H-NOX Influences Biofilm Formation, Central Metabolism, and Quorum Sensing in Paracoccus denitrificans.

The transition from planktonic to biofilm growth in bacteria is often accompanied by greater resistance to antibiotics and other stressors, as well as distinct alterations in physical traits, genetic activity, and metabolic restructuring. In many species, the heme nitric oxide/oxygen binding proteins (H-NOX) play an important role in this process, although the signaling mechanisms and pathways in which they participate are quite diverse and largely unknown. In Paracoccus denitrificans, deletion of the hnox gene results in a severe biofilm-deficient phenotype. Quantitative proteomics was used to assemble a comprehensive data set of P. denitrificans proteins showing altered abundance of those involved in several important metabolic pathways. Further, decreased levels of pyruvate and elevated levels of C16 homoserine lactone were detected for the Δhnox strain, associating the biofilm deficiency with altered central carbon metabolism and quorum sensing, respectively. These results expand our knowledge of the important role of H-NOX signaling in biofilm formation.

Structure of the turnover-ready state of an ancestral respiratory complex I.

Respiratory complex I is pivotal for cellular energy conversion, harnessing energy from NADH:ubiquinone oxidoreduction to drive protons across energy-transducing membranes for ATP synthesis. Despite detailed structural information on complex I, its mechanism of catalysis remains elusive due to lack of accompanying functional data for comprehensive structure-function analyses. Here, we present the 2.3-Å resolution structure of complex I from the α-proteobacterium Paracoccus denitrificans, a close relative of the mitochondrial progenitor, in phospholipid-bilayer nanodiscs. Three eukaryotic-type supernumerary subunits (NDUFS4, NDUFS6 and NDUFA12) plus a novel L-isoaspartyl-O-methyltransferase are bound to the core complex. Importantly, the enzyme is in a single, homogeneous resting state that matches the closed, turnover-ready (active) state of mammalian complex I. Our structure reveals the elements that stabilise the closed state and completes P. denitrificans complex I as a unified platform for combining structure, function and genetics in mechanistic studies.

Identification and Characterization of an R-M System in Paracoccus denitrifican DYTN-1 to Improve the Plasmid Conjugation Transfer Efficiency.

Paracoccus denitrificans has been identified as a representative strain with heterotrophic nitrification-aerobic 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.

Multiple levels of transcriptional regulation control glycolate metabolism in Paracoccus denitrificans.

The hydroxyacid glycolate is a highly abundant carbon source in the environment. Glycolate is produced by unicellular photosynthetic organisms and excreted at petagram scales to the environment, where it serves as growth substrate for heterotrophic bacteria. In microbial metabolism, glycolate is first oxidized to glyoxylate by the enzyme glycolate oxidase. The recently described ß-hydroxyaspartate cycle (BHAC) subsequently mediates the carbon-neutral assimilation of glyoxylate into central metabolism in ubiquitous Alpha- and Gammaproteobacteria. Although the reaction sequence of the BHAC was elucidated in Paracoccus denitrificans, little is known about the regulation of glycolate and glyoxylate assimilation in this relevant alphaproteobacterial model organism. Here, we show that regulation of glycolate metabolism in P. denitrificans is surprisingly complex, involving two regulators, the IclR-type transcription factor BhcR that acts as an activator for the BHAC gene cluster, and the GntR-type transcriptional regulator GlcR, a previously unidentified repressor that controls the production of glycolate oxidase. Furthermore, an additional layer of regulation is exerted at the global level, which involves the transcriptional regulator CceR that controls the switch between glycolysis and gluconeogenesis in P. denitrificans. Together, these regulators control glycolate metabolism in P. denitrificans, allowing the organism to assimilate glycolate together with other carbon substrates in a simultaneous fashion, rather than sequentially. Our results show that the metabolic network of Alphaproteobacteria shows a high degree of flexibility to react to the availability of multiple substrates in the environment.IMPORTANCEAlgae perform ca. 50% of the photosynthetic carbon dioxide fixation on our planet. In the process, they release the two-carbon molecule glycolate. Due to the abundance of algae, massive amounts of glycolate are released. Therefore, this molecule is available as a source of carbon for bacteria in the environment. Here, we describe the regulation of glycolate metabolism in the model organism Paracoccus denitrificans. This bacterium uses the recently characterized ß-hydroxyaspartate cycle to assimilate glycolate in a carbon- and energy-efficient manner. We found that glycolate assimilation is dynamically controlled by three different transcriptional regulators: GlcR, BhcR, and CceR. This allows P. denitrificans to assimilate glycolate together with other carbon substrates in a simultaneous fashion. Overall, this flexible and multi-layered regulation of glycolate metabolism in P. denitrificans represents a resource-efficient strategy to make optimal use of this globally abundant molecule under fluctuating environmental conditions.

