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Volume 150, Issue 1

Development of an insertional expression vector system for AM1 and generation of null mutants lacking and/or Free

Abstract

Over the past few years, the genetic ‘toolkit’ available for use with AM1 has expanded significantly. Here a further advance is presented and demonstrated, an insertional expression system that allows expression of genes from a stable, unmarked chromosomal locus. This system has been used to better understand the role of the tetrahydrofolate (HF) pathway in methylotrophy. Previously, it has not been possible to generate null mutants lacking either (encoding an NADP-dependent methylene-HF/methylene-tetrahydromethanopterin dehydrogenase) or (encoding methenyl-HF cyclohydrolase). An unmarked strain was generated that expressed the analogous gene (encoding a bifunctional NADP-dependent methylene-HF dehydrogenase/methenyl-HF cyclohydrolase) from CM4. In this strain, null mutants could be obtained that grew normally on multicarbon substrates but were defective for growth on C substrates. Additionally, null mutants of and/or could also be generated in the wild-type by supplementing the succinate medium with formate. These strains were unable to grow on C compounds but were not methanol-sensitive. These approaches have demonstrated that the apparent essentiality of and is due to the need for formyl-HF for biosynthesis of purines and other compounds, and have provided clear genetic evidence that the HF pathway is required for methylotrophy.

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Keyword(s): gfp/GFP, green fluorescent protein , H4F, tetrahydrofolate , H4MPT, tetrahydromethanopterin , MCS, multiple-cloning site , P prefix, promoter and t prefix, terminator of transcription
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2004-01-01
2025-04-27
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References

