Marker Exchange Mutagenesis of mxaF , Encoding the Large Subunit of the Mxa Methanol Dehydrogenase, in Methylosinus trichosporium OB3b

Journal Article · Mon Dec 28 00:00:00 EST 2015 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/AEM.03615-15· OSTI ID:1470729

Farhan Ul Haque, Muhammad ^[1]; Gu, Wenyu ^[2]; DiSpirito, Alan A. ^[3]; Semrau, Jeremy D. ^[2]

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1. Univ. of Michigan, Ann Arbor, MI (United States). Dept. of Civil and Environmental Engineering; Univ. of Michigan, Ann Arbor, MI (United States).
2. Univ. of Michigan, Ann Arbor, MI (United States). Dept. of Civil and Environmental Engineering
3. Iowa State Univ., Ames, IA (United States). Roy J. Carver Dept of Biochemistry, Biophysics and Molecular Biology

Here, methanotrophs have remarkable redundancy in multiple steps of the central pathway of methane oxidation to carbon dioxide. For example, it has been known for over 30 years that two forms of methane monooxygenase, responsible for oxidizing methane to methanol, exist in methanotrophs, i.e., soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), and that expression of these two forms is controlled by the availability of copper. Specifically, sMMO expression occurs in the absence of copper, while pMMO expression increases with increasing copper concentrations. More recently, it was discovered that multiple forms of methanol dehydrogenase (MeDH), Mxa MeDH and Xox MeDH, also exist in methanotrophs and that the expression of these alternative forms is regulated by the availability of cerium. That is, expression of Xox MeDH increases in the presence of cerium, while Mxa MeDH expression decreases in the presence of cerium. As it had been earlier concluded that pMMO and Mxa MeDH form a supercomplex in which electrons from Mxa MeDH are back donated to pMMO to drive the initial oxidation of methane, we speculated that Mxa MeDH could be rendered inactive through marker-exchange mutagenesis but growth on methane could still be possible if cerium was added to increase the expression of Xox MeDH under sMMO-expressing conditions. Here we report that mxaF, encoding the large subunit of Mxa MeDH, could indeed be knocked out in Methylosinus trichosporium OB3b, yet growth on methane was still possible, so long as cerium was added. Interestingly, growth of this mutant occurred in both the presence and the absence of copper, suggesting that Xox MeDH can replace Mxa MeDH regardless of the form of MMO expressed.

• Research Organization: Univ. of Michigan, Ann Arbor, MI (United States).
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
• Grant/Contract Number: SC0006630
• OSTI ID: 1470729
• Journal Information: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 5 Vol. 82; ISSN 0099-2240
• Publisher: American Society for Microbiology
• Country of Publication: United States
• Language: English

Related Subjects

54 ENVIRONMENTAL SCIENCES
59 BASIC BIOLOGICAL SCIENCES