Respiration and growth of Paracoccus denitrificans R-1 with nitrous oxide as an electron acceptor.

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.

Choline degradation in Paracoccus denitrificans: identification of sources of formaldehyde.

Paracoccus denitrificans is a facultative methylotroph that can grow on methanol and methylamine as sole sources of carbon and energy. Both are oxidized to formaldehyde and then to formate, so growth on C1 substrates induces the expression of genes encoding enzymes required for the oxidation of formaldehyde and formate. This induction involves a histidine kinase response regulator pair (FlhSR) that is likely triggered by formaldehyde. Catabolism of some complex organic substrates (e.g., choline and L-proline betaine) also generates formaldehyde. Thus, flhS and flhR mutants that fail to induce expression of the formaldehyde catabolic enzymes cannot grow on methanol, methylamine, and choline. Choline is oxidized to glycine via glycine betaine, dimethylglycine, and sarcosine. By exploring flhSR growth phenotypes and the activities of a promoter and enzyme known to be upregulated by formaldehyde, we identify the oxidative demethylations of glycine betaine, dimethylglycine, and sarcosine as sources of formaldehyde. Growth on glycine betaine, dimethylglycine, and sarcosine is accompanied by the production of up to three, two, and one equivalents of formaldehyde, respectively. Genetic evidence implicates two orthologous monooxygenases in the oxidation of glycine betaine. Interestingly, one of these appears to be a bifunctional enzyme that also oxidizes L-proline betaine (stachydrine). We present preliminary evidence to suggest that growth on L-proline betaine induces expression of a formaldehyde dehydrogenase distinct from the enzyme induced during growth on other formaldehyde-generating substrates.IMPORTANCEThe bacterial degradation of one-carbon compounds (methanol and methylamine) and some complex multi-carbon compounds (e.g., choline) generates formaldehyde. Formaldehyde is toxic and must be removed, which can be done by oxidation to formate and then to carbon dioxide. These oxidations provide a source of energy; in some species, the CO2 thus generated can be assimilated into biomass. Using the Gram-negative bacterium Paracoccus denitrificans as the experimental model, we infer that oxidation of choline to glycine generates up to three equivalents of formaldehyde, and we identify the three steps in the catabolic pathway that are responsible. Our work sheds further light on metabolic pathways that are likely important in a variety of environmental contexts.

Genome-scale metabolic model of the versatile bacterium Paracoccus denitrificans Pd1222.

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.

Functional Degeneracy in Paracoccus denitrificans Pd1222 Is Coordinated via RamB, Which Links Expression of the Glyoxylate Cycle to Activity of the Ethylmalonyl-CoA Pathway.

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.

DksA, ppGpp, and RegAB Regulate Nitrate Respiration in Paracoccus denitrificans.

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.

The NtrYX Two-Component System of Paracoccus denitrificans Is Required for the Maintenance of Cellular Iron Homeostasis and for a Complete Denitrification under Iron-Limited Conditions.

Denitrification consists of the sequential reduction of nitrate to nitrite, nitric oxide, nitrous oxide, and dinitrogen. Nitrous oxide escapes to the atmosphere, depending on copper availability and other environmental factors. Iron is also a key element because many proteins involved in denitrification contain iron-sulfur or heme centers. The NtrYX two-component regulatory system mediates the responses in a variety of metabolic processes, including denitrification. A quantitative proteomic analysis of a Paracoccus denitrificans NtrY mutant grown under denitrifying conditions revealed the induction of different TonB-dependent siderophore transporters and proteins related to iron homeostasis. This mutant showed lower intracellular iron content than the wild-type strain, and a reduced growth under denitrifying conditions in iron-limited media. Under iron-rich conditions, it releases higher concentrations of siderophores and displayes lower nitrous oxide reductase (NosZ) activity than the wild-type, thus leading to nitrous oxide emission. Bioinformatic and qRT-PCR analyses revealed that NtrYX is a global transcriptional regulatory system that responds to iron starvation and, in turn, controls expression of the iron-responsive regulators fur, rirA, and iscR, the denitrification regulators fnrP and narR, the nitric oxide-responsive regulator nnrS, and a wide set of genes, including the cd1-nitrite reductase NirS, nitrate/nitrite transporters and energy electron transport proteins.

Substitution of the sole tryptophan of the cupredoxin, amicyanin, with 5-hydroxytryptophan alters fluorescence properties and energy transfer to the type 1 copper site.

