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Paracoccus denitrificans has been identified as a representative strain with heterotrophic nitrificationaerobic denitrification capabilities (HN-AD), and demonstrates strong denitrification proficiency. Previously, we isolated the DYTN-1 strain from activated sludge, and it has showcased remarkable nitrogen removal abilities and genetic editability, which positions P. denitrificans DYTN-1 as a promising chassis cell for synthetic biology engineering, with versatile pollutant degradation capabilities. However, the strain's low stability in plasmid conjugation transfer efficiency (PCTE) hampers gene editing efficacy, and is attributed to its restriction modification system (R-M system). To overcome this limitation, we characterized the R-M system in P. denitrificans DYTN-1 and identified a DNA endonuclease and 13 DNA methylases, with the DNA endonuclease identified as HNH endonuclease. Subsequently, we developed a plasmid artificial modification approach to enhance conjugation transfer efficiency, which resulted in a remarkable 44-fold improvement in single colony production. This was accompanied by an increase in the frequency of positive colonies from 33.3% to 100%. Simultaneously, we cloned, expressed, and characterized the speculative HNH endonuclease capable of degrading unmethylated DNA at 30°C without specific cutting site preference. Notably, the impact of DNA methylase M9 modification on the plasmid was discovered, significantly impeding the cutting efficiency of the HNH endonuclease. This revelation unveils a novel R-M system in P. denitrificans and sheds light on protective mechanisms employed against exogenous DNA invasion. These findings pave the way for future engineering endeavors aimed at enhancing the DNA editability of P. denitrificans.

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In the nitrogen biogeochemical cycle, the reduction of nitrous oxide (N2O) to N2 by N2O reductase, which is encoded by nosZ gene, is the only biological pathway for N2O consumption. In this study, we successfully isolated a strain of denitrifying Paracoccus denitrificans R-1 from sewage treatment plant sludge. This strain has strong N2O reduction capability, and the average N2O reduction rate was 5.10 ± 0.11 × 10-9 µmol·h-1·cell-1 under anaerobic condition in a defined medium. This reduction was accompanied by the stoichiometric consumption of acetate over time when N2O served as the sole electron acceptor and the reduction can yield energy to support microbial growth, suggesting that microbial N2O reduction is related to the energy generation process. Genomic analysis showed that the gene cluster encoding N2O reductase of P. denitrificans R-1 was composed of nosR, nosZ, nosD, nosF, nosY, nosL, and nosZ, which was identified as that in other strains in clade I. Respiratory inhibitors test indicated that the pathway of electron transport for N2O reduction was different from that of the traditional electron transport chain for aerobic respiration. Cu2+, silver nanoparticles, O2, and acidic conditions can strongly inhibit the reduction, whereas NO3- or NH4+ can promote it. These findings suggest that modular N2O reduction of P. denitrificans R-1 is linked to the electron transport and energy conservation, and dissimilatory N2O reduction is a form of microbial anaerobic respiration. IMPORTANCE: Nitrous oxide (N2O) is a potent greenhouse gas and contributor to ozone layer destruction, and atmospheric N2O has increased steadily over the past century due to human activities. The release of N2O from fixed N is almost entirely controlled by microbial N2O reductase activities. Here, we investigated the ability to obtain energy for the growth of Paracoccus denitrificans R-1 by coupling the oxidation of various electron donors to N2O reduction. The modular N2O reduction process of denitrifying microorganism not only can consume N2O produced by itself but also can consume the external N2O generated from biological or abiotic pathways under suitable condition, which should be critical for controlling the release of N2O from ecosystems into the atmosphere.

