Summary Bacterial denitrification is a respiratory process that is a major source and sink of the potent greenhouse gas nitrous oxide. Many denitrifying bacteria can adjust to life in both oxic and anoxic environments through differential expression of their respiromes in response to environmental signals such as oxygen, nitrate and nitric oxide. We used steady‐state oxic and anoxic chemostat cultures to demonstrate that the switch from aerobic to anaerobic metabolism is brought about by changes in the levels of expression of relatively few genes, but this is sufficient to adjust the configuration of the respirome to allow the organism to efficiently respire nitrate without the significant release of intermediates, such as nitrous oxide. The regulation of the denitrification respirome in strains deficient in the transcription factors FnrP, Nnr and NarR was explored and revealed that these have both inducer and repressor activities, possibly due to competitive binding at similar DNA binding sites. This may contribute to the fine tuning of expression of the denitrification respirome and so adds to the understanding of the regulation of nitrous oxide emission by denitrifying bacteria in response to different environmental signals.

Nitrite, in equilibrium with free nitrous acid (FNA), can inhibit both aerobic and anaerobic growth of microbial communities through bactericidal activities that have considerable potential for control of microbial growth in a range of water systems. There has been much focus on the effect of nitrite/FNA on anaerobic metabolism and so, to enhance understanding of the metabolic impact of nitrite/FNA on aerobic metabolism, a study was undertaken with a model denitrifying bacterium Paracoccus denitrificans PD1222. Extracellular nitrite inhibits aerobic growth of P. denitrificans in a pH-dependent manner that is likely to be a result of both nitrite and free nitrous acid (pKa = 3.25) and subsequent reactive nitrogen oxides generated from the intracellular passage of FNA into P. denitrificans. Increased expression of a gene encoding a flavohemoglobin protein (Fhp) (Pden_1689) was observed in response to extracellular nitrite. Construction and analysis of a deletion mutant established Fhp to be involved in endowing nitrite/FNA resistance at high extracellular nitrite concentrations. Global transcriptional analysis confirmed nitrite-dependent expression of fhp and indicated that P. denitrificans expressed a number of stress response systems associated with protein, DNA and lipid repair. It is therefore suggested that nitrite causes a pH-dependent stress response that is due to the production of associated reactive nitrogen species, such as nitric oxide from the internalisation of FNA.

We investigated the effects of trace metal additions on microbial nitrogen (N) and carbon (C) cycling using freshwater wetland sediment microcosms amended with micromolar concentrations of copper (Cu), molybdenum (Mo), iron (Fe), and all combinations thereof. In addition to monitoring inorganic N transformations (NO 3 − , NO 2 − , N 2 O, NH 4 +) and carbon mineralization (CO 2 , CH 4), we tracked changes in functional gene abundance associated with denitrification (nirS, nirK, nosZ), dissimilatory nitrate reduction to ammonium (DNRA; nrfA), and methanogenesis (mcrA). With regards to N cycling, greater availability of Cu led to more complete denitrification (i.e., less N 2 O accumulation) and a higher abundance of the nirK and nosZ genes, which encode for Cu-dependent reductases. In contrast, we found sparse biochemical evidence of DNRA activity and no consistent effect of the trace metal additions on nrfA gene abundance. With regards to C mineralization, CO 2 production was unaffected, but the amendments stimulated net CH 4 production and Mo additions led to increased mcrA gene abundance. These findings demonstrate that trace metal effects on sediment microbial physiology can impact community-level function. We observed direct and indirect effects on both N and C biogeochemistry that resulted in increased production of greenhouse gasses, which may have been mediated through the documented changes in microbial community composition and shifts in functional group abundance. Overall, this work supports a more nuanced consideration of metal effects on environmental microbial communities that recognizes the key role that metal limitation plays in microbial physiology.

We investigated the effects of trace metal additions on microbial nitrogen and carbon cycling using freshwater wetland sediment microcosms amended with μM concentrations of copper (Cu), molybdenum (Mo), iron (Fe), and all combinations. In addition to monitoring inorganic nitrogen transformations (NO3−, NO2−, N2O, NH4+) and carbon mineralization (CO2, CH4), we tracked changes in functional gene abundance associated with denitrification (nirS, nirK, nosZ), DNRA (nrfA), and methanogenesis (mcrA). Greater availability of Cu led to more complete denitrification (i.e., less N2O accumulation) and a higher abundance of the nirK and nosZ genes, which encode for Cu-dependent reductases. We found sparse evidence of DNRA activity and no consistent effect on CO2 production. Contrary, net CH4 production was stimulated by the trace metal amendments and the Mo additions, in particular, led to increased mcrA gene abundance. Taken together, these findings demonstrate that trace metal effects on microbial physiology, which have heretofore only been studied in pure culture, can impact community-level function. We observed direct and indirect effects on both nitrogen and carbon biogeochemistry that culminated in increased production of greenhouse gasses, and the shifts in functional group abundance that we documented suggest these responses may have been mediated through changes in microbial community composition. Overall, this work supports a more holistic consideration of metal effects on environmental microbial communities that recognizes the key role that metal limitation plays in microbial physiology.

Neonicotinoids (NEO) represent the main class of insecticides currently in use, with thiamethoxam (THX) and clothianidin (CLO) primarily applied agriculturally. With few comprehensive studies having been performed with non-target amphibians, the aim was to investigate potential biomarker responses along an adverse outcome pathway of NEO exposure, whereby data were collected on multiple biological hierarchies. Juvenile African clawed frogs, Xenopus laevis, were exposed to commercial formulations of THX and CLO at high (100 ppm) and low (20 ppm) concentrations of the active ingredient. Mortality, growth, development, liver metabolic enzyme activity, and gene expression endpoints were quantified. Tadpoles (n > 1000) from NF 47 through tail resorption stage (NF 66) were exposed to NEO or to NEO-free media treatments. Liver cell reductase activity and cytotoxicity were quantified by flow cytometry. Compared to control reference gene expressions, levels of expression for NEO receptor subunits, cell structure, function, and decontamination processes were measured by RT-qPCR by using liver and brain. Mortality in THX high was 21.5% compared to the control (9.1%); the metabolic conversion of THX to CLO may explain these results. The NF 57 control tadpoles were heavier, longer, and more developed than the others. The progression of development from NF 57–66 was reduced by THX low, and weight gain was impaired. Liver reductases were highest in the control (84.1%), with low NEO exhibiting the greatest reductions; the greatest cytotoxicity was seen with THX high. More transcriptional activity was noted in brains than in livers. Results affirm the utility of a study approach that considers multiple complexities in ecotoxicological studies with non-target amphibians, underscoring the need for simultaneously considering NEO concentration-response relationships with both whole-organism and biomarker endpoints.