16]  G. Ditta,et al.  Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. , 1985, Plasmid.

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

[18]  J. R. Quayle,et al.  Microbial growth on C1 compounds. I. Isolation and characterization of Pseudomonas AM 1. , 1961, The Biochemical journal.

[19]  J. R. Quayle,et al.  Microbial growth on C1 compounds. II. Synthesis of cell constituents by methanol- and formate-grown Pseudomonas AM 1, and methanol-grown Hyphomicrobium vulgare. , 1961, The Biochemical journal.

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

[21]  C. Anthony,et al.  The methanol oxidation genes mxaFJGIR (S) ACKLD in Methylobacterium extorquens. , 1997, FEMS microbiology letters.

[22]  S. Porwollik,et al.  The 52 degrees-55 degrees segment of the Bacillus subtilis chromosome: a region devoted to purine uptake and metabolism, and containing the genes cotA, gabP and guaA and the pur gene cluster within a 34960 bp nucleotide sequence. , 1996, Microbiology.

[23]  J. Murrell,et al.  Microbial growth on C[1] compounds , 1993 .

[24]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

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

[26]  ScienceDirect,et al.  FEMS microbiology letters , 1977 .

[27]  W. Harder,et al.  A rapid and specific enrichment procedure for Hyphomicrobium spp. , 1972, Antonie van Leeuwenhoek.