Response characteristics of nirS-type denitrifier Paracoccus denitrificans under florfenicol stress.

Florfenicol (FF) is widely used in aquaculture and can interfere with denitrification when released into natural ecosystems. The aim of this study was to analyze the response characteristics of nirS-type denitrifier Paracoccus denitrificans under FF stress and further mine antibiotic-responsive factors in aquatic environment. Phenotypic analysis revealed that FF delayed the nitrate removal with a maximum inhibition value of 82.4% at exponential growth phase, leading to nitrite accumulation reached to 21.9-fold and biofilm biomass decreased by ~38.6%, which were due to the lower bacterial population count (P < 0.01). RNA-seq transcriptome analyses indicated that FF treatment decreased the expression of nirS, norB, nosD and nosZ genes that encoded enzymes required for NO2- to N2 conversion from 1.02- to 2.21-fold (P < 0.001). Furthermore, gene associated with the flagellar system FlgL was also down-regulated by 1.03-fold (P < 0.001). Moreover, 10 confirmed sRNAs were significantly induced, which regulated a wide range of metabolic pathways and protein expression. Interestingly, different bacteria contained the same sRNAs means that sRNAs can spread between them. Overall, this study suggests that the denitrification of nirS-type denitrifiers can be hampered widely by FF and the key sRNAs have great potential to be antibiotic-responsive factors.

Tetracycline-induced effects on the nitrogen transformations in sediments: Roles of adsorption behavior and bacterial activity.

Nitrification and denitrification are the most important nitrogen transformation processes in the environment. Recently, due to widespread use, antibiotics have been reported to lead to environmental risks. Tetracycline (TC) is one of the most extensively used antibiotics in many areas. However, its reported effects on nitrogen transformations were conflicting in previous studies. In this study, the effects of TC on nitrogen transformations in sediment were investigated by analyzing TC transport and bacterial activity. It was found that the adsorption of TC onto the sediment was favorable and spontaneous, with adsorption capacity 54.3 mg/kg. The adsorption kinetics of TC onto the sediment and the isotherm fitted the Elvoich and Freundlich models, respectively, indicating that the adsorption was a chemisorption process, including electrostatic interactions and chemical bonding between TC and the sediment. TC showed no effect on nitrification in the sediment, but significantly inhibited the reduction of nitrate and nitrite during denitrification, consistent with observations made for the model denitrifier Paracoccus denitrificans under TC stress. Mechanistic study indicated that TC at 130 µg/g-cell inhibited 50.7% of P. denitrificans growth and 61.6% of cell viability. Meanwhile, the catalytic activities of the key denitrifying enzymes, nitrate reductase (NAR) and nitrite reductase (NIR), decreased to 29.1% and 68.0% of the control levels when the TC concentration was 130 µg/g-cell, suggesting that NAR was more sensitive to the TC than NIR, which contributed to a delay in nitrite accumulation.

Inhibition of 1, 4-dioxane on the denitrification process by altering the viability and metabolic activity of Paracoccus denitrificans.

1,4-Dioxane is an emerging pollutant, which widely exists in natural environments and poses potential risks to the living organisms. However, its effect on the denitrification process is still unknown. In this study, the effects of 1,4-dioxane on the denitrification process were therefore investigated by using Paracoccus denitrificans as the model denitrifier. The obtained results showed that the exposure of 1,4-dioxane exhibited remarkable lag or inhibition on the denitrification process, especially with high dose. In the control without 1,4-dioxane exposure, Paracoccus denitrificans showed high denitrification efficiency (98.5%). However, the efficiency decreased to 78.5, 63.9, and 9.3% with 0.50, 0.75, and 1.0% (v/v) 1,4-dioxane dose, respectively. The dose-induced inhibition of denitrification by 1,4-dioxane could be partially attributed to the negative effects on proliferation and viability of functional microorganisms by conjugating and disrupting the cell membranes. Furthermore, 1,4-dioxane caused biotoxicity to the intracellular activities of denitrifiers via disturbing carbon source utilization and interfering the key enzymes responsible for glycolysis. The decrease of microbial viability and activity inevitably resulted in the decline of key enzymes (NAR, NIR, NOR, and N2OR) closely related with denitrification process, which could be the direct reason for the decrease of denitrification performance.

