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

Abstract

bMethanotrophs 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. In methanotrophy, the central oxidation pathway of methane is well-known, with methane first being oxidized to methanol by methane monooxygenase (MMO) and methanol further being converted to formaldehyde by methanol dehydrogenase (MeDH). Subsequently, formaldehyde can be combined with either tetrahydrofolate or tetrahydromethanopterin to form methylene tetrahydrofolate and methylene tetrahydromethanopterin, respectively. Methylene tetrahydrofolate can then be assimilated into biomass via the serine cycle. Methylene tetrahydromethanopterin is oxidized to formate, which can be either converted to carbon dioxide via formate dehydrogenase or reduced to methylene tetrahydrofolate. It is speculated that such redundancy for the conversion of formaldehyde exists to control the buildup of this toxic intermediate (1)(2)(3)(4)(5)(6)(7)(8)(9).What is less readily discerned from this simple pathway, however, is that substantial redundancy exists for many steps of the transformation of methane to carbon dioxide and not just the condensation of formaldehyde with either methylene tetrahydrofolate or methylene tetrahydromethanopterin. For example, the first step of methane oxidation (conversion of methane to methanol) can be carried out by two different forms of MMO that are regulated by the availability of copper, i....

Metals and Methanotrophy

“…In rapid succession, a number of papers documented that the expression of Mxa-MeDH and Xox-MeDH in a variety of methanotrophs and methylotrophs was strongly dependent on the availability of REEs, primarily light REEs (124)(125)(126)(127)(128). In the absence of REEs, little expression of xox genes was observed, while the expression of mxa genes was high.…”

Efficient Counterselection for Methylococcus capsulatus (Bath) by Using a Mutated pheS Gene

“…We confirmed the physiological modification of the ΔxoxF mutant by growth experiments. Mxa MDH and Xox MDH require calcium and a rare earth element (such as cerium), respectively, for enzymatic activity (15,(27)(28)(29)(30). When Xox MDH is inactive, M. capsulatus (Bath) depends on Mxa MDH for methanol oxidation.…”

“…In counterselection for gene disruption in methanotrophs, the sacB gene is employed as the only available marker (8,9,(12)(13)(14)(15)(16). Levansucrase, encoded by the sacB gene, converts sucrose into levan, which accumulates in the periplasmic space, thereby causing cell death in Gram-negative bacteria (17).…”

Isolation and Genomic Characterization of a Proteobacterial Methanotroph Requiring Lanthanides

“…S3a and b). A previous study reported that the lanthanide dependency of the ΔmxaF mutant of M. trichosporium OB3b became more conspicuous with repeated subcultures, possibly due to the alleviation of the carryover of lanthanides (Farhan Ul Haque et al, 2016). Hence, each culture was further subjected to repeated subcultures.…”