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Nitrite oxidoreductase (NXR) is the key enzyme responsible for the oxidation of NO2 to NO3 in nitrite-oxidizing bacteria. For the first time a molecular approach for targeting the nxrA gene was developed, encoding the catalytic subunit of the NXR, to study diversity of Nitrobacter-like organisms based on the phylogeny of nxrA gene sequences in soils. NxrA sequences of the Nitrobacter strains analysed (Nitrobacter hamburgensis, Nitrobacter vulgaris, Nitrobacter winogradskyi, Nitrobacter alkalicus) by PCR, cloning and sequencing revealed the occurrence of multiple copies of nxrA genes in these strains. The copy number and similarity varied among strains. The diversity of Nitrobacter-like nxrA sequences was explored in three soils (a French permanent pasture soil, a French fallow soil, and an African savannah soil) using a cloning and sequencing approach. Most nxrA sequences found in these soils (84%) differed from nxrA sequences obtained from Nitrobacter strains. Moreover, the phylogenetic distribution and richness of nxrAlike sequences was extremely variable depending on soil type. This nxrA tool extends the panel of functional genes available for studying bacteria involved in the nitrogen cycle.

Nitrobacter winogradskyi Nb-255 is a nitrite-oxidizing bacterium that can grow solely on nitrite (NO2−) as a source of energy and nitrogen. In most natural situations, NO2− oxidation is coupled closely to ammonium (NH4+) oxidation by bacteria and archaea and, conceptually, N. winogradskyi can save energy using NH4+ to meet its N-biosynthetic requirements. Interestingly, NH4+ delayed the growth of N. winogradskyi when at concentrations higher than 35 mM, but grew well at concentrations below 25 mM NH4+ while adjusting the expression of 24% of its genes. Notable genes that changed in expression included those with roles in nitrogen and carbon assimilation. Contrary to expectations, higher expression of glutamate synthase (GOGAT), instead of glutamate dehydrogenase, was detected at higher NH4+ concentration. Genes in assimilatory NO2− metabolism and the degradation of glycogen and biofilm/motility were downregulated when N. winogradskyi was grown in the presence of NH4+. Nitrobacter winogradskyi grown in medium with 25 mM NH4+ upregulated genes in post-translational modification, protein turnover, biogenesis and chaperons. The data suggest that N. winogradskyi physiology is modified in the presence of NH4+ and is likely to be modified during coupled nitrification with NH3 oxidizers. Nitrobacter winogradskyi is a nitrite-oxidizing bacterium that grows solely on nitrite. In the presence of ammonium, its physiology changes likely in a manner as during coupled nitrification with ammonia oxidizers.

Copper nanoparticles (CuNPs) are increasingly used in agriculture either alone or in combination with pesticides. Recognizing the potential hazards of CuNPs in soil environments, our study evaluated their effects on the metabolic activity of Nitrobacter winogradskyi ATCC 2539, a chemolithoautotrophic bacterium crucial for the nitrification process, which involves the oxidation of nitrite to nitrate in soil ecosystems. This study evaluated the effects of concentration ranges of CuNPs (2.5 to 162.7 mg L−1), CuONPs (3.2 to 203.6 mg L−1), and various pesticides (iprodione, carbendazim, and 2,4-D) and their derivatives (3,5-dichloroaniline, catechol, and 2,4-dichlorophenol) at concentrations ranging from 0.04 to 2.56 mM. CuSO4 was also used as a control for comparative purposes. Our findings indicated that the CuNPs significantly inhibited the metabolic activity of N. winogradskyi, resulting in a reduction of up to 95% at concentrations of ≥2.5 mg L−1. The CuONPs were less toxic, while the pesticides and their derivatives generally showed lower toxicity. Notably, combinations of CuNPs with pesticides or their derivatives maintained high toxicity levels comparable to those of the CuNPs alone. According to the Loewe additivity model, these effects were largely additive and primarily associated with CuNPs or CuONPs. Protein profiling using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry (MS) revealed that carbendazim induced noticeable changes in protein profiles. These findings underscore the detrimental impacts of CuNPs and CuONPs on the metabolic activity of N. winogradskyi, posing a considerable risk to the health of agricultural soils. Overall, this research provides crucial insights into the risks associated with using CuNPs in agriculture, particularly regarding their potential threat to nitrifying microorganisms in soils.