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Paracoccus denitrificans has a classical cytochrome-dependent electron transport chain and two alternative oxidases. The classical transport chain is very similar to that in eukaryotic mitochondria. Thus, P. denitrificans can serve as a model of the mammalian mitochondrion that may be more tractable in elucidating mechanisms of regulation of energy production than are mitochondria. In a previous publication we reported detailed studies on respiration in P. denitrificans grown aerobically on glucose or malate. We noted that P. denitrificans has large stores of lactate under various growth conditions. This is surprising because P. denitrificans lacks an NAD+-dependent lactate dehydrogenase. The aim of this study was to investigate the mechanisms of lactate oxidation in P. denitrificans. We found that the bacterium grows well on either d-lactate or l-lactate. Growth on lactate supported a rate of maximum respiration that was equal to that of cells grown on glucose or malate. We report proteomic, metabolomic, and biochemical studies that establish that the metabolism of lactate by P. denitrificans is mediated by two non-NAD+-dependent lactate dehydrogenases. One prefers d-lactate over l-lactate (D-iLDH) and the other prefers l-lactate (L-iLDH). We cloned and produced the D-iLDH and characterized it. The Km for d-lactate was 34 µM, and for l-lactate it was 3.7 mM. Pyruvate was not a substrate, rendering the reaction unidirectional with lactate being converted to pyruvate for entry into the TCA cycle. The intracellular lactate was ∼14 mM such that both isomers could be metabolized by the enzyme. The enzyme has 1 FAD per molecule and utilizes a quinone rather than NAD + as an electron acceptor. D-iLDH provides a direct entry of lactate reducing equivalents into the cytochrome chain, potentially explaining the high respiratory capacity of P. denitrificans in the presence of lactate.

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A genome scale metabolic model of the bacterium Paracoccus denitrificans has been constructed. The model containing 972 metabolic genes, 1,371 reactions, and 1,388 unique metabolites has been reconstructed. The model was used to carry out quantitative predictions of biomass yields on 10 different carbon sources under aerobic conditions. Yields on C1 compounds suggest that formate is oxidized by a formate dehydrogenase O, which uses ubiquinone as redox co-factor. The model also predicted the threshold methanol/mannitol uptake ratio, above which ribulose biphosphate carboxylase has to be expressed in order to optimize biomass yields. Biomass yields on acetate, formate, and succinate, when NO3- is used as electron acceptor, were also predicted correctly. The model reconstruction revealed the capability of P. denitrificans to grow on several non-conventional substrates such as adipic acid, 1,4-butanediol, 1,3-butanediol, and ethylene glycol. The capacity to grow on these substrates was tested experimentally, and the experimental biomass yields on these substrates were accurately predicted by the model.IMPORTANCEParacoccus denitrificans has been broadly used as a model denitrifying organism. It grows on a large portfolio of carbon sources, under aerobic and anoxic conditions. These characteristics, together with its amenability to genetic manipulations, make P. denitrificans a promising cell factory for industrial biotechnology. This paper presents and validates the first functional genome-scale metabolic model for P. denitrificans, which is a key tool to enable P. denitrificans as a platform for metabolic engineering and industrial biotechnology. Optimization of the biomass yield led to accurate predictions in a broad scope of substrates.

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Introduction: The ζ subunit is a potent inhibitor of the F1FO-ATPase of Paracoccus denitrificans (PdF1FO-ATPase) and related α-proteobacteria different from the other two canonical inhibitors of bacterial (ε) and mitochondrial (IF1) F1FO-ATPases. ζ mimics mitochondrial IF1 in its inhibitory N-terminus, blocking the PdF1FO-ATPase activity as a unidirectional pawl-ratchet and allowing the PdF1FO-ATP synthase turnover. ζ is essential for the respiratory growth of P. denitrificans, as we showed by a Δζ knockout. Given the vital role of ζ in the physiology of P. denitrificans, here, we assessed the evolution of ζ across the α-proteobacteria class. Methods: Through bioinformatic, biochemical, molecular biology, functional, and structural analyses of several ζ subunits, we confirmed the conservation of the inhibitory N-terminus of ζ and its divergence toward its C-terminus. We reconstituted homologously or heterologously the recombinant ζ subunits from several α-proteobacteria into the respective F-ATPases, including free-living photosynthetic, facultative symbiont, and intracellular facultative or obligate parasitic α-proteobacteria. Results and discussion: The results show that ζ evolved, preserving its inhibitory function in free-living α-proteobacteria exposed to broad environmental changes that could compromise the cellular ATP pools. However, the ζ inhibitory function was diminished or lost in some symbiotic α-proteobacteria where ζ is non-essential given the possible exchange of nutrients and ATP from hosts. Accordingly, the ζ gene is absent in some strictly parasitic pathogenic Rickettsiales, which may obtain ATP from the parasitized hosts. We also resolved the NMR structure of the ζ subunit of Sinorhizobium meliloti (Sm-ζ) and compared it with its structure modeled in AlphaFold. We found a transition from a compact ordered non-inhibitory conformation into an extended α-helical inhibitory N-terminus conformation, thus explaining why the Sm-ζ cannot exert homologous inhibition. However, it is still able to inhibit the PdF1FO-ATPase heterologously. Together with the loss of the inhibitory function of α-proteobacterial ε, the data confirm that the primary inhibitory function of the α-proteobacterial F1FO-ATPase was transferred from ε to ζ and that ζ, ε, and IF1 evolved by convergent evolution. Some key evolutionary implications on the endosymbiotic origin of mitochondria, as most likely derived from α-proteobacteria, are also discussed.

