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Plasmid-dependent methylotrophy in thermotolerant Bacillus methanolicus
Trygve Brautaset1, Øyvind M Jakobsen M, Michael C Flickinger
1Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim. SINTEF Applied Chemistry, SINTEF, N-7043 Trondheim, Norway. trygve.brautaset@biotech.ntnu.no
Journal of Bacteriology
|February 20, 2004
Summary
Bacillus methanolicus uses methanol as a carbon source, facilitated by genes on plasmid pBM19. This plasmid is essential for methanol utilization, revealing plasmid-linked methylotrophy in this bacterium.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Bacillus methanolicus is a thermotolerant bacterium that efficiently utilizes methanol as a sole carbon source.
- It is classified as a restrictive methylotroph, with limited known pathways for sugar utilization.
- Methanol metabolism is critical for its growth, particularly at optimal temperatures of 50°C.
Purpose of the Study:
- To characterize the plasmid pBM19 isolated from B. methanolicus MGA3.
- To identify genes on pBM19 involved in methanol assimilation.
- To investigate the role of pBM19 in enabling methylotrophy in B. methanolicus.
Main Methods:
- DNA sequencing and characterization of the 19,167-bp plasmid pBM19.
- Identification and analysis of genes encoded by pBM19, including methanol dehydrogenase (mdh) and genes for the ribulose monophosphate pathway.
- Construction of a shuttle vector (pTB1.9) for genetic manipulation and transformation of B. methanolicus.
- Curing B. methanolicus of pBM19 and assessing the impact on methanol utilization.
Main Results:
- Plasmid pBM19 contains the methanol dehydrogenase (mdh) gene and five other genes (pfk, rpe, tkt, glpX, fba) involved in methanol assimilation via the ribulose monophosphate pathway.
- B. methanolicus strains cured of pBM19 lost the ability to grow on methanol.
- Reintroduction of the mdh gene alone did not restore methanol growth, indicating the necessity of other pBM19-encoded genes.
- All 13 screened thermotolerant B. methanolicus strains harbored similar plasmids.
Conclusions:
- Plasmid pBM19 is essential for methanol utilization in B. methanolicus, hosting key genes for methylotrophy.
- This study reports the first instance of plasmid-linked methylotrophy in microorganisms.
- The findings necessitate a re-evaluation of genetic manipulation strategies for B. methanolicus and advance the understanding of methylotroph biology.