Comprehensive analysis of transcriptional and proteomic profiling reveals silver nanoparticles-induced toxicity to bacterial denitrification.

Although the toxicity of silver nanoparticles (Ag NPs or nanosilver) to model bacteria has been reported, the effects of Ag NPs on microbial denitrification under anoxic conditions and the mechanism of Ag NPs induced-toxicity to denitrification remain unclear. In this study, the effects of Ag NPs on Paracoccus denitrificans under anoxic conditions were investigated, and the mechanism was explored by analyzing the transcriptional and proteomic responses of bacteria to Ag NPs. The presence of 5mg/L Ag NPs led to excessive nitrate accumulation (232.5 versus 5.3mg/L) and increased nitrous oxide emission. Transcriptional analysis indicated that Ag NPs restrained the expression of key genes related to denitrification. Specifically, the genes involved in denitrifying catalytic reduction and electron transfer were significantly down-regulated. Moreover, the expression of the genes responsible for polyhydroxybutyrate synthesis was enhanced, which was adverse to denitrification. Proteomic profiling revealed that the syntheses of the proteins involved in catalytic process, electron transfer, and metabolic process were inhibited by Ag NPs. The activities of nitrate reductase and nitrite reductase in the presence of 5mg/L Ag NPs were only 42% and 61% of those in the control, respectively, indicating the inhibition of denitrifying enzymes. These results improve understanding of the inhibitory mechanism of Ag NPs toward bacterial denitrification.

The effects of fulvic acid on microbial denitrification: promotion of NADH generation, electron transfer, and consumption.

The heterotrophic denitrification requires the participation of electrons which are derived from direct electron donor (usually nicotinamide adenine dinucleotide (NADH)), and the electrons are transferred via electron transport system in denitrifiers and then consumed by denitrifying enzymes. Despite the reported electron transfer ability of humic substances (HS), the influences of fulvic acid (FA), an ubiquitous major component of HS, on promoting NADH generation, electron transfer, and consumption in denitrification process have never been reported. The presence of FA, compared with the control, was found not only significantly improved the total nitrogen (TN) removal efficiency (99.9 % versus 74.8 %) but remarkably reduced the nitrite accumulation (0.2 against 43.8 mg/L) and N2O emission (0.003 against 0.240 mg nitrogen/mg TN removed). The mechanisms study showed that FA increased the metabolism of carbon source via glycolysis and tricarboxylic acid (TCA) cycle pathways to produce more available NADH. FA also facilitated the electron transfer activities from NADH to denitrifying enzymes via complex I and complex III in electron transport system, which improved the reduction of nitrate and accelerated the transformations of nitrite and N2O, and lower nitrite and N2O accumulations were therefore observed. In addition, the consumption of electrons in denitrification was enhanced due to FA stimulating the synthesis and the catalytic activity of key denitrifying enzymes, especially nitrite reductase and N2O reductase. It will provide an important new insight into the potential effect of FA on microbial denitrification metabolism process and even nitrogen cycle in nature niches.

Alteration of intracellular protein expressions as a key mechanism of the deterioration of bacterial denitrification caused by copper oxide nanoparticles.

The increasing production and utilization of copper oxide nanoparticles (CuO NPs) result in the releases into the environment. However, the influence of CuO NPs on bacterial denitrification, one of the most important pathways to transform nitrate to dinitrogen in environment, has seldom been studied. Here we reported that CuO NPs caused a significant alteration of key protein expressions of a model denitrifier, Paracoccus denitrificans, leading to severe inhibition to denitrification. Total nitrogen removal efficiency was decreased from 98.3% to 62.1% with the increase of CuO NPs from 0.05 to 0.25 mg/L. Cellular morphology and integrity studies indicated that nanoparticles entered the cells. The proteomic bioinformatics analysis showed that CuO NPs caused regulation of proteins involved in nitrogen metabolism, electron transfer and substance transport. The down-regulation of GtsB protein (responsible for glucose transport) decreased the production of NADH (electron donor for denitrification). Also, the expressions of key electron-transfer proteins (including NADH dehydrogenase and cytochrome) were suppressed by CuO NPs, which adversely affected electrons transfer for denitrification. Further investigation revealed that CuO NPs significantly inhibited the expressions and catalytic activities of nitrate reductase and nitrite reductase. These results provided a fundamental understanding of the negative influences of CuO NPs on bacterial denitrification.