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Sulfamethoxazole (SMX), as one of the most widely used sulfonamide antibiotics, has been frequently detected in the aqueous environment, posing potential risks to the environment and human health. Although microbial degradation methods have been widely applied, some issues remain, including low degradation efficiency and poor environmental adaptability. In this regard, constructing efficient degrading bacteria by metabolic engineering is an ideal solution to these challenges. In this study, we used Paracoccus denitrificans DYTN-1, a superior nitrogen removal environment strain, as chassis to construct an SMX degradation pathway, obtaining a new bacteria for simultaneous degradation of SMX and removal of ammonia nitrogen. In doing this, we first identified and characterized four native promoters of P. denitrificans DYTN-1 with gradient strength to control the expression of the SMX degradation pathway. After degradation pathway expression level optimization and FMN reductase optimization, SMX degradation efficiency was significantly improved. The constructed P. d-pIAB4-PCS-sutR strain exhibited superior co-degradation of SMX and ammonia nitrogen contaminants with degradation rates of 44% and 71%, respectively. This study could pave the way for SMX degradation engineered strain design and evolution of environmental bioremediation. IMPORTANCE The abuse of sulfamethoxazole (SMX) had led to an increased accumulation in the environment, resulting in the disruption of the structure of microbial communities, further disrupting the bio-degradation process of other pollutants, such as ammonia nitrogen. To solve this challenge, we first identified and characterized four native promoters of Paracoccus denitrificans DYTN-1 with gradient strength to control the expression of the SMX degradation pathway. Then SMX degradation efficiency was significantly improved with degradation pathway expression level optimization and FMN reductase optimization. Finally, the superior nitrogen removal environment strain, P. denitrificans DYTN-1, obtained an SMX degradation function. This pioneering study of metabolic engineering to enhance the SMX degradation in microorganisms could pave the way for designing the engineered strains of SMX and nitrogen co-degradation and the environmental bioremediation.

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Nitrification inhibitors are recognized as a key approach that decreases the denitrification process to inhibit the loss of nitrogen to the atmosphere in the form of N2O. Targeting denitrification microbes directly could be one of the mitigation approaches. However, minimal attempts have been devoted towards the development of denitrification inhibitors. In this study, we aimed to investigate the molecular docking behavior of the nitrous oxide reductase (N2OR) and nitrite reductase (NIR) involved in the microbial denitrification pathway. Specifically, in silico screening was performed to detect the inhibitors of nitrous oxide reductase (N2OR) and nitrite reductase (NIR) using the PatchDock tool. Additionally, a toxicity analysis based on insecticide-likeness, Bee-Tox screening, and a STITCH analysis were performed using the SwissADME, Bee-Tox, and pkCSM free online servers, respectively. Among the twenty-two compounds tested, nine ligands were predicted to comply well with the TICE rule. Furthermore, the Bee-Tox screening revealed that none of the selected 22 ligands exhibited toxicity on honey bees. The STITCH analysis showed that two ligands, namely procyanidin B2 and thiocyanate, have interactions with both the Paracoccus denitrificans and Hyphomicrobium denitrificans microbial proteins. The molecular docking results indicated that ammonia exhibited the second least atomic contact energy (ACE) of -15.83 kcal/mol with Paracoccus denitrificans nitrous oxide reductase (N2OR) and an ACE of -15.20 kcal/mol with Hyphomicrobium denitrificans nitrite reductase (NIR). The inhibition of both the target enzymes (N2OR and NIR) supports the view of a low denitrification property and suggests the potential future applications of natural/synthetic compounds as significant nitrification inhibitors.

