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

ABSTRACTMethanotrophs 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 thatmxaF, encoding the large subunit of Mxa MeDH, could indeed be knocked out inMethylosinus trichosporiumOB3b, 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.

Chloromethane-Dependent Expression of the cmu Gene Cluster of Hyphomicrobium chloromethanicum

ABSTRACT The methylotrophic bacterium Hyphomicrobium chloromethanicum CM2 can utilize chloromethane (CH3Cl) as the sole carbon and energy source. Previously genes cmuB, cmuC, cmuA, and folD were shown to be essential for the growth of Methylobacterium chloromethanicum on CH3Cl. These CH3Cl-specific genes were subsequently detected in H. chloromethanicum. Transposon and marker exchange mutagenesis studies were carried out to identify the genes essential for CH3Cl metabolism in H. chloromethanicum. New developments in genetic manipulation of Hyphomicrobium are presented in this study. An electroporation protocol has been optimized and successfully applied for transformation of mutagenesis plasmids into H. chloromethanicum to generate stable CH3Cl-negative mutants. Both transposon and marker exchange mutageneses were highly applicable for genetic analysis of Hyphomicrobium. A reliable and reproducible selection procedure for screening of CH3Cl utilization-negative mutants has also been developed. Mutational inactivation of cmuB, cmuC, or hutI resulted in strains that were unable to utilize CH3Cl or to express the CH3Cl-dependent polypeptide CmuA. Reverse transcription-PCR analysis indicated that cmuB, cmuC, cmuA, fmdB, paaE, hutI, and metF formed a single cmuBCA-metF operon and were coregulated and coexpressed in H. chloromethanicum. This finding led to the conclusion that, in cmuB and cmuC mutants, impaired expression of cmuA was likely to be due to a polar effect of the defective gene (cmuB or cmuC) located upstream (5′) of cmuA. The detrimental effect of mutation in hutI on the upstream (5′)-located cmuA is not clear but indicated that all the genes located within the cmuBCA-metF operon are coordinately expressed. Expression of the cmuBCA-metF transcript was also shown to be strictly CH3Cl inducible and was not repressed by the alternative C1 substrate methanol. Sequence analysis of a transposon mutant (D20) led to the discovery of the previously undetected hutI and metF genes located 3′ of the paaE gene in H. chloromethanicum. MetF, a putative methylene-tetrahydrofolate reductase, had 27% identity to MetF from M. chloromethanicum. Mutational and transcriptional analysis data indicated that, in H. chloromethanicum, CH3Cl is metabolized via a corrinoid-specific (cmuA) and tetrahydrofolate-dependent (metF, purU, folD) methyltransfer system.

rpoN, mmoR and mmoG, genes involved in regulating the expression of soluble methane monooxygenase in Methylosinus trichosporium OB3b

The methanotrophic bacterium Methylosinus trichosporium OB3b converts methane to methanol using two distinct forms of methane monooxygenase (MMO) enzyme: a cytoplasmic soluble form (sMMO) and a membrane-bound form (pMMO). The transcription of these two operons is known to proceed in a reciprocal fashion with sMMO expressed at low copper-to-biomass ratios and pMMO at high copper-to-biomass ratios. Transcription of the smmo operon is initiated from a σ N promoter 5′ of mmoX. In this study the genes encoding σ N (rpoN) and a typical σ N-dependent transcriptional activator (mmoR) were cloned and sequenced. mmoR, a regulatory gene, and mmoG, a gene encoding a GroEL homologue, lie 5′ of the structural genes for the sMMO enzyme. Subsequent mutation of rpoN and mmoR by marker-exchange mutagenesis resulted in strains Gm1 and JS1, which were unable to express functional sMMO or initiate transcription of mmoX. An rpoN mutant was also unable to fix nitrogen or use nitrate as sole nitrogen source, indicating that σ N plays a role in both nitrogen and carbon metabolism in Ms. trichosporium OB3b. The data also indicate that mmoG is transcribed in a σ N- and MmoR-independent manner. Marker-exchange mutagenesis of mmoG revealed that MmoG is necessary for smmo gene transcription and activity and may be an MmoR-specific chaperone required for functional assembly of transcriptionally competent MmoR in vivo. The data presented allow the proposal of a more complete model for copper-mediated regulation of smmo gene expression.