Planktonic and biofilm-grown nitrogen-cycling bacteria exhibit different susceptibilities to copper nanoparticles.

Proper characterization of nanoparticle (NP) interactions with environmentally relevant bacteria under representative conditions is necessary to enable their sustainable manufacture, use, and disposal. Previous nanotoxicology research based on planktonic growth has not adequately explored biofilms, which serve as the predominant mode of bacterial growth in natural and engineered environments. Copper nanoparticle (Cu-NP) impacts on biofilms were compared with respective planktonic cultures of the ammonium-oxidizing Nitrosomonas europaea, nitrogen-fixing Azotobacter vinelandii, and denitrifying Paracoccus denitrificans using a suite of independent toxicity diagnostics. Median inhibitory concentration (IC50) values derived from adenosine triphosphate (ATP) for Cu-NPs were lower in N. europaea biofilms (19.6 ± 15.3 mg/L) than in planktonic cells (49.0 ± 8.0 mg/L). However, in absorbance-based growth assays, compared with unexposed controls, N. europaea growth rates in biofilms were twice as resilient to inhibition than those in planktonic cultures. Similarly, relative to unexposed controls, growth rates and yields of P. denitrificans in biofilms exposed to Cu-NPs were 40-fold to 50-fold less inhibited than those in planktonic cells. Physiological evaluation of ammonium oxidation and nitrate reduction suggested that biofilms were also less inhibited by Cu-NPs than planktonic cells. Furthermore, functional gene expression for ammonium oxidation (amoA) and nitrite reduction (nirK) showed lower inhibition by NPs in biofilms relative to planktonic-grown cells. These results suggest that biofilms mitigate NP impacts, and that nitrogen-cycling bacteria in wastewater, wetlands, and soils might be more resilient to NPs than planktonic-based assessments suggest.

Carboxyl-modified single-walled carbon nanotubes negatively affect bacterial growth and denitrification activity.

Single-walled carbon nanotubes (SWNTs) have been used in a wide range of fields, and the surface modification via carboxyl functionalization can further improve their physicochemical properties. However, whether carboxyl-modified SWNT poses potential risks to microbial denitrification after its release into the environment remains unknown. Here we present the possible effects of carboxyl-modified SWNT on the growth and denitrification activity of Paracoccus denitrificans (a model denitrifying bacterium). It was found that carboxyl-modified SWNT were present both outside and inside the bacteria, and thus induced bacterial growth inhibition at the concentrations of 10 and 50 mg/L. After 24 h of exposure, the final nitrate concentration in the presence of 50 mg/L carboxyl-modified SWNT was 21-fold higher than that in its absence, indicating that nitrate reduction was substantially suppressed by carboxyl-modified SWNT. The transcriptional profiling revealed that carboxyl-modified SWNT led to the transcriptional activation of the genes encoding ribonucleotide reductase in response to DNA damage and also decreased the gene expressions involved in glucose metabolism and energy production, which was an important reason for bacterial growth inhibition. Moreover, carboxyl-modified SWNT caused the significant down-regulation and lower activity of nitrate reductase, which was consistent with the decreased efficiency of nitrate reduction.

Prediction and biochemical demonstration of a catabolic pathway for the osmoprotectant proline betaine.