  1. Attwood M. M., Harder W. 1972; A rapid and specific enrichment procedure for Hyphomicrobium spp. Antonie Van Leeuwenhoek 38:369–377 [CrossRef]
     [Google Scholar]
  2. Chambers S. P., Prior S. E., Barstow D. A., Minton N. P. 1988; The pMTL nic− cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68:139–149 [CrossRef]
     [Google Scholar]
  3. Chistoserdov A. Y., Chistoserdova L. V., McIntire W. S., Lidstrom M. E. 1994; Genetic organization of the mau gene cluster in Methylobacterium extorquens AM1: complete nucleotide sequence and generation and characteristics of mau mutants. J Bacteriol 176:4052–4065
     [Google Scholar]
  4. Chistoserdova L., Vorholt J. A., Thauer R. K., Lidstrom M. E. 1998; C1 transfer enzymes and coenzymes linking methylotrophic bacteria and methanogenic Archaea. Science 281:99–102 [CrossRef]
     [Google Scholar]
  5. Chistoserdova L., Chen S. W., Lapidus A., Lidstrom M. E. 2003; Methylotrophy in Methylobacterium extorquens AM1 from a genomic point of view. J Bacteriol 185:2980–2987 [CrossRef]
     [Google Scholar]
  6. Chistoserdova L. V., Lidstrom M. E. 1994a; 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. J Bacteriol 176:7398–7404
     [Google Scholar]
  7. Chistoserdova L. V., Lidstrom M. E. 1994b; Genetics of the serine cycle in Methylobacterium extorquens AM1: identification of sgaA and mtdA and sequences of sgaA, hprA, and mtdA. J Bacteriol 176:1957–1968
     [Google Scholar]
  8. Chistoserdova L. V., Lidstrom M. E. 1996; Molecular characterization of a chromosomal region involved in the oxidation of acetyl-CoA to glyoxylate in the isocitrate-lyase-negative methylotroph Methylobacterium extorquens AM1. . Microbiology 142:1459–1468 [CrossRef]
     [Google Scholar]
  9. Figurski D. H., Helinski D. R. 1979; Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76:1648–1652 [CrossRef]
     [Google Scholar]
  10. Hagemeier C. H., Chistoserdova L., Lidstrom M. E., Thauer R. K., Vorholt J. A. 2000; Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1. . Eur J Biochem 267:3762–3769 [CrossRef]
     [Google Scholar]
  11. Kalb V. F., Bernlohr R. W. 1977; A new spectrophotometric assay for protein in cell extracts. Anal Biochem 82:362–371 [CrossRef]
     [Google Scholar]
  12. Kataeva I. M., Golovleva L. A. 1990; Catechol 2,3-dioxygenases from Pseudomonas aeruginosa 2x. Methods Enzymol 188:115–121
     [Google Scholar]
  13. Large P. J., Peel D., Quayle J. R. 1961; Microbial growth on C1 compounds. 2. Synthesis of cell constituents by methanol- and formate-grown Pseudomonas AM1, and methanol-grown Hyphomicrobium vulgare. Biochem J 81:470–479
     [Google Scholar]
  14. Laukel M., Chistoserdova L., Lidstrom M. E., Vorholt J. A. 2003; The tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1: purification and properties. Eur J Biochem 270:325–333 [CrossRef]
     [Google Scholar]
  15. Lidstrom M. E. 2001; Aerobic methylotrophic prokaryotes. In The Prokaryotes, posting date 2 November, 2001 Edited by Dworkin M. http://141.150.157.117:8080/prokPUB/chaprender/jsp/showchap.jsp?chapnum=300&initsec=01_00
     [Google Scholar]
  16. Marison I. W., Attwood M. M. 1982; A possible alternative mechanism for the oxidation of formaldehyde to formate. J Gen Microbiol 128:1441–1446
     [Google Scholar]
  17. Marx C. J., Lidstrom M. E. 2001; Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria. Microbiology 147:2065–2075
     [Google Scholar]
  18. Marx C. J., Lidstrom M. E. 2002; Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria. BioTechniques 33:1062–1067
     [Google Scholar]
  19. Marx C. J., Laukel M., Vorholt J. A., Lidstrom M. E. 2003a; Purification of the formate-tetrahydrofolate ligase from Methylobacterium extorquens AM1 and demonstration of its requirement for methylotrophic growth. J Bacteriol 185:7169–7175 [CrossRef]
     [Google Scholar]
  20. Marx C. J., O'Brien B. N., Breezee J., Lidstrom M. E. 2003b; Novel methylotrophy genes of Methylobacterium extorquens AM1 identified by using transposon mutagenesis including a putative dihydromethanopterin reductase. J Bacteriol 185:669–673 [CrossRef]
     [Google Scholar]
  21. Nagy P. L., Marolewski A., Benkovic S. J., Zalkin H. 1995; Formyltetrahydrofolate hydrolase: a regulatory enzyme that functions to balance pools of tetrahydrofolate and one-carbon tetrahydrofolate adducts in Escherichia coli. J Bacteriol 177:1292–1298
     [Google Scholar]
  22. Nunn D. N., Lidstrom M. E. 1986; Isolation and complementation analysis of 10 methanol oxidation mutant classes and identification of the methanol dehydrogenase structural gene of Methylobacterium sp. strain AM1. . J Bacteriol 166:581–590
     [Google Scholar]
  23. Palmeros B., Wild J., Szybalski W., Le Borgne S., Hernandez-Chavez G., Gosset G., Valle F., Bolivar F. 2000; A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria. Gene 247:255–264 [CrossRef]
     [Google Scholar]
  24. Pomper B. K., Vorholt J. A. 2001; Characterization of the formyltransferase from Methylobacterium extorquens AM1. . Eur J Biochem 268:4769–4775 [CrossRef]
     [Google Scholar]
  25. Pomper B. K., Vorholt J. A., Chistoserdova L., Lidstrom M. E., Thauer R. K. 1999; A methenyl tetrahydromethanopterin cyclohydrolase and a methenyl tetrahydrofolate cyclohydrolase in Methylobacterium extorquens AM1. . Eur J Biochem 261:475–480 [CrossRef]
     [Google Scholar]
  26. Pomper B. K., Saurel O., Milon A., Vorholt J. A. 2002; Generation of formate by the formyltransferase/hydrolase complex (Fhc) from Methylobacterium extorquens AM1. . FEBS Lett 523:133–137 [CrossRef]
     [Google Scholar]
  27. Purdy D., O'Keeffe T. A., Elmore M., Herbert M., McLeod A., Bokori-Brown M., Ostrowski A., Minton N. P. 2002; Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier. Mol Microbiol 46:439–452 [CrossRef]
     [Google Scholar]
  28. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  29. Simon R., Priefer U., Puhler A. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1:784–791 [CrossRef]
     [Google Scholar]
  30. Studer A., McAnulla C., Buchele R., Leisinger T., Vuilleumier S. 2002; Chloromethane-induced genes define a third C1 utilization pathway inMethylobacterium chloromethanicum CM4. . J Bacteriol 184:3476–3484 [CrossRef]
     [Google Scholar]
  31. Toyama H., Anthony C., Lidstrom M. E. 1998; Construction of insertion and deletion mxa mutants of Methylobacterium extorquens AM1 by electroporation. FEMS Microbiol Lett 166:1–7 [CrossRef]
     [Google Scholar]
  32. Vannelli T., Messmer M., Studer A., Vuilleumier S., Leisinger T. 1999; A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane. Proc Natl Acad Sci U S A 96:4615–4620 [CrossRef]
     [Google Scholar]
  33. Vorholt J. A. 2002; Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria. Arch Microbiol 178:239–249 [CrossRef]
     [Google Scholar]
  34. Vorholt J. A., Chistoserdova L., Lidstrom M. E., Thauer R. K. 1998; The NADP-dependent methylene tetrahydromethanopterin dehydrogenase in Methylobacterium extorquens AM1. . J Bacteriol 180:5351–5356
     [Google Scholar]
  35. Vorholt J. A., Marx C. J., Lidstrom M. E., Thauer R. K. 2000; Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol. J Bacteriol 182:6645–6650 [CrossRef]
     [Google Scholar]
  36. Whitaker J. R., Granum P. E. 1980; An absolute method for protein determination based on the difference at 235 and 280 nm. Anal Biochem 109:156–159 [CrossRef]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.26587-0
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Development of an insertional expression vector system for Methylobacterium extorquens AM1 and generation of null mutants lacking mtdA and/or fch
Microbiology 150, 9 (2004); https://doi.org/10.1099/mic.0.26587-0
/content/journal/micro/10.1099/mic.0.26587-0
/content/journal/micro/10.1099/mic.0.26587-0
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