Physiological Influence of Fe and Cu Availability on Nitrogen Isotope Fractionation during Ammonia Oxidation.

Microbially mediated cycling processes play central roles in regulating the speciation and availability of nitrogen, a vital nutrient with wide implications for agriculture, water quality, wastewater treatment, ecosystem health, and climate change. Ammonia oxidation, the first and rate-limiting step of nitrification, is carried out by bacteria (AOB) and archaea (AOA) that require the trace metal micronutrients copper (Cu) and iron (Fe) for growth and metabolic catalysis. While stable isotope analyses for constraining nitrogen cycling are commonly used, it is unclear whether metal availability may modulate expression of stable isotope fractionation during ammonia oxidation, by varying growth or through regulation of metabolic metalloenzymes. We present the first study examining the influence of Fe and Cu availability on the kinetic nitrogen isotope effect in ammonia oxidation (15εAO). We report a general independence of 15εAO from the growth rate in AOB, except at a low temperature (10 °C). With AOA Nitrosopumilus maritimus SCM1, however, 15εAO decreases nonlinearly at lower oxidation rates. We examine assumptions involved in the interpretation of 15εAO values and suggest these dynamics may arise from physiological constraints that push the system toward isotopic equilibrium. These results suggest important links between isotope fractionation and environmental constraints on physiology in these key N cycling microorganisms.

Ratio of Electron Donor to Acceptor Influences Metabolic Specialization and Denitrification Dynamics in Pseudomonas aeruginosa in a Mixed Carbon Medium.

Denitrifying microbes sequentially reduce nitrate (NO3 -) to nitrite (NO2 -), NO, N2O, and N2 through enzymes encoded by nar, nir, nor, and nos. Some denitrifiers maintain the whole four-gene pathway, but others possess partial pathways. Partial denitrifiers may evolve through metabolic specialization whereas complete denitrifiers may adapt toward greater metabolic flexibility in nitrogen oxide (NOx -) utilization. Both exist within natural environments, but we lack an understanding of selective pressures driving the evolution toward each lifestyle. Here we investigate differences in growth rate, growth yield, denitrification dynamics, and the extent of intermediate metabolite accumulation under varying nutrient conditions between the model complete denitrifier Pseudomonas aeruginosa and a community of engineered specialists with deletions in the denitrification genes nar or nir. Our results in a mixed carbon medium indicate a growth rate vs. yield tradeoff between complete and partial denitrifiers, which varies with total nutrient availability and ratios of organic carbon to NOx -. We found that the cultures of both complete and partial denitrifiers accumulated nitrite and that the metabolic lifestyle coupled with nutrient conditions are responsible for the extent of nitrite accumulation.

H-Aquil: a chemically defined cell culture medium for trace metal studies in Vibrios and other marine heterotrophic bacteria.

A variety of trace metals, including prominently iron (Fe) are necessary for marine microorganisms. Chemically defined medium recipes have been used for several decades to study phytoplankton, but similar methods have not been adopted as widely in studies of marine heterotrophic bacteria. Medium recipes for these organisms frequently include tryptone, casamino acids, as well as yeast and animal extracts. These components introduce unknown concentrations of trace elements and organic compounds, complicating metal speciation. Minimal medium recipes utilizing known carbon and nitrogen sources do exist but often have high background trace metal concentrations. Here we present H-Aquil, a version of the phytoplankton medium Aquil adapted for marine heterotrophic bacteria. This medium consists of artificial seawater supplemented with a carbon source, phosphate, amino acids, and vitamins. As in Aquil, trace metals are controlled using the synthetic chelator EDTA. We also address concerns of EDTA toxicity, showing that concentrations up to 100 µM EDTA do not lead to growth defects in the copiotrophic bacterium Vibrio harveyi or the oligotrophic bacterium Candidatus Pelagibacter ubique HTCC1062, a member of the SAR11 clade. H-Aquil is used successfully to culture species of Vibrio, Phaeobacter, and Silicibacter, as well as several environmental isolates. We report a substantial decrease in growth rate between cultures grown with or without added Fe, making the medium suitable for conducting Fe-limitation studies in a variety of marine heterotrophic bacteria.