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Metabolic degeneracy describes the phenomenon that cells can use one substrate through different metabolic routes, while metabolic plasticity, refers to the ability of an organism to dynamically rewire its metabolism in response to changing physiological needs. A prime example for both phenomena is the dynamic switch between two alternative and seemingly degenerate acetyl-CoA assimilation routes in the alphaproteobacterium Paracoccus denitrificans Pd1222: the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). The EMCP and the GC each tightly control the balance between catabolism and anabolism by shifting flux away from the oxidation of acetyl-CoA in the tricarboxylic acid (TCA) cycle toward biomass formation. However, the simultaneous presence of both the EMCP and GC in P. denitrificans Pd1222 raises the question of how this apparent functional degeneracy is globally coordinated during growth. Here, we show that RamB, a transcription factor of the ScfR family, controls expression of the GC in P. denitrificans Pd1222. Combining genetic, molecular biological and biochemical approaches, we identify the binding motif of RamB and demonstrate that CoA-thioester intermediates of the EMCP directly bind to the protein. Overall, our study shows that the EMCP and the GC are metabolically and genetically linked with each other, demonstrating a thus far undescribed bacterial strategy to achieve metabolic plasticity, in which one seemingly degenerate metabolic pathway directly drives expression of the other. IMPORTANCE Carbon metabolism provides organisms with energy and building blocks for cellular functions and growth. The tight regulation between degradation and assimilation of carbon substrates is central for optimal growth. Understanding the underlying mechanisms of metabolic control in bacteria is of importance for applications in health (e.g., targeting of metabolic pathways with new antibiotics, development of resistances) and biotechnology (e.g., metabolic engineering, introduction of new-to-nature pathways). In this study, we use the alphaproteobacterium P. denitrificans as model organism to study functional degeneracy, a well-known phenomenon of bacteria to use the same carbon source through two different (competing) metabolic routes. We demonstrate that two seemingly degenerate central carbon metabolic pathways are metabolically and genetically linked with each other, which allows the organism to control the switch between them in a coordinated manner during growth. Our study elucidates the molecular basis of metabolic plasticity in central carbon metabolism, which improves our understanding of how bacterial metabolism is able to partition fluxes between anabolism and catabolism.

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The effective and cheap remediation of ammonia (NH+4) and multiple heavy metals from landfill leachate is currently a grand challenge. In this study, Paracoccus denitrificans AC-3, a bacterial strain capable of heterotrophic nitrification aerobic denitrification (HNAD) and carbonate precipitation, exhibited good tolerance to a variety of heavy metals and could remove 99.70% of NH+4, 99.89% of zinc (Zn2+), 97.42% of cadmium (Cd2+) and 46.19% of nickel (Ni2+) simultaneously after 24 h of incubation. The conversion pathway of NH+4 by strain AC-3 was dominated by assimilation (84.68%), followed by HNAD (14.93%), and the increase in environmental pH was mainly dependent on assimilation rather than HNAD. Calcium (Ca2+) primarily played four roles in heavy metal mineralization: (ⅰ) improving bacterial tolerance to heavy metals; (ⅱ) ensuring the HNAD capacity of strain AC-3; (ⅲ) co-precipitating with heavy metals; and (ⅳ) precipitating into calcite to adsorb heavy metals. The heavy metals removal mechanisms were mainly calcite adsorption and formation of carbonate and hydroxide precipitation for Zn2+, co-precipitation for Cd2+, and adsorption for Ni2+. The Zn2+, Cd2+, and Ni2+ precipitates displayed unique morphologies. This research provided a promising biological resource for the simultaneous remediation of NH+4 and heavy metals from landfill leachate.