UNLABELLED: Through the use of genetic, enzymatic, metabolomic, and structural analyses, we have discovered the catabolic pathway for proline betaine, an osmoprotectant, in Paracoccus denitrificans and Rhodobacter sphaeroides. Genetic and enzymatic analyses showed that several of the key enzymes of the hydroxyproline betaine degradation pathway also function in proline betaine degradation. Metabolomic analyses detected each of the metabolic intermediates of the pathway. The proline betaine catabolic pathway was repressed by osmotic stress and cold stress, and a regulatory transcription factor was identified. We also report crystal structure complexes of the P. denitrificans HpbD hydroxyproline betaine epimerase/proline betaine racemase with l-proline betaine and cis-hydroxyproline betaine. IMPORTANCE: At least half of the extant protein annotations are incorrect, and the errors propagate as the number of genome sequences increases exponentially. A large-scale, multidisciplinary sequence- and structure-based strategy for functional assignment of bacterial enzymes of unknown function has demonstrated the pathway for catabolism of the osmoprotectant proline betaine.

Copper control of bacterial nitrous oxide emission and its impact on vitamin B12-dependent metabolism.

Global agricultural emissions of the greenhouse gas nitrous oxide (N2O) have increased by around 20% over the last 100 y, but regulation of these emissions and their impact on bacterial cellular metabolism are poorly understood. Denitrifying bacteria convert nitrate in soils to inert di-nitrogen gas (N2) via N2O and the biochemistry of this process has been studied extensively in Paracoccus denitrificans. Here we demonstrate that expression of the gene encoding the nitrous oxide reductase (NosZ), which converts N2O to N2, is regulated in response to the extracellular copper concentration. We show that elevated levels of N2O released as a consequence of decreased cellular NosZ activity lead to the bacterium switching from vitamin B12-dependent to vitamin B12-independent biosynthetic pathways, through the transcriptional modulation of genes controlled by vitamin B12 riboswitches. This inhibitory effect of N2O can be rescued by addition of exogenous vitamin B12.

Reduction of Fe(III)EDTA in a NOx scrubber liquor by a denitrifying bacterium and the effects of inorganic sulfur compounds on this process.

Biological reduction of Fe(III)EDTA is one of the key steps in nitrogen oxides removal in the integrated approach of metal chelate absorption combined with microbial reduction. Paracoccus denitrificans ZGL1 was used as a model bacterium to evaluate the process of Fe(III)EDTA reduction by such microorganisms that could carry out the simultaneous reduction of NO chelated by Fe(II)EDTA (Fe(II)EDTA-NO) and Fe(III)EDTA. Enzymes analysis indicated Fe(III)EDTA reductase of ZGL1 was located both in the membrane and cytoplasmic fractions. Glucose was identified as the most efficient electron donor for Fe(III)EDTA reduction. Better reduction performance was obtained with higher initial cell concentration corresponding to a specific reduction rate of 8.7 µmol h(-1) mg protein(-1). The presence of sulfate and thiosulfate had no influences on both cell growth and Fe(III)EDTA reduction. Fe(III)EDTA reduction rate and cell growth could be inhibited by addition of sulfite mainly due to its direct and indirect toxic effects.

Sodium influence on energy transduction by complexes I from Escherichia coli and Paracoccus denitrificans.

The nature of the ions that are translocated by Escherichia coli and Paracoccus denitrificans complexes I was investigated. We observed that E. coli complex I was capable of proton translocation in the same direction to the established deltapsi, showing that in the tested conditions, the coupling ion is the H(+). Furthermore, Na(+) transport to the opposite direction was also observed, and, although Na(+) was not necessary for the catalytic or proton transport activities, its presence increased the latter. We also observed H(+) translocation by P. denitrificans complex I, but in this case, H(+) transport was not influenced by Na(+) and also Na(+) transport was not observed. We concluded that E. coli complex I has two energy coupling sites (one Na(+) independent and the other Na(+) dependent), as previously observed for Rhodothermus marinus complex I, whereas the coupling mechanism of P. denitrificans enzyme is completely Na(+) independent. This work thus shows that complex I energy transduction by proton pumping and Na(+)/H(+) antiporting is not exclusive of the R. marinus enzyme. Nevertheless, the Na(+)/H(+) antiport activity seems not to be a general property of complex I, which may be correlated with the metabolic characteristics of the organisms.