Amicyanin is a type 1 copper protein with a single tryptophan residue. Using genetic code expansion, the tryptophan was selectively replaced with the unnatural amino acid, 5-hydroxytryptophan (5-HTP). The 5-HTP substituted amicyanin exhibited absorbance at 300-320 nm, characteristic of 5-HTP and not seen in native amicyanin. The fluorescence emission maximum in 5-HTP substituted amicyanin is redshifted from 318 nm in native amicyanin to 331 nm and to 348 nm in the unfolded protein. The fluorescence quantum yield of 5-HTP substituted amicyanin mutant was much less than that of native amicyanin. Differences in intrinsic fluorescence are explained by differences in the excited states of tryptophan versus 5-HTP and the intraprotein environment. The substitution of tryptophan with 5-HTP did not affect the visible absorbance and redox potential of the copper, which is 10 Å away. In amicyanin and other cupredoxins, an unexplained quenching of the intrinsic fluorescence by the bound copper is observed. However, the fluorescence of 5-HTP substituted amicyanin is not quenched by the copper. It is shown that the mechanism of quenching in native amicyanin is Förster, or fluorescence, resonance energy transfer (FRET). This does not occur in 5-HTP substituted amicyanin because the fluorescence quantum yield is significantly lower and the red-shift of fluorescence emission maximum decreases overlap with the near UV absorbance of copper. Characterization of the distinct fluorescence properties of 5-HTP relative to tryptophan in amicyanin provides a basis for spectroscopic interrogation of the protein microenvironment using 5-HTP, and long-distance interactions with transition metals.

Effect of pH on the denitrification proteome of the soil bacterium Paracoccus denitrificans PD1222.

Denitrification is a respiratory process by which nitrate is reduced to dinitrogen. Incomplete denitrification results in the emission of the greenhouse gas nitrous oxide and this is potentiated in acidic soils, which display reduced denitrification rates and high N2O/N2 ratios compared to alkaline soils. In this work, impact of pH on the proteome of the soil denitrifying bacterium Paracoccus denitrificans PD1222 was analysed with nitrate as sole energy and nitrogen source under anaerobic conditions at pH ranging from 6.5 to 7.5. Quantitative proteomic analysis revealed that the highest difference in protein representation was observed when the proteome at pH 6.5 was compared to the reference proteome at pH 7.2. However, this difference in the extracellular pH was not enough to produce modification of intracellular pH, which was maintained at 6.5 ± 0.1. The biosynthetic pathways of several cofactors relevant for denitrification and nitrogen assimilation like cobalamin, riboflavin, molybdopterin and nicotinamide were negatively affected at pH 6.5. In addition, peptide representation of reductases involved in nitrate assimilation and denitrification were reduced at pH 6.5. Data highlight the strong negative impact of pH on NosZ synthesis and intracellular copper content, thus impairing active NosZ assembly and, in turn, leading to elevated nitrous oxide emissions.

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.

Functional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor.

Bioconversion of peptidyl amino acids into enzyme cofactors is an important post-translational modification. Here, we report a flavoprotein, essential for biosynthesis of a protein-derived quinone cofactor, cysteine tryptophylquinone, contained in a widely distributed bacterial enzyme, quinohemoprotein amine dehydrogenase. The purified flavoprotein catalyzes the single-turnover dihydroxylation of the tryptophylquinone-precursor, tryptophan, in the protein substrate containing triple intra-peptidyl crosslinks that are pre-formed by a radical S-adenosylmethionine enzyme within the ternary complex of these proteins. Crystal structure of the peptidyl tryptophan dihydroxylase reveals a large pocket that may dock the protein substrate with the bound flavin adenine dinucleotide situated close to the precursor tryptophan. Based on the enzyme-protein substrate docking model, we propose a chemical reaction mechanism of peptidyl tryptophan dihydroxylation catalyzed by the flavoprotein monooxygenase. The diversity of the tryptophylquinone-generating systems suggests convergent evolution of the peptidyl tryptophan-derived cofactors in different proteins.

Regulation of ATP hydrolysis by the ε subunit, ζ subunit and Mg-ADP in the ATP synthase of Paracoccus denitrificans.