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Sustainable nitrogen cycle is an essential biogeochemical process that ensures ecosystem safety and byproduct greenhouse gas nitrous oxide reduction. Antimicrobials are always co-occurring with anthropogenic reactive nitrogen sources. However, their impacts on the ecological safety of microbial nitrogen cycle remain poorly understood. Here, a denitrifying bacterial strain Paracoccus denitrificans PD1222 was exposed to a widespread broad-spectrum antimicrobial triclocarban (TCC) at environmental concentrations. The denitrification was hindered by TCC at 25 µg L-1 and was completely inhibited once the TCC concentration exceeded 50 µg L-1. Importantly, the accumulation of N2O at 25 µg L-1 of TCC was 813 times as much as the control group without TCC, which attributed to the significantly downregulated expression of nitrous oxide reductase and the genes related to electron transfer, iron, and sulfur metabolism under TCC stress. Interestingly, combining TCC-degrading denitrifying Ochrobactrum sp. TCC-2 with strain PD1222 promoted the denitrification process and mitigated N2O emission by 2 orders of magnitude. We further consolidated the importance of complementary detoxification by introducing a TCC-hydrolyzing amidase gene tccA from strain TCC-2 into strain PD1222, which successfully protected strain PD1222 against the TCC stress. This study highlights an important link between TCC detoxification and sustainable denitrification and suggests a necessity to assess the ecological risks of antimicrobials in the context of climate change and ecosystem safety.

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The periplasmic (NAP) and membrane-associated (Nar) nitrate reductases of Paracoccus denitrificans are responsible for nitrate reduction under aerobic and anaerobic conditions, respectively. Expression of NAP is elevated in cells grown on a relatively reduced carbon and energy source (such as butyrate); it is believed that NAP contributes to redox homeostasis by coupling nitrate reduction to the disposal of excess reducing equivalents. Here, we show that deletion of either dksA1 (one of two dksA homologs in the P. denitrificans genome) or relA/spoT (encoding a bifunctional ppGpp synthetase and hydrolase) eliminates the butyrate-dependent increase in nap promoter and NAP enzyme activity. We conclude that ppGpp likely signals growth on a reduced substrate and, together with DksA1, mediates increased expression of the genes encoding NAP. Support for this model comes from the observation that nap promoter activity is increased in cultures exposed to a protein synthesis inhibitor that is known to trigger ppGpp synthesis in other organisms. We also show that, under anaerobic growth conditions, the redox-sensing RegAB two-component pair acts as a negative regulator of NAP expression and as a positive regulator of expression of the membrane-associated nitrate reductase Nar. The dksA1 and relA/spoT genes are conditionally synthetically lethal; the double mutant has a null phenotype for growth on butyrate and other reduced substrates while growing normally on succinate and citrate. We also show that the second dksA homolog (dksA2) and relA/spoT have roles in regulation of expression of the flavohemoglobin Hmp and in biofilm formation. IMPORTANCE Paracoccus denitrificans is a metabolically versatile Gram-negative bacterium that is used as a model for studies of respiratory metabolism. The organism can utilize nitrate as an electron acceptor for anaerobic respiration, reducing it to dinitrogen via nitrite, nitric oxide, and nitrous oxide. This pathway (known as denitrification) is important as a route for loss of fixed nitrogen from soil and as a source of the greenhouse gas nitrous oxide. Thus, it is important to understand those environmental and genetic factors that govern flux through the denitrification pathway. Here, we identify four proteins and a small molecule (ppGpp) which function as previously unknown regulators of expression of enzymes that reduce nitrate and oxidize nitric oxide.