Quorum sensing (QS) is a widespread process in bacteria used to coordinate gene expression with cell density, diffusion dynamics, and spatial distribution through the production of diffusible chemical signals. To date, most studies on QS have focused on model bacteria that are amenable to genetic manipulation and capable of high growth rates, but many environmentally important bacteria have been overlooked. For example, representatives of proteobacteria that participate in nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, produce QS signals called acyl-homoserine lactones (AHLs). Nitrification emits nitrogen oxide gases (NO, NO 2 , and N 2 O), which are potentially hazardous compounds that contribute to global warming. Despite considerable interest in nitrification, the purpose of QS in the physiology/ecology of nitrifying bacteria is poorly understood. Through a quorum quenching approach, we investigated the role of QS in a well-studied AHL-producing nitrite oxidizer, Nitrobacter winogradskyi . We added a recombinant AiiA lactonase to N. winogradskyi cultures to degrade AHLs to prevent their accumulation and to induce a QS-negative phenotype and then used mRNA sequencing (mRNA-Seq) to identify putative QS-controlled genes. Our transcriptome analysis showed that expression of nirK and nirK cluster genes ( ncgABC ) increased up to 19.9-fold under QS-proficient conditions (minus active lactonase). These data led to us to query if QS influenced nitrogen oxide gas fluxes in N. winogradskyi . Production and consumption of NO x increased and production of N 2 O decreased under QS-proficient conditions. Quorum quenching transcriptome approaches have broad potential to identify QS-controlled genes and phenotypes in organisms that are not genetically tractable. IMPORTANCE Bacterial cell-cell signaling, or quorum sensing (QS), is a method of bacterial communication and gene regulation that is well studied in bacteria. However, little is known about the purpose of QS in many environmentally important bacteria. Here, we demonstrate quorum quenching coupled with mRNA-Seq to identify QS-controlled genes and phenotypes in Nitrobacter winogradskyi , a nitrite-oxidizing bacterium. Nitrite oxidizers play an important role in the nitrogen cycle though their participation in nitrification, the aerobic oxidation of ammonia to nitrate via nitrite. Our quorum quenching approach revealed that QS influences production and consumption of environmentally important nitrogen oxide gases (NO, NO 2 , and N 2 O) in N. winogradskyi . This study demonstrated a novel technique for studying QS in difficult-to-work-with microorganisms and showed that nitrite oxidizers might also contribute to nitrification-dependent production of nitrogen oxide gases that contribute to global warming. Bacterial cell-cell signaling, or quorum sensing (QS), is a method of bacterial communication and gene regulation that is well studied in bacteria. However, little is known about the purpose of QS in many environmentally important bacteria. Here, we demonstrate quorum quenching coupled with mRNA-Seq to identify QS-controlled genes and phenotypes in Nitrobacter winogradskyi , a nitrite-oxidizing bacterium. Nitrite oxidizers play an important role in the nitrogen cycle though their participation in nitrification, the aerobic oxidation of ammonia to nitrate via nitrite. Our quorum quenching approach revealed that QS influences production and consumption of environmentally important nitrogen oxide gases (NO, NO 2 , and N 2 O) in N. winogradskyi . This study demonstrated a novel technique for studying QS in difficult-to-work-with microorganisms and showed that nitrite oxidizers might also contribute to nitrification-dependent production of nitrogen oxide gases that contribute to global warming.