F1FO-ATP synthase is a crucial metabolic enzyme that uses the proton motive force from respiration to regenerate ATP. For maximum thermodynamic efficiency ATP synthesis should be fully reversible, but the enzyme from Paracoccus denitrificans catalyzes ATP hydrolysis at far lower rates than it catalyzes ATP synthesis, an effect often attributed to its unique ζ subunit. Recently, we showed that deleting ζ increases hydrolysis only marginally, indicating that other common inhibitory mechanisms such as inhibition by the C-terminal domain of the ε subunit (ε-CTD) or Mg-ADP may be more important. Here, we created mutants lacking the ε-CTD, and double mutants lacking both the ε-CTD and ζ subunit. No substantial activation of ATP hydrolysis was observed in any of these strains. Instead, hydrolysis in even the double mutant strains could only be activated by oxyanions, the detergent lauryldimethylamine oxide, or a proton motive force, which are all considered to release Mg-ADP inhibition. Our results establish that P. denitrificans ATP synthase is regulated by a combination of the ε and ζ subunits and Mg-ADP inhibition.

nosX is essential for whole-cell N2O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase.

Nitrous oxide (N2O) is a potent greenhouse gas that is produced naturally as an intermediate during the process of denitrification carried out by some soil bacteria. It is consumed by nitrous oxide reductase (N2OR), the terminal enzyme of the denitrification pathway, which catalyses a reduction reaction to generate dinitrogen. N2OR contains two important copper cofactors (CuA and CuZ centres) that are essential for activity, and in copper-limited environments, N2OR fails to function, contributing to rising levels of atmospheric N2O and a major environmental challenge. Here we report studies of nosX, one of eight genes in the nos cluster of the soil dwelling α-proteobaterium Paraccocus denitrificans. A P. denitrificans ΔnosX deletion mutant failed to reduce N2O under both copper-sufficient and copper-limited conditions, demonstrating that NosX plays an essential role in N2OR activity. N2OR isolated from nosX-deficient cells was found to be unaffected in terms of the assembly of its copper cofactors, and to be active in in vitro assays, indicating that NosX is not required for the maturation of the enzyme; in particular, it plays no part in the assembly of either of the CuA and CuZ centres. Furthermore, quantitative Reverse Transcription PCR (qRT-PCR) studies showed that NosX does not significantly affect the expression of the N2OR-encoding nosZ gene. NosX is a homologue of the FAD-binding protein ApbE from Pseudomonas stutzeri, which functions in the flavinylation of another N2OR accessory protein, NosR. Thus, it is likely that NosX is a system-specific maturation factor of NosR, and so is indirectly involved in maintaining the reaction cycle of N2OR and cellular N2O reduction.

Efficient total nitrogen removal from wastewater by Paracoccus denitrificans DYTN-1.

Bioaugmentation is an effective treatment method to reduce nitrogenous pollutants from wastewater. A strain of DYTN-1, which could effectively remove TN from sewage, was isolated from the sludge of a wastewater treatment plant and was identified as Paracoccus denitrificans. The TN in wastewater reduced to <20 mg l-1 within 12 h under optimal conditions by free cells of P. denitrificans DYTN-1. To enhance the removal of TN, P. denitrificans DYTN-1 cells were immobilized in sodium alginate (SA) using different divalent metal ions as cross-linking agents. It was found that the immobilized P. denitrificans DYTN-1 cells could reduce the TN concentration from 100 to below 20 mg l-1 within 8 h. After the optimization of an orthogonal experiment, the immobilized P. denitrificans DYTN-1 cells could reduce the TN concentration from 100 mg l-1 to below 20 mg l-1 within 1 h and significantly reduce the fermentation cycle. These findings would provide an economical and effective method for the removal of total nitrogen in wastewater by immobilized cells of P. denitrificans DYTN-1. SIGNIFICANCE AND IMPACT OF THE STUDY: We identified a new Paracoccus denitrificans strain (DYTN-1) for removal of the total nitrogen in wastewater. The total nitrogen could be removed effectively by P. denitrificans DYTN-1 within 12 h in wastewater. Using sodium alginate as the carrier and Ba2+ as cross-linking agent, the immobilized P. denitrificans DYTN-1 cells could improve the removal efficiency of total nitrogen in wastewater and significantly reduce the fermentation cycle. The assay has provided an economical and effective method for the removal of total nitrogen in wastewater by immobilized cell.

A Central Small RNA Regulatory Circuit Controlling Bacterial Denitrification and N2O Emissions.