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The Pden_5119 protein oxidizes NADH with oxygen under mediation by the bound flavin mononucleotide (FMN) and may be involved in the maintenance of the cellular redox pool. In biochemical characterization, the curve of the pH-rate dependence was bell-shaped with pKa1 = 6.6 and pKa2 = 9.2 at 2 µM FMN while it contained only a descending limb pKa of 9.7 at 50 µM FMN. The enzyme was found to undergo inactivation by reagents reactive with histidine, lysine, tyrosine, and arginine. In the first three cases, FMN exerted a protective effect against the inactivation. X-ray structural analysis coupled with site-directed mutagenesis identified three amino acid residues important to the catalysis. Structural and kinetic data suggest that His-117 plays a role in the binding and positioning of the isoalloxazine ring of FMN, Lys-82 fixes the nicotinamide ring of NADH to support the proS-hydride transfer, and Arg-116 with its positive charge promotes the reaction between dioxygen and reduced flavin.

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Paracoccus denitrificans can adapt to complex environmental changes and sRNAs play crucial roles during this process. This work aim to identify antibiotic-induced sRNA that regulated denitrification and explored its potential for functional enhancement of this process. Target prediction indicated complementary base pairing between the denitrifying gene nosZ and the sRNA Pda200. Anaerobic culture of P. denitrificans ATCC 19367 in the presence of florfenicol (FF) resulted in significant decreases in nosZ and Pda200 gene expression (p < 0.01). Two additional denitrifiers isolated from contaminated sediment were co-cultured with ATCC 19367 to generate a consortium. And an inducible Pda200 expression strain was also added. The results revealed that Pda200 significantly enhanced napA, napB and norB expression in different types of denitrifiers under FF condition (p < 0.05 âˆ¼ 0.001). This study identified the sRNA Pda200 as a novel positive regulator of denitrification, which may realize the efficient treatment of antibiotic-contaminated wastewater by microbial agents.

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Proton-translocating Fo×F1-ATPase/synthase that catalyzes synthesis and hydrolysis of ATP is commonly considered to be a reversibly functioning complex. We have previously shown that venturicidin, a specific Fo-directed inhibitor, blocks the synthesis and hydrolysis of ATP with a significant difference in the affinity [Zharova, T. V. and Vinogradov, A. D. (2017) Biochim. Biophys. Acta, 1858, 939-944]. In this paper, we have studied in detail inhibition of Fo×F1-ATPase/synthase by venturicidin in tightly coupled membranes of Paracoccus denitrificans under conditions of membrane potential generation. ATP hydrolysis was followed by the ATP-dependent succinate-supported NAD+ reduction (potential-dependent reverse electron transfer) catalyzed by the respiratory chain complex I. It has been demonstrated that membrane energization did not affect the affinity of Fo×F1-ATPase/synthase for venturicidin. The dependence of the residual ATP synthase activity on the concentration of venturicidin approximated a linear function, whereas the dependence of ATP hydrolysis was sigmoidal: at low inhibitor concentrations venturicidin strongly inhibited ATP synthesis without decrease in the rate of ATP hydrolysis. A model is proposed suggesting that ATP synthesis and ATP hydrolysis are catalyzed by two different forms of Fo×F1.

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Bacterial biofilms are ubiquitous in natural environments and play an essential role in bacteria's environmental adaptability. Quorum sensing (QS), as the main signaling mechanism bacteria used for cell-to-cell communication, plays a key role in bacterial biofilm formation. However, little is known about the role of QS circuit in the N-transformation type strain, Paracoccus denitrificans, especially for the regulatory protein PdeR. In this study, we found the overexpression of pdeR promoted bacterial aggregation and biofilm formation. Through RNA-seq analysis, we demonstrated that PdeR is a global regulator which could regulate 656 genes expression, involved in multiple metabolic pathways. Combined with transcriptome as well as biochemical experiments, we found the overexpressed pdeR mainly promoted the intracellular degradation of amino acids and fatty acids, as well as siderophore biosynthesis and transportation, thus providing cells enough energy and iron for biofilm development. These results revealed the underlying mechanism for PdeR in biofilm formation of P. denitrificans, adding to our understanding of QS regulation in biofilm development.

