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Methylococcus capsulatus strain Bath, a methane-oxidizing bacterium, and ammonia-oxidizing bacteria (AOB) carry out the first step of nitrification, the oxidation of ammonia to nitrite, through the intermediate hydroxylamine. AOB use hydroxylamine oxidoreductase (HAO) to produce nitrite. M. capsulatus Bath was thought to oxidize hydroxylamine with cytochrome P460 (cytL), until the recent discovery of an hao gene in its genome. We used quantitative PCR analyses of cDNA from M. capsulatus Bath incubated with CH(4) or CH(4) plus 5 mM (NH(4))(2)SO(4) to determine whether cytL and hao transcript levels change in response to ammonia. While mRNA levels for cytL were not affected by ammonia, hao mRNA levels increased by 14.5- and 31-fold in duplicate samples when a promoter proximal region of the transcript was analyzed, and by sixfold when a region at the distal end of the transcript was analyzed. A conserved open reading frame, orf2, located 3' of hao in all known AOB genomes and in M. capsulatus Bath, was cotranscribed with hao and showed increased mRNA levels in the presence of ammonia. These data led to designating this gene pair as haoAB, with the role of haoB still undefined. We also determined mRNA levels for additional genes that encode proteins involved in N-oxide detoxification: cytochrome c'-beta (CytS) and nitric oxide (NO) reductase (NorCB). Whereas cytS mRNA levels increased in duplicate samples by 28.5- and 40-fold in response to ammonia, the cotranscribed norC-norB mRNA did not increase. Our results strongly suggest that M. capsulatus Bath possesses a functional, ammonia-responsive HAO involved in nitrification.

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Traditionally, methane (CH4) emission from terrestrial plants is thought to originate from belowground microbial metabolism under anaerobic conditions, with subsequent transport to the atmosphere through stems. However, a recent study reported aerobic CH4 emission from plants by an unrecognized process, a result that has since been questioned. We investigated CH4 emissions under aerobic conditions from aboveground tissues of 44 species indigenous to the temperate Inner Mongolia steppe. Ten herbaceous hydrophytes (wetland-adapted plants) were examined, two of which--Glyceria spiculosa and Scirpus yagara--emitted CH4 from stems but not from detached leaves. Of 34 xerophytes (arid-adapted plants) examined, 7 out of 9 shrub species emitted CH4 from detached leaves but not stems, whereas none of 25 herbaceous xerophytes emitted CH4. The herbaceous hydrophyte, S. yagara, emitted highly 13C-depleted CH4, suggesting a microbial origin. Achillea frigida exhibited the highest CH4 emission rates among the shrubs and continuously emitted relatively 13C-enriched CH4 from detached leaves, indicating that CH4 was derived directly from plant tissues under aerobic conditions. Because woody species are relatively rare in the Inner Mongolia steppe, aerobic, plant-derived CH4 emission is probably negligible in this region. Our results may imply a larger role for aerobic CH4 production in upland ecosystems dominated by woody species or in ecosystems where woody encroachment is occurring as a result of global change.

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Unprecedented agricultural intensification and increased crop yield will be necessary to feed the burgeoning world population, whose global food demand is projected to double in the next 50 years. Although grain production has doubled in the past four decades, largely because of the widespread use of synthetic nitrogenous fertilizers, pesticides, and irrigation promoted by the "Green Revolution," this rate of increased agricultural output is unsustainable because of declining crop yields and environmental impacts of modern agricultural practices. The last 20 years have seen diminishing returns in crop yield in response to increased application of fertilizers, which cannot be completely explained by current ecological models. A common strategy to reduce dependence on nitrogenous fertilizers is the production of leguminous crops, which fix atmospheric nitrogen via symbiosis with nitrogen-fixing rhizobia bacteria, in rotation with nonleguminous crops. Here we show previously undescribed in vivo evidence that a subset of organochlorine pesticides, agrichemicals, and environmental contaminants induces a symbiotic phenotype of inhibited or delayed recruitment of rhizobia bacteria to host plant roots, fewer root nodules produced, lower rates of nitrogenase activity, and a reduction in overall plant yield at time of harvest. The environmental consequences of synthetic chemicals compromising symbiotic nitrogen fixation are increased dependence on synthetic nitrogenous fertilizer, reduced soil fertility, and unsustainable long-term crop yields.

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To determine whether repeated, long-term NH(4) (+) fertilization alters the enzymatic function of the atmospheric CH(4) oxidizer community in soil, we examined CH(4) uptake kinetics in temperate pine and hardwood forest soils amended with 150 kg N ha(-1) y(-1) as NH(4)NO(3) for more than a decade. The highest rates of atmospheric CH(4) consumption occurred in the upper 5 cm mineral soil of the control plots. In contrast to the results of several previous studies, surface organic soils in the control plots also exhibited high consumption rates. Fertilization decreased in situ CH(4) consumption in the pine and hardwood sites relative to the control plots by 86% and 49%, respectively. Fertilization increased net N mineralization and relative nitrification rates and decreased CH(4) uptake most dramatically in the organic horizon, which contributed substantially to the overall decrease in field flux rates. In all cases, CH(4) oxidation followed Michaelis-Menten kinetics, with apparent K(m) (K(m(app))) values typical of high-affinity soil CH(4) oxidizers. Both K(m(app)) and V(max(app)) were significantly lower in fertilized soils than in unfertilized soils. The physiology of the methane consumer community in the fertilized soils was distinct from short-term responses to NH(4) (+) addition. Whereas the immediate response to NH(4) (+) was an increase in K(m(app)), resulting from apparent enzymatic substrate competition, the long-term response to fertilization was a community-level shift to a lower K(m(app)), a possible adaptation to diminish the competitiveness of NH(4) (+) for enzyme active sites.

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