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Journal of Bacteriology, March 2004, p. 1229-1238, Vol. 186, No. 5
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.5.1229-1238.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus

Trygve Brautaset,1* Øyvind M. Jakobsen,1,2 Michael C. Flickinger,3 Svein Valla,1 and Trond E. Ellingsen2

Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim,1 SINTEF Applied Chemistry, SINTEF, N-7043 Trondheim, Norway,2 BioTechnology Institute, Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, Minnesota 551083

Received 8 October 2003/ Accepted 20 November 2003

Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50°C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs.

* Corresponding author. Mailing address: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway. Phone: 47 73 59 86 89. Fax: 47 73 59 12 83. E-mail: trygve.brautaset{at}biotech.ntnu.no.

Journal of Bacteriology, March 2004, p. 1229-1238, Vol. 186, No. 5
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.5.1229-1238.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jakobsen, O. M., Brautaset, T., Degnes, K. F., Heggeset, T. M. B., Balzer, S., Flickinger, M. C., Valla, S., Ellingsen, T. E. (2009). Overexpression of Wild-Type Aspartokinase Increases L-Lysine Production in the Thermotolerant Methylotrophic Bacterium Bacillus methanolicus. Appl. Environ. Microbiol. 75: 652-661 [Abstract] [Full Text]  
  • Jakobsen, O. M., Benichou, A., Flickinger, M. C., Valla, S., Ellingsen, T. E., Brautaset, T. (2006). Upregulated Transcription of Plasmid and Chromosomal Ribulose Monophosphate Pathway Genes Is Critical for Methanol Assimilation Rate and Methanol Tolerance in the Methylotrophic Bacterium Bacillus methanolicus.. J. Bacteriol. 188: 3063-3072 [Abstract] [Full Text]  


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