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The novel Paracoccus denitrificans AC-3 strain was isolated and displayed outstanding purification capability for high concentrations of ammonia nitrogen (NH4+-N) and calcium (Ca2+). Meanwhile, the strain exhibited excellent environmental adaptability within a wide pH range and high levels of NH4+-N and Ca2+. Nitrogen balance analysis demonstrated that the pathways of NH4+-N removal consisted of 80.12% assimilation and 16.60% heterotrophic nitrification aerobic denitrification (HNAD). In addition, Ca2+ was removed by forming calcium carbonate (CaCO3) with carbonate (CO32-) and bicarbonate (HCO3-). CO32-and HCO3- were obtained from carbon dioxide (CO2) hydration, which was catalyzed by carbonic anhydrase (CA) secreted by strain AC-3. The alkaline environment for carbonate precipitation was provided by CA and HNAD. The resulting CaCO3 existed in the form of calcite and exhibited a unique morphology and elemental composition.

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Paracoccus denitrificans ArsH is encoded by two identical genes located in two distinct putative arsenic resistance (ars) operons. Escherichia coli-produced recombinant N-His6-ArsH was characterized both structurally and kinetically. The X-ray structure of ArsH revealed a flavodoxin-like domain and motifs for the binding of flavin mononucleotide (FMN) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). The protein catalyzed FMN reduction by NADPH via ternary complex mechanism. At a fixed saturating FMN concentration, it acted as an NADPH-dependent organoarsenic reductase displaying ping-pong kinetics. A 1:1 enzymatic reaction of phenylarsonic acid with the reduced form of FMN (FMNH2) and formation of phenylarsonous acid were observed. Growth experiments with P. denitrificans and E. coli revealed increased toxicity of phenylarsonic acid to cells expressing arsH, which may be related to in vivo conversion of pentavalent As to more toxic trivalent form. ArsH expression was upregulated not only by arsenite, but also by redox-active agents paraquat, tert-butyl hydroperoxide and diamide. A crucial role is played by the homodimeric transcriptional repressor ArsR, which was shown in in vitro experiments to monomerize and release from the DNA-target site. Collectively, our results establish ArsH as responsible for enhancement of organo-As(V) toxicity and demonstrate redox control of ars operon.

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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.

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Strain Z195 was isolated and identified as Paracoccus denitrificans. Z195 exhibited efficient aerobic denitrification and carbon removal abilities, and removed 93.74% of total nitrogen (TN) and 97.81% of total organic carbon.71.88% of nitrogen was lost as gaseous products.13C-metabolic flux analysis revealed that 95% and 132% of the carbon fluxes entered the Entner-Doudoroff (ED) pathway and tricarboxylic acid (TCA) cycle, respectively. Electrons produced by carbon metabolism markedly promoted the processes of nitrogen metabolism process and aerobic respiration. A response surface methodology model demonstrated that the optimal conditions for the maximum TN removal were a C/N ratio of 7.47, shaking speed of 108 rpm, temperature of 31 °C and initial pH of 8.02. Additionally, the average TN and chemical oxygen demand removal efficiencies of raw wastewater were 89% and 91%, respectively. The results give new insight for understanding metabolic flux analysis of aerobic denitrifying bacteria.

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Most bacteria form biofilms, which are thick multicellular communities covered in extracellular matrix. Biofilms can become thick enough to be even observed by the naked eye, and biofilm formation is a tightly regulated process. Paracoccus denitrificans is a non-motile, Gram-negative bacterium that forms a very thin, unique biofilm. A key factor in the biofilm formed by this bacterium is a large surface protein named biofilm-associated protein A (BapA), which was recently reported to be regulated by cyclic diguanosine monophosphate (cyclic-di-GMP or c-di-GMP). Cyclic-di-GMP is a major second messenger involved in biofilm formation in many bacteria. Though cyclic-di-GMP is generally reported as a positive regulatory factor in biofilm formation, it represses biofilm formation in P. denitrificans. Furthermore, quorum sensing (QS) represses biofilm formation in this bacterium, which is also reported as a positive regulator of biofilm formation in most bacteria. The QS signal used in P. denitrificans is hydrophobic and is delivered through membrane vesicles. Studies on QS show that P. denitrificans can potentially form a thick biofilm but maintains a thin biofilm under normal growth conditions. In this review, we discuss the peculiarities of biofilm formation by P. denitrificans with the aim of deepening the overall understanding of bacterial biofilm formation and functions.

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