Methylotenera mobilis is a recently described bacterium that is a prominent member of the active methylotroph community in Lake Washington sediment. More recent experiments suggested that M. mobilis and related bacteria carry out denitrification linked to methanol oxidation, in the presence of oxygen, resulting in emission of N2O. This is a novel process that may be an important part of the nitrogen cycle in lakes. Genomic analysis of M. mobilis identified few traditional components of the classic denitrification pathway, suggesting involvement of alternative enzymes. The bacterium also lacks a traditional methanol dehydrogenase. This phenomenon is interesting for a number of reasons. First, this is the first case of denitrification by a member of the Methylophilaceae family; second, oxygen-dependent denitrification is unusual in the microbial world; third, denitrification by Methylotenera and related organisms may represent an important biogeochemical process that has been overlooked, accounting for a portion of N2O released into the atmosphere from the environments active in C1 metabolism, such as freshwater lakes. The major goal of this project is to understand the biochemistry and the physiology of denitrification by Methylotenera and to uncover the specific nature of coupling of denitrification to methanol oxidation. This work will present new paradigms of both methylotrophy and denitrification and will provide new insights into the connection between the global cycles of carbon and nitrogen. M. mobilis will be the model system. The specific objectives are 1) identification of genes, enzymes and pathways involved in denitrification and methanol oxidation, 2) testing the functions of the genes predicted to be involved in both methanol oxidation and denitrification, via mutation, and 3) purification and characterization of one or more key enzymes involved in denitrification by M. mobilis, the primary candidate being a novel periplasmic nitrate reductase. The intellectual merit of this proposal is in obtaining biochemical insights into methylotrophy linked to denitrification, transforming the paradigms of both processes and broadening the knowledge on energy-generating pathways in microbes in general.
Broader Impacts:
This project will involve graduate, undergraduate and high school students, with special emphasis on the members of underrepresented groups. Established partnerships with local schools and colleges will provide the public outreach. The data will be broadly disseminated via educational materials, conference presentations, scientific and popular publications and the dedicated web site. The societal impact will be in providing biochemical and genomic insights into a novel, previously overlooked biogeochemical process that potentially contributes to climate change.
Intellectual merit: As part of this project, we made significant advances in understanding methylotrophy and nitrogen metabolism in novel bacteria named Methylotenera, and more broadly in bacteria representing the family Methylophilaceae. The major advances are as follows. (1) We elucidated the components of the denitrification pathway in Methylotenera mobilis strain JLW8, our main model, via mutant analysis, followed by enzyme activity measurements and metabolite detection, uncovering the pathway’s involvement in nitrogen assimilation, but finding the pathway not essential for energy generation. (2) Trough mutant analysis, we defined the roles of two protein homologs, XoxF1 and XoxF2, as novel methanol dehydrogenase enzymes, and demonstrated their involvement in methanol metabolism. (3) Using next-generation sequencing-enabled metagenomics, we obtained evidence for Methylotenera being involved in metabolism of methane, likely through cooperation with methane-oxidizing species, Methylobacter, with major implications for understanding the methane cycle. (4) Trough comparative transcriptomic analysis of a number of divergent Methylophilaceae, we uncovered alternative metabolic strategies utilized by closely related species in environmental conditions, suggesting niche-specific or condition-specific roles for different ecotypes of Methylophilaceae. (5) We isolated, characterized and sequenced genomes of a number of Methylophilaceae, identifying five novel ecotypes and identifying alternative configurations of carbon and nitrogen metabolism modules. (6) To directly address the proposed cooperative behavior between Methylophilaceae and Methylococcaceae, we carried out microcosm manipulations and identified the major Methylophilaceae partners in these relationships. Overall, the findings from this project provide a dramatically new and complex outlook at genotypic, phenotypic and metabolic diversity of Methylophilaceae, and suggest an important role for Methylophilaceae in global carbon and nitrogen cycling and specifically in conversion of methane, as part of synergistic community metabolism. A total of fifteen manuscripts have resulted from this project, including invited reviews, book chapters and general interest manuscripts. Data were presented at numerous national and international conferences. Broader impacts: Two post-doctoral fellows, two graduate students (one female, one visiting international student), one international scholar from a developing country (female), two undergraduate students (both female) and three high school students (two female) have been trained as part of this project. One former postdoctoral fellow is now in a colleague’s lab, gaining additional experience before starting his independent career. The second postdoctoral fellow has returned to the country of origin (Russia) to carry out independent research as a PI, funded by the Russian Federation. One former graduate student is employed in a University system. The visiting graduate student is returning to the country of origin (Russia) to complete his PhD program. One former undergraduate student is now graduate student of Biomedical Informatics at the Oregon Health & Science University. The second undergraduate student will be starting graduate school with a focus on bioengineering. One former high school is currently a sophomore at MIT (with fellowship), at the department of Biological Engineering. The second high school student is a freshwoman at University of Notre Dame, with major in biology and minor in bioengineering. The third high school student has been accepted to a number of top-tier colleges, and she is in the process of making her final decision. Results from this project have been included into courses taught to graduate, undergraduate and high school students and have been broadly disseminated through conference presentations and through primary research publications, review articles and book chapters.