Direct demonstration of half-of-the-sites reactivity in the dimeric cytochrome bc1 complex: enzyme with one inactive monomer is fully active but unable to activate the second ubiquinol oxidation site in response to ligand binding at the ubiquinone reduction site.

We previously proposed that the dimeric cytochrome bc(1) complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc(1) monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289-22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc(1) complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10-16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c(1) by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.

Inducible-expression plasmid for Rhodobacter sphaeroides and Paracoccus denitrificans.

We have developed a stable isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible-expression plasmid, pIND4, which allows graduated levels of protein expression in the alphaproteobacteria Rhodobacter sphaeroides and Paracoccus denitrificans. pIND4 confers kanamycin resistance and combines the stable replicon of pMG160 with the lacI(q) gene from pYanni3 and the lac promoter, P(A1/04/03), from pJBA24.

Inhibition of bacterial oxidases by formamide and analogs.

The enzymatic activity of Paracoccus denitrificans cytochrome c oxidase (COX) and Escherichia coli cytochrome b(o) ubiquinol oxidase (QOX) was determined in the presence of formamide, N,N-dimethyl formamide and N,N-dimethyl acetamide. Formamide was found to inhibit the enzyme activity of the oxidases most significantly, whereas the other two compounds inhibited the activity to a lesser extent. The effects of formamide and analogs on enzyme activity were very similar for COX and QOX, indicating that the mechanism of inhibition might be the same for both of these oxidases. The inhibition kinetics followed a non-competitive mechanism. Studies using proteoliposomes of COX and QOX containing the electron entry site of the enzyme directed towards the outside of the vesicles showed that the effect of inhibition by formamide was higher when the inhibitor was present on the outside of the proteoliposome compared to when it was present only in the aqueous core. This indicates that inhibition of enzyme activity by formamide possibly predominantly involves blocking of the water exit pathway in the oxidases.

Phosphate removal under denitrifying conditions by Brachymonas sp. strain P12 and Paracoccus denitrificans PP15.

In this study, we used the denitrifying phosphorus-removing bacterium Brachymonas sp. strain P12 to investigate the enhanced biologic phosphorus-removal (EBPR) mechanism involved with polyhydroxybutyrate (PHB), glycogen, and phosphorus uptake in the presence of acetate under anoxic or aerobic conditions. The results showed that excess acetate concentration and aerobic cultivation can enhance PHB formation efficiency and that PHB formation might be stimulated by glycogenolysis of the cellular glycogen. The efficiency of the uptake of anoxic phosphorus was greater when PHB production was lower. The EBPR mechanism of Brachymonas sp. strain P12 for PHB, phosphorus, and glycogen was similar to the conventional anaerobic-aerobic (or anaerobic-anoxic) EBPR models, but these models were developed under anoxic or aerobic conditions only, without an anaerobic stage. The anoxic or aerobic log phase of growth is divided into two main phases: the early log phase, in which acetate and glycogen are consumed to supply enough energy and reducing power for PHB formation and cell growth (phosphorus assimilation), and the late log phase, which ends the simultaneous degradation of PHB and remaining acetate for polyphosphate accumulation. Glycogenolysis plays a significant role in the alternate responses between PHB formation and phosphorus uptake under anoxic or aerobic conditions. After the application of the denitrifying phosphorus-removing bacterium Brachymonas sp. strain P12, aerobic cultivation increases the level of PHB production, and anoxic cultivation further increases phosphorus uptake.

Energy coupling to nitrate uptake into the denitrifying cells of Paracoccus denitrificans.