Nitrosomonas europaea and Nitrobacter winogradskyi were grown singly and in co-culture in chemostats to probe for physiological differences between the two growth conditions. Co-culture growth medium containing 60 mM NH 4 + resulted in a cell density (0.20–0.29 OD 600 ) greater than the sum of the densities in single chemostat cultures, i.e., 0.09–0.14 OD 600 for N.   europaea with 60 mM NH 4 + and 0.04–0.06 OD 600 for N.   winogradskyi with 60 mM NO 2 − . The NO 2 − - and NH 4 + -dependent O 2 uptake rates, qRT–PCR, and microscopic observations indicated that in co-culture, N.   europaea contributed ~0.20 OD 600 (~80 %) and N.   winogradskyi ~0.05 OD 600 (~20 %). In co-culture, the transcriptomes showed that the mRNA levels of 773 genes in N.   europaea (30.2 % of the genes) and of 372 genes in N.   winogradskyi (11.8 % of the genes) changed significantly. Total cell growth and the analysis of the transcriptome revealed that in co-culture, N.   europaea benefits more than N.   winogradskyi .

The conditions present in both in vitro and in vivo ecosystems determine the microbial population harbouring it. One commonly accepted theory is that a species with a high substrate affinity and low growth rate (k-strategist) will win the competition against a second species with a lower substrate affinity and higher growth rate (r-strategist) if both species are subjected to low substrate concentrations. In this study two nitrite oxidizing bacteria (NOB), Nitrospira defluvii (k-strategist) and Nitrobacter vulgaris (r-strategist), were cultivated in a continuous reactor systems. The minimal hydraulic retention time (HRT) required for maintaining the slower growing Nitrospira was first determined. A reactor containing Nitrobacter was set to the same HRT and Nitrospira was injected to evaluate the effect of the dilution rate on the competition between both species. By following the microbial population dynamics with qPCR analysis, it was shown that not only the substrate affinity drives the competition between k- and r-strategists but also the dilution rate. Experimental data and numerical simulations both revealed that the washout of Nitrobacter was significantly delayed at dilution rates close to the μmax of Nitrospira. The competition could be even reverted towards Nitrobacter (r-strategist) despite of low nitrite concentrations and dilution rates lower than the μmax of Nitrospira.

The inhibitory effects of free ammonia (FA) and free nitrous acid (FNA) on the catabolic and anabolic processes of Nitrosomonas and Nitrobacter were investigated using a method that allows decoupling the growth and energy generation processes. Lab-scale sequencing batch reactors (SBRs) were operated for the enrichment of Nitrosomonas and Nitrobacter. Fluorescent In-Situ Hybridization (FISH) analysis showed that the reactors were 82% and 73% enriched with Nitrosomonas and Nitrobacter, respectively. Batch tests were carried out to measure the oxygen uptake rate (OUR) by the enriched cultures at various FA and FNA levels, in the presence (OURwithCO2) or absence (OURwithoutCO2) of inorganic carbon (CO2, HCO3− and CO32−). FA up to 16.0 mgNH3-N.L−1 was not found to have any inhibitory effect on either the catabolic or anabolic processes of the Nitrosomonas culture, but both these processes were inhibited by FNA. While an FNA level of 0.40–0.63 mgHNO2-N.L−1 inhibited the energy production capability of Nitrosomonas by 50%, the growth process of the culture was completely inhibited by FNA at a concentration of 0.40 mgHNO2-N.L−1. Both FA and FNA were found to have strong inhibition on the anabolic processes of Nitrobacter, but with limited inhibitory effects on the catabolism of this culture. The biosynthesis of Nitrobacter was totally inhibited at an FA level of 6.0 mgNH3-N.L−1 (or above) or an FNA level of 0.02 mgHNO2-N.L−1 (or above). At the same level of FA, the energy production capability of Nitrobacter was only inhibited by 12%, whereas an FNA level of up to 0.024 mgHNO2-N.L−1 did not show any inhibition on the energy production of Nitrobacter. Further, these inhibitory effects appears to be much stronger on Nitrobacter than on Nitrosomonas, supporting that FA and FNA inhibition may play a major role in the elimination of nitrite oxidizing bacteria in processes treating wastewater containing a high level of nitrogen.

