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. 1999 Aug;152(4):1429–1437. doi: 10.1093/genetics/152.4.1429

Isolation of acetate auxotrophs of the methane-producing archaeon Methanococcus maripaludis by random insertional mutagenesis.

W Kim 1, W B Whitman 1
PMCID: PMC1460683  PMID: 10430573

Abstract

To learn more about autotrophic growth of methanococci, we isolated nine conditional mutants of Methanococcus maripaludis after transformation of the wild type with a random library in pMEB.2, a suicide plasmid bearing the puromycin-resistance cassette pac. These mutants grew poorly in mineral medium and required acetate or complex organic supplements such as yeast extract for normal growth. One mutant, JJ104, was a leaky acetate auxotroph. A plasmid, pWDK104, was recovered from this mutant by electroporation of a plasmid preparation into Escherichia coli. Transformation of wild-type M. maripaludis with pWDK104 produced JJ104-1, a mutant with the same phenotype as JJ104, thus establishing that insertion of pWDK104 into the genome was responsible for the phenotype. pWDK104 contained portions of the methanococcal genes encoding an ABC transporter closely related to MJ1367-MJ1368 of M. jannaschii. Because high levels of molybdate, tungstate, and selenite restored growth to wild-type levels, this transporter may be specific for these oxyanions. A second acetate auxotroph, JJ117, had an absolute growth requirement for either acetate or cobalamin, and wild-type growth was observed only in the presence of both. Cobinamide, 5', 6'-dimethylbenzimidazole, and 2-aminopropanol did not replace cobalamin. This phenotype was correlated with tandem insertions in the genome but not single insertions and appeared to have resulted from an indirect effect on cobamide metabolism. Plasmids rescued from other mutants contained portions of ORFs denoted in M. jannaschii as endoglucanase (MJ0555), transketolase (MJ0681), thiamine biosynthetic protein thiI (MJ0931), and several hypothetical proteins (MJ1031, MJ0835, and MJ0835.1).

