Significance: A great challenge in microbial ecology lies in determining the underlying mechanisms that drive interactions in complex natural communities. In this study we used bacterial isolates from Lake Washington sediment that are important for the utilization of the greenhouse gas methane. We show how simple cocultures can be used to identify mechanisms involved in cross-feeding in microbial communities; these mechanisms cannot be deduced from pure cultures alone. We demonstrate that the presence of one species alters gene expression and metabolism in another species such that the second species excretes a carbon and energy source to sustain the cross-fed species. This mode of cross-feeding based on partner-induced altered gene expression may have important implications for microbial interactions in the environment.

Many methylotrophic taxa harbor two distinct methanol dehydrogenase (MDH) systems for oxidizing methanol to formaldehyde: the well-studied calcium-dependent MxaFI type and the more recently discovered lanthanide-containing XoxF type. MxaFI has traditionally been accepted as the major functional MDH in bacteria that contain both enzymes. However, in this study, we present evidence that, in a type I methanotroph, Methylomicrobium buryatense , XoxF is likely the primary functional MDH in the environment. The addition of lanthanides increases xoxF expression and greatly reduces mxa expression, even under conditions in which calcium concentrations are almost 100-fold higher than lanthanide concentrations. Mutations in genes encoding the MDH enzymes validate our finding that XoxF is the major functional MDH, as XoxF mutants grow more poorly than MxaFI mutants under unfavorable culturing conditions. In addition, mutant and transcriptional analyses demonstrate that the lanthanide-dependent MDH switch operating in methanotrophs is mediated in part by the orphan response regulator MxaB, whose gene transcription is itself lanthanide responsive. IMPORTANCE Aerobic methanotrophs, bacteria that oxidize methane for carbon and energy, require a methanol dehydrogenase enzyme to convert methanol into formaldehyde. The calcium-dependent enzyme MxaFI has been thought to primarily carry out methanol oxidation in methanotrophs. Recently, it was discovered that XoxF, a lanthanide-containing enzyme present in most methanotrophs, can also oxidize methanol. In a methanotroph with both MxaFI and XoxF, we demonstrate that lanthanides transcriptionally control genes encoding the two methanol dehydrogenases, in part by controlling expression of the response regulator MxaB. Lanthanides are abundant in the Earth's crust, and we demonstrate that micromolar amounts of lanthanides are sufficient to suppress MxaFI expression. Thus, we present evidence that XoxF acts as the predominant methanol dehydrogenase in a methanotroph.

Methane-utilizing bacteria (methanotrophs) are capable of growth on methane and are attractive systems for bio-catalysis. However, the application of natural methanotrophic strains to large-scale production of value-added chemicals/biofuels requires a number of physiological and genetic alterations. An accurate metabolic model coupled with flux balance analysis can provide a solid interpretative framework for experimental data analyses and integration. A stoichiometric flux balance model of Methylomicrobium buryatense strain 5G(B1) was constructed and used for evaluating metabolic engineering strategies for biofuels and chemical production with a methanotrophic bacterium as the catalytic platform. The initial metabolic reconstruction was based on whole-genome predictions. Each metabolic step was manually verified, gapfilled, and modified in accordance with genome-wide expression data. The final model incorporates a total of 841 reactions (in 167 metabolic pathways). Of these, up to 400 reactions were recruited to produce 118 intracellular metabolites. The flux balance simulations suggest that only the transfer of electrons from methanol oxidation to methane oxidation steps can support measured growth and methane/oxygen consumption parameters, while the scenario employing NADH as a possible source of electrons for particulate methane monooxygenase cannot. Direct coupling between methane oxidation and methanol oxidation accounts for most of the membrane-associated methane monooxygenase activity. However the best fit to experimental results is achieved only after assuming that the efficiency of direct coupling depends on growth conditions and additional NADH input (about 0.1–0.2 mol of incremental NADH per one mol of methane oxidized). The additional input is proposed to cover loss of electrons through inefficiency and to sustain methane oxidation at perturbations or support uphill electron transfer. Finally, the model was used for testing the carbon conversion efficiency of different pathways for C1-utilization, including different variants of the ribulose monophosphate pathway and the serine cycle. We demonstrate that the metabolic model can provide an effective tool for predicting metabolic parameters for different nutrients and genetic perturbations, and as such, should be valuable for metabolic engineering of the central metabolism of M. buryatense strains.

Methane is a feedstock of interest for the future, both from natural gas and from renewable biogas sources. Methanotrophic bacteria have the potential to enable commercial methane bioconversion to value-added products such as fuels and chemicals. A strain of interest for such applications is Methylomicrobium buryatense 5GB1, due to its robust growth characteristics. However, to take advantage of the potential of this methanotroph, it is important to generate comprehensive bioreactor-based datasets for different growth conditions to compare bioprocess parameters. Datasets of growth parameters, gas utilization rates, and products (total biomass, extracted fatty acids, glycogen, excreted acids) were obtained for cultures of M. buryatense 5GB1 grown in continuous culture under methane limitation and O2 limitation conditions. Additionally, experiments were performed involving unrestricted batch growth conditions with both methane and methanol as substrate. All four growth conditions show significant differences. The most notable changes are the high glycogen content and high formate excretion for cells grown on methanol (batch), and high O2:CH4 utilization ratio for cells grown under methane limitation. The results presented here represent the most comprehensive published bioreactor datasets for a gamma-proteobacterial methanotroph. This information shows that metabolism by M. buryatense 5GB1 differs significantly for each of the four conditions tested. O2 limitation resulted in the lowest relative O2 demand and fed-batch growth on methane the highest. Future studies are needed to understand the metabolic basis of these differences. However, these results suggest that both batch and continuous culture conditions have specific advantages, depending on the product of interest.

The rapid increase of the potent greenhouse gas methane in the atmosphere creates great urgency to develop and deploy technologies for methane mitigation. One approach to removing methane is to use bacteria for which methane is their carbon and energy source (methanotrophs). Such bacteria naturally convert methane to CO ₂ and biomass, a value-added product and a cobenefit of methane removal. Typically, methanotrophs grow best at around 5,000 to 10,000 ppm methane, but methane in the atmosphere is 1.9 ppm. Air above emission sites such as landfills, anaerobic digestor effluents, rice paddy effluents, and oil and gas wells contains elevated methane in the 500 ppm range. If such sites are targeted for methane removal, technology harnessing aerobic methanotroph metabolism has the potential to become economically and environmentally viable. The first step in developing such methane removal technology is to identify methanotrophs with enhanced ability to grow and consume methane at 500 ppm and lower. We report here that some existing methanotrophic strains grow well at 500 ppm methane, and one of them, Methylotuvimicrobium buryatense 5GB1C, consumes such low methane at enhanced rates compared to previously published values. Analyses of bioreactor-based performance and RNAseq-based transcriptomics suggest that this ability to utilize low methane is based at least in part on extremely low non-growth-associated maintenance energy and on high methane specific affinity. This bacterium is a candidate to develop technology for methane removal at emission sites. If appropriately scaled, such technology has the potential to slow global warming by 2050.