This study deals with the effects of the agents that dissipate the individual components of the proton motive force (short-chain fatty acids, nigericin, and valinomycin) upon the methyl viologen-coupled nitrate reductase activity in intact cells. Substitution of butyrate or acetate for chloride in Tris-buffered assay media resulted in a marked inhibition at pH 7. In a Tris--chloride buffer of neutral pH, the reaction was almost fully inhibitable by nigericin. Alkalinisation increased the IC(50) value for nigericin and decreased the maximal inhibition attained. Both types of inhibitions could be reversed by the permeabilisation of cells or by the addition of nitrite, and that caused by nigericin disappeared at high extracellular concentrations of potassium. These data indicate that nitrate transport step relies heavily on the pH gradient at neutral pH. Since the affinity of cells for nitrate was strongly diminished by imposing an inside-positive potassium (or lithium) diffusion potential at alkaline external pH, a potential dependent step may be of significance in the transporter cycle under these conditions. Experiments with sodium-depleted media provided no hints for Na(+) as a possible H(+) substitute.

Lucigenin and coelenterazine as superoxide probes in mitochondrial and bacterial membranes.

The chemiluminescent superoxide indicators lucigenin and coelenterazine were compared in rat liver submitochondrial particles and cytoplasmic membranes from Paracoccus denitrificans. Qualitative monitoring is possible with both probes, but quantitative work with lucigenin is hampered by its dependence on one-electron reduction before the photon-emitting reaction. Therefore, calibration of measurements on complex I, capable of efficient lucigenin prereduction with reduced nicotinamide adenine dinucleotide, against xanthine oxidase, which in the presence of hypoxanthine is not able to reduce the probe to a significant rate compared to complex I, may give results in error by one order of magnitude. Coelenterazine, although susceptible of storage-dependent high background chemiluminescence, does not require prereduction and is thus a more reliable probe.

Fine-tuned regulation by oxygen and nitric oxide of the activity of a semi-synthetic FNR-dependent promoter and expression of denitrification enzymes in Paracoccus denitrificans.

In Paracoccus denitrificans at least three fumarate and nitrate reductase regulator (FNR)-like proteins [FnrP, nitrite and nitric oxide reductases regulator (NNR) and NarR] control the expression of several genes necessary for denitrifying growth. To gain more insight into this regulation, beta-galactosidase activity from a plasmid carrying the lacZ gene fused to the Escherichia coli melR promoter with the consensus FNR-binding (FF) site was examined. Strains defective in the fnrP gene produced only very low levels of beta-galactosidase, indicating that FnrP is the principal activator of the FF promoter. Anoxic beta-galactosidase levels were much higher relative to those under oxic growth and were strongly dependent on the nitrogen electron acceptor used, maximal activity being promoted by N(2)O. Additions of nitrate or nitroprusside lowered beta-galactosidase expression resulting from an oxic to micro-oxic switch. These results suggest that the activity of FnrP is influenced not only by oxygen, but also by other factors, most notably by NO concentration. Observations of nitric oxide reductase (NOR) activity in a nitrite-reductase-deficient strain and in cells treated with haemoglobin provided evidence for dual regulation of the synthesis of this enzyme, partly independent of NO. Both regulatory modes were operative in the FnrP-deficient strain, but not in the NNR-deficient strain, suggesting involvement of the NNR protein. This conclusion was further substantiated by comparing the respective NOR promoter activities.

Succinate:quinone oxidoreductase in the bacteria Paracoccus denitrificans and Bacillus subtilis.

An overview of the present knowledge about succinate:quinone oxidoreductase in Paracoccus denitrificans and Bacillus subtilis is presented. P. denitrificans contains a monoheme succinate:ubiquinone oxidoreductase that is similar to that of mammalian mitochondria with respect to composition and sensitivity to carboxin. Results obtained with carboxin-resistant P. denitrificans mutants provide information about quinone-binding sites on the enzyme and the molecular basis for the resistance. B. subtilis contains a diheme succinate:menaquinone oxidoreductase whose activity is dependent on the electrochemical gradient across the cytoplasmic membrane. Data from studies of mutant variants of the B. subtilis enzyme combined with available crystal structures of a similar enzyme, Wolinella succinogenes fumarate reductase, substantiate a proposed explanation for the mechanism of coupling between quinone reductase activity and transmembrane potential.