The Impact of Nitrite on Aerobic Growth of Paracoccus denitrificans PD1222

The effect of nitrite stress induced in Paracoccus denitrificans PD1222 was examined using additions of sodium nitrite to an aerobic bacterial culture. Nitrite generates a strong stress response in P. denitrificans, causing growth inhibition. This is dependent on both the concentration of nitrite present and the pH. The pH dependent effect of nitrite growth inhibition is likely a result of nitrite and free nitrous acid (FNA; pKa = 3.16) and subsequent reactive nitrogen oxides, generated from the intracellular passage of FNA into P. denitrificans. A flavohemoglobin (fhb; Pd1689) and its associated NsrR family, transcriptional regulator (Pd1690), were transcribed above a ≥2 fold expression filter (p ≤ 0.05) at 95% significance in qRT-PCR and a type II microarray transcriptional analysis at 12.5 mM nitrite in batch culture. Additionally, >25 fold expression of the flavohemoglobin was confirmed by qRT-PCR in continuous culture with nitrite. A deletion mutant determined fhb to be involved in conveying nitrite resistance at high nitrite concentrations and is linked to a stimulation of biomass generated by the presence of nitrite. The cytochrome ba3 oxidase was found to be associated with nitrite in transcriptional analysis, suggesting the uncoupling of the protonmotive force caused by the transport of FNA across the membrane, and subsequent dissociation in the cytoplasm were reduced by a method of counterbalance. No nitrate accumulation was seen and nitrous oxide levels were above that observed for atmospheric background levels. The microarray analysis was used to confirm that in batch growth at 12.5 mM nitrite addition, P. denitrificans shows an overall stress response associated with protein, DNA and lipid repair, with the addition of fhb detoxification and action of the cytochrome ba3 oxidase. It is therefore suggested that nitrite presents a pHdependent stress response in P. denitrificans, likely due to the production of associated reactive nitrogen species such as NO from the internalisation of FNA and the uncoupling of the protonmotive force

Trace Metal Availability Affects Greenhouse Gas Emissions and Microbial Functional Group Abundance in Freshwater Wetland Sediments

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 (NO3–, NO2–, N2O, NH4+) and carbon mineralization (CO2, CH4), 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 N2O 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, CO2 production was unaffected, but the amendments stimulated net CH4 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

The metabolic impact of extracellular nitrite on aerobic metabolism of Paracoccus denitrificans

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 (FNA) (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 the 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 NO from the internalisation of FNA

Juvenile African Clawed Frogs (Xenopus laevis) Express Growth, Metamorphosis, Mortality, Gene Expression, and Metabolic Changes When Exposed to Thiamethoxam and Clothianidin

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