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Selected References

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  1. Balch W. E., Fox G. E., Magrum L. J., Woese C. R., Wolfe R. S. Methanogens: reevaluation of a unique biological group. Microbiol Rev. 1979 Jun;43(2):260–296. doi: 10.1128/mr.43.2.260-296.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berghofer Y., Klein A. Insertional Mutations in the Hydrogenase vhc and frc Operons Encoding Selenium-Free Hydrogenases in Methanococcus voltae. Appl Environ Microbiol. 1995 May;61(5):1770–1775. doi: 10.1128/aem.61.5.1770-1775.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blank C. E., Kessler P. S., Leigh J. A. Genetics in methanogens: transposon insertion mutagenesis of a Methanococcus maripaludis nifH gene. J Bacteriol. 1995 Oct;177(20):5773–5777. doi: 10.1128/jb.177.20.5773-5777.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bult C. J., White O., Olsen G. J., Zhou L., Fleischmann R. D., Sutton G. G., Blake J. A., FitzGerald L. M., Clayton R. A., Gocayne J. D. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science. 1996 Aug 23;273(5278):1058–1073. doi: 10.1126/science.273.5278.1058. [DOI] [PubMed] [Google Scholar]
  5. Chen H. P., Marsh E. N. How enzymes control the reactivity of adenosylcobalamin: effect on coenzyme binding and catalysis of mutations in the conserved histidine-aspartate pair of glutamate mutase. Biochemistry. 1997 Jun 24;36(25):7884–7889. doi: 10.1021/bi970169y. [DOI] [PubMed] [Google Scholar]
  6. Daniels L., Belay N., Rajagopal B. S. Assimilatory reduction of sulfate and sulfite by methanogenic bacteria. Appl Environ Microbiol. 1986 Apr;51(4):703–709. doi: 10.1128/aem.51.4.703-709.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gardner W. L., Whitman W. B. Expression vectors for Methanococcus maripaludis: overexpression of acetohydroxyacid synthase and beta-galactosidase. Genetics. 1999 Aug;152(4):1439–1447. doi: 10.1093/genetics/152.4.1439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gernhardt P., Possot O., Foglino M., Sibold L., Klein A. Construction of an integration vector for use in the archaebacterium Methanococcus voltae and expression of a eubacterial resistance gene. Mol Gen Genet. 1990 Apr;221(2):273–279. doi: 10.1007/BF00261731. [DOI] [PubMed] [Google Scholar]
  9. Gorris L. G., van der Drift C. Cofactor contents of methanogenic bacteria reviewed. Biofactors. 1994 May;4(3-4):139–145. [PubMed] [Google Scholar]
  10. Horlacher R., Xavier K. B., Santos H., DiRuggiero J., Kossmann M., Boos W. Archaeal binding protein-dependent ABC transporter: molecular and biochemical analysis of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis. J Bacteriol. 1998 Feb;180(3):680–689. doi: 10.1128/jb.180.3.680-689.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jarrell K. F., Bayley D. P., Florian V., Klein A. Isolation and characterization of insertional mutations in flagellin genes in the archaeon Methanococcus voltae. Mol Microbiol. 1996 May;20(3):657–666. doi: 10.1046/j.1365-2958.1996.5371058.x. [DOI] [PubMed] [Google Scholar]
  12. Jovell R. J., Macario A. J., Conway de Macario E. ABC transporters in Archaea: two genes encoding homologs of the nucleotide-binding components in the methanogen Methanosarcina mazei S-6. Gene. 1996 Oct 3;174(2):281–284. doi: 10.1016/0378-1119(96)00249-1. [DOI] [PubMed] [Google Scholar]
  13. Ladapo J., Whitman W. B. Method for isolation of auxotrophs in the methanogenic archaebacteria: role of the acetyl-CoA pathway of autotrophic CO2 fixation in Methanococcus maripaludis. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5598–5602. doi: 10.1073/pnas.87.15.5598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Larson J. L., Hershberger C. L. Insertion of plasmids into the chromosome of Streptomyces griseofuscus. Plasmid. 1990 May;23(3):252–256. doi: 10.1016/0147-619x(90)90058-k. [DOI] [PubMed] [Google Scholar]
  15. Law J., Buist G., Haandrikman A., Kok J., Venema G., Leenhouts K. A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes. J Bacteriol. 1995 Dec;177(24):7011–7018. doi: 10.1128/jb.177.24.7011-7018.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Leloup L., Ehrlich S. D., Zagorec M., Morel-Deville F. Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes. Appl Environ Microbiol. 1997 Jun;63(6):2117–2123. doi: 10.1128/aem.63.6.2117-2123.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marsh E. N., Holloway D. E. Cloning and sequencing of glutamate mutase component S from Clostridium tetanomorphum. Homologies with other cobalamin-dependent enzymes. FEBS Lett. 1992 Sep 28;310(2):167–170. doi: 10.1016/0014-5793(92)81321-c. [DOI] [PubMed] [Google Scholar]
  18. Nakashita H, Watanabe K, Hara O, Hidaka T, Seto H. Studies on the biosynthesis of bialaphos. Biochemical mechanism of C-P bond formation: discovery of phosphonopyruvate decarboxylase which catalyzes the formation of phosphonoacetaldehyde from phosphonopyruvate. J Antibiot (Tokyo) 1997 Mar;50(3):212–219. [PubMed] [Google Scholar]
  19. SAITO H., MIURA K. I. PREPARATION OF TRANSFORMING DEOXYRIBONUCLEIC ACID BY PHENOL TREATMENT. Biochim Biophys Acta. 1963 Aug 20;72:619–629. [PubMed] [Google Scholar]
  20. Sandbeck K. A., Leigh J. A. Recovery of an integration shuttle vector from tandem repeats in Methanococcus maripaludis. Appl Environ Microbiol. 1991 Sep;57(9):2762–2763. doi: 10.1128/aem.57.9.2762-2763.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schwartz D., Recktenwald J., Pelzer S., Wohlleben W. Isolation and characterization of the PEP-phosphomutase and the phosphonopyruvate decarboxylase genes from the phosphinothricin tripeptide producer Streptomyces viridochromogenes Tü494. FEMS Microbiol Lett. 1998 Jun 15;163(2):149–157. doi: 10.1111/j.1574-6968.1998.tb13039.x. [DOI] [PubMed] [Google Scholar]
  22. Shieh J., Whitman W. B. Autotrophic acetyl coenzyme A biosynthesis in Methanococcus maripaludis. J Bacteriol. 1988 Jul;170(7):3072–3079. doi: 10.1128/jb.170.7.3072-3079.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Smith D. R., Doucette-Stamm L. A., Deloughery C., Lee H., Dubois J., Aldredge T., Bashirzadeh R., Blakely D., Cook R., Gilbert K. Complete genome sequence of Methanobacterium thermoautotrophicum deltaH: functional analysis and comparative genomics. J Bacteriol. 1997 Nov;179(22):7135–7155. doi: 10.1128/jb.179.22.7135-7155.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Whitman W. B., Sohn S., Kuk S., Xing R. Role of Amino Acids and Vitamins in Nutrition of Mesophilic Methanococcus spp. Appl Environ Microbiol. 1987 Oct;53(10):2373–2378. doi: 10.1128/aem.53.10.2373-2378.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Whitman W. B., Sohn S., Kuk S., Xing R. Role of Amino Acids and Vitamins in Nutrition of Mesophilic Methanococcus spp. Appl Environ Microbiol. 1987 Oct;53(10):2373–2378. doi: 10.1128/aem.53.10.2373-2378.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wilting R., Schorling S., Persson B. C., Böck A. Selenoprotein synthesis in archaea: identification of an mRNA element of Methanococcus jannaschii probably directing selenocysteine insertion. J Mol Biol. 1997 Mar 7;266(4):637–641. doi: 10.1006/jmbi.1996.0812. [DOI] [PubMed] [Google Scholar]
  27. Xavier K. B., Martins L. O., Peist R., Kossmann M., Boos W., Santos H. High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis. J Bacteriol. 1996 Aug;178(16):4773–4777. doi: 10.1128/jb.178.16.4773-4777.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

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