Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named 'Candidatus Methylomirabilis oxyfera', was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that 'M. oxyfera' bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis.

Nitrous oxide (N2O) is an environmentally important atmospheric trace gas because it is an effective greenhouse gas and it leads to ozone depletion through photo-chemical nitric oxide (NO) production in the stratosphere. Mitigating its steady increase in atmospheric concentration requires an understanding of the mechanisms that lead to its formation in natural and engineered microbial communities. N2O is formed biologically from the oxidation of hydroxylamine (NH2OH) or the reduction of nitrite (NO−2) to NO and further to N2O. Our review of the biological pathways for N2O production shows that apparently all organisms and pathways known to be involved in the catabolic branch of microbial N-cycle have the potential to catalyze the reduction of NO−2 to NO and the further reduction of NO to N2O, while N2O formation from NH2OH is only performed by ammonia oxidizing bacteria (AOB). In addition to biological pathways, we review important chemical reactions that can lead to NO and N2O formation due to the reactivity of NO−2, NH2OH, and nitroxyl (HNO). Moreover, biological N2O formation is highly dynamic in response to N-imbalance imposed on a system. Thus, understanding NO formation and capturing the dynamics of NO and N2O build-up are key to understand mechanisms of N2O release. Here, we discuss novel technologies that allow experiments on NO and N2O formation at high temporal resolution, namely NO and N2O microelectrodes and the dynamic analysis of the isotopic signature of N2O with quantum cascade laser absorption spectroscopy (QCLAS). In addition, we introduce other techniques that use the isotopic composition of N2O to distinguish production pathways and findings that were made with emerging molecular techniques in complex environments. Finally, we discuss how a combination of the presented tools might help to address important open questions on pathways and controls of nitrogen flow through complex microbial communities that eventually lead to N2O build-up.

Most microorganisms live in environments where nutrients are limited and fluctuate over time. Cells respond to nutrient fluctuations by sensing and adapting their physiological state. Recent studies suggest phenotypic heterogeneity(1) in isogenic populations as an alternative strategy in fluctuating environments, where a subpopulation of cells express a function that allows growth under conditions that might arise in the future(2-9). It is unknown how environmental factors such as nutrient limitation shape phenotypic heterogeneity in metabolism and whether this allows cells to respond to nutrient fluctuations. Here, we show that substrate limitation increases phenotypic heterogeneity in metabolism, and this heterogeneity allows cells to cope with substrate fluctuations. We subjected the N2-fixing bacterium Klebsiella oxytoca to different levels of substrate limitation and substrate shifts, and obtained time-resolved single-cell measurements of metabolic activities using nanometre-scale secondary ion mass spectrometry (NanoSIMS). We found that the level of NH4(+) limitation shapes phenotypic heterogeneity in N2 fixation. In turn, the N2 fixation rate of single cells during NH4(+) limitation correlates positively with their growth rate after a shift to NH4(+) depletion, experimentally demonstrating the benefit of heterogeneity. The results indicate that phenotypic heterogeneity is a general solution to two important ecological challenges-nutrient limitation and fluctuations-that many microorganisms face.

The intestinal microbiota features intricate metabolic interactions involving the breakdown and reuse of host- and diet-derived nutrients. The competition for these resources can limit pathogen growth. Nevertheless, some enteropathogenic bacteria can invade this niche through mechanisms that remain largely unclear. Using a mouse model for Salmonella diarrhea and a transposon mutant screen, we discovered that initial growth of Salmonella Typhimurium (S. Tm) in the unperturbed gut is powered by S. Tm hyb hydrogenase, which facilitates consumption of hydrogen (H2), a central intermediate of microbiota metabolism. In competitive infection experiments, a hyb mutant exhibited reduced growth early in infection compared to wild-type S. Tm, but these differences were lost upon antibiotic-mediated disruption of the host microbiota. Additionally, introducing H2-consuming bacteria into the microbiota interfered with hyb-dependent S. Tm growth. Thus, H2 is an Achilles' heel of microbiota metabolism that can be subverted by pathogens and might offer opportunities to prevent infection.

Permeable or sandy sediments cover the majority of the seafloor on continental shelves worldwide, but little is known about their role in the coastal nitrogen cycle. We investigated the rates and controls of nitrogen loss at a sand flat (Janssand) in the central German Wadden Sea using multiple experimental approaches, including the nitrogen isotope pairing technique in intact core incubations, slurry incubations, a flow-through stirred retention reactor and microsensor measurements. Results indicate that permeable Janssand sediments are characterized by some of the highest potential denitrification rates (X0.19 mmol N m À2 h À1 ) in the marine environment. Moreover, several lines of evidence showed that denitrification occurred under oxic conditions. In intact cores, microsensor measurements showed that the zones of nitrate/nitrite and O 2 consumption overlapped. In slurry incubations conducted with 15 NO 3 À enrichment in gas-impermeable bags, denitrification assays revealed that N 2 production occurred at initial O 2 concentrations of up to B90 lM. Initial denitrification rates were not substantially affected by O 2 in surficial (0-4 cm) sediments, whereas rates increased by twofold with O 2 depletion in the at 4-6 cm depth interval. In a well mixed, flow-through stirred retention reactor (FTSRR), 29 N 2 and 30 N 2 were produced and O 2 was consumed simultaneously, as measured online using membrane inlet mass spectrometry. We hypothesize that the observed high denitrification rates in the presence of O 2 may result from the adaptation of denitrifying bacteria to recurrent tidally induced redox oscillations in permeable sediments at Janssand.