Many studies have assessed the responses of soil microbial functional groups to increases in atmospheric CO sub(2) or N deposition alone and more rarely in combination. However, the effects of elevated CO sub(2) and N on the (de)coupling between different microbial functional groups (e.g., different groups of nitrifiers) have been barely studied, despite potential consequences for ecosystem functioning. Here, we investigated the short-term combined effects of elevated CO sub(2) and N supply on the abundances of the four main microbial groups involved in soil nitrification: ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (belonging to the genera Nitrobacter and Nitrospira) in grassland mesocosms. AOB and AOA abundances responded differently to the treatments: N addition increased AOB abundance, but did not alter AOA abundance. Nitrobacter and Nitrospira abundances also showed contrasted responses to the treatments: N addition increased Nitrobacter abundance, but decreased Nitrospira abundance. Our results support the idea of a niche differentiation between AOB and AOA, and between Nitrobacter and Nitrospira. AOB and Nitrobacter were both promoted at high N and C conditions (and low soil water content for Nitrobacter), while AOA and Nitrospira were favored at low N and C conditions (and high soil water content for Nitrospira). In addition, Nitrobacter abundance was positively correlated to AOB abundance and Nitrospira abundance to AOA abundance. Our results suggest that the couplings between ammonia and nitrite oxidizers are influenced by soil N availability. Multiple environmental changes may thus elicit rapid and contrasted responses between and among the soil ammonia and nitrite oxidizers due to their different ecological requirements.

The widespread application of plastics and its eventual degradation to micro-sized or nano-sized plastics has led to several environmental concerns. Moreover, nanoplastics can easily cascade through the food chain accumulating in the aquatic organisms. Thus, our study focussed on investigating the hazardous impact of nano-sized plastics on aquatic species including Nitrobacter vulgaris , Scenedesmus sp , and Daphnia magna . Various concentrations of polystyrene nanoplastics ranging from 0.01 mg/L to 100 mg/L were tested against Nitrobacter vulgaris , Scenedesmus sp , and Daphnia magna . The minimum inhibitory concentration of polystyrene nanoplastics in Nitrobacter vulgaris was found to be 25 mg/L, and in Daphnia magna , the median lethal concentration 50 was observed to be 64.02 mg/L. Exposure of Scenedesmus sp with increasing nanoplastic concentrations showed a significant decrease in total protein ( p  < 0.001), and chlorophyll content ( p  < 0.01), whereas the lipid peroxidation increased ( p  < 0.001) significantly. Similarly , Nitrobacter vulgaris and Daphnia magna showed a significant decrease in catalase activity ( p  < 0.001) and an increase in lipid peroxidation levels ( p  < 0.01). Concomitant with lipid peroxidation results, decreased superoxide dismutase levels ( p  < 0.01) and protein concentrations ( p  < 0.01) were observed in Daphnia magna . Besides, the increasing concentration of polystyrene nanoplastics displayed an elevated mortality rate in Scenedesmus sp ( p  < 0.001) and Nitrobacter vulgaris ( p  < 0.01). Further, scanning electron microscopy analysis substantiated the morphological alterations in Nitrobacter vulgaris and Scenedesmus sp on exposure to polystyrene nanoplastics. Graphical Abstract

Microcosms were set up to evaluate the effect of nitrification inhibitors (DCD, c-PTiO, and NaClO3) on the abundance and expression of ammonia-oxidizing bacteria (AOB) and archaea (AOA), as well as the nitrite-oxidizing bacteria (NOB) Nitrospira and Nitrobacter. Both DCD and NaClO3 inhibited the net nitrification rate, while c-PTiO had no significant effects, and NaClO3 had a much greater inhibitory effect (> 60%) in all soils than DCD. No significant changes in total microbial abundance were observed with DCD and NaClO3. DCD limited only the growth of AOB; however, NaClO3 inhibited growth of both AOA and Nitrospira-NOB with no significant effects on AOB and Nitrobacter-NOB. Probably NaClO3 inhibited both ammonia oxidation and nitrite oxidation. This is the first report to reveal the inhibitory effects of NaClO3 on a specific nitrification process, helping to clarify the ecological niche of nitrifiers and the potential of nitrification inhibitors applied to soil.