Global atmospheric loading of the climate-active gas nitrous oxide (N2O) continues to increase. A significant proportion of anthropogenic N2O emissions arises from microbial transformation of nitrogen-based fertilizers during denitrification, making microbial N2O emissions a key target for greenhouse gas reduction strategies. The genetic, physiological, and environmental regulation of microbially mediated N2O flux is poorly understood and therefore represents a critical knowledge gap in the development of successful mitigation approaches. We have previously mapped the transcriptional landscape of the model soil-denitrifying bacterium Paracoccus denitrificans Here, we show that a single bacterial small RNA (sRNA) can control the denitrification rate of P. denitrificans by stalling denitrification at nitrite reduction to limit production of downstream pathway intermediates and N2O emissions. Overexpression of sRNA-29 downregulates nitrite reductase and limits NO and N2O production by cells. RNA sequencing (RNA-seq) analysis revealed 53 genes that are controlled by sRNA-29, one of which is a previously uncharacterized GntR-type transcriptional regulator. Overexpression of this regulator phenocopies sRNA-29 overexpression and allows us to propose a model whereby sRNA-29 enhances levels of the regulator to repress denitrification under appropriate conditions. Our identification of a new regulatory pathway controlling the core denitrification pathway in bacteria highlights the current chasm in knowledge regarding genetic regulation of this pivotal biogeochemical process, which needs to be closed to support future biological and chemical N2O mitigation strategies.IMPORTANCE N2O is an important greenhouse gas and a major cause of ozone depletion. Denitrifying bacteria play vital roles in the production and consumption of N2O in many environments. Complete denitrification consists of the conversion of a soluble N-oxyanion, nitrate (NO3-), to an inert gaseous N-oxide, dinitrogen (N2). Incomplete denitrification can occur if conditions are prohibitive, for example, under conditions of low soil copper concentrations, leading to emission of N2O rather than N2 Although enzymatically well characterized, the genetic drivers that regulate denitrification in response to environmental and physiological cues are not fully understood. This study identified a new regulatory sRNA-based control mechanism for denitrification in the model denitrifying bacterium P. denitrificans Overexpression of this sRNA slows the rate of denitrification. This report highlights that there are gaps in understanding the regulation of this important pathway which need to be filled if strategies for N2O mitigation can be rationally and carefully developed.

Dynamic Metabolic Rewiring Enables Efficient Acetyl Coenzyme A Assimilation in Paracoccus denitrificans.

During growth, microorganisms have to balance metabolic flux between energy and biosynthesis. One of the key intermediates in central carbon metabolism is acetyl coenzyme A (acetyl-CoA), which can be either oxidized in the citric acid cycle or assimilated into biomass through dedicated pathways. Two acetyl-CoA assimilation strategies in bacteria have been described so far, the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). Here, we show that Paracoccus denitrificans uses both strategies for acetyl-CoA assimilation during different growth stages, revealing an unexpected metabolic complexity in the organism's central carbon metabolism. The EMCP is constitutively expressed on various substrates and leads to high biomass yields on substrates requiring acetyl-CoA assimilation, such as acetate, while the GC is specifically induced on these substrates, enabling high growth rates. Even though each acetyl-CoA assimilation strategy alone confers a distinct growth advantage, P. denitrificans recruits both to adapt to changing environmental conditions, such as a switch from succinate to acetate. Time-resolved single-cell experiments show that during this switch, expression of the EMCP and GC is highly coordinated, indicating fine-tuned genetic programming. The dynamic metabolic rewiring of acetyl-CoA assimilation is an evolutionary innovation by P. denitrificans that allows this organism to respond in a highly flexible manner to changes in the nature and availability of the carbon source to meet the physiological needs of the cell, representing a new phenomenon in central carbon metabolism.IMPORTANCE Central carbon metabolism provides organisms with energy and cellular building blocks during growth and is considered the invariable "operating system" of the cell. Here, we describe a new phenomenon in bacterial central carbon metabolism. In contrast to many other bacteria that employ only one pathway for the conversion of the central metabolite acetyl-CoA, Paracoccus denitrificans possesses two different acetyl-CoA assimilation pathways. These two pathways are dynamically recruited during different stages of growth, which allows P. denitrificans to achieve both high biomass yield and high growth rates under changing environmental conditions. Overall, this dynamic rewiring of central carbon metabolism in P. denitrificans represents a new strategy compared to those of other organisms employing only one acetyl-CoA assimilation pathway.

Insights into the mode of flavin mononucleotide binding and catalytic mechanism of bacterial chromate reductases: A molecular dynamics simulation study.

Enzymes from natural sources protect the environment via complex biological mechanisms, which aid in reductive immobilization of toxic metals including chromium. Nevertheless, progress was being made in elucidating high-resolution crystal structures of reductases and their binding with flavin mononucleotide (FMN) to understand the underlying mechanism of chromate reduction. Therefore, herein, we employed molecular dynamics (MD) simulations, principal component analysis (PCA), and binding free energy calculations to understand the dynamics behavior of these enzymes with FMN. Six representative chromate reductases in monomeric and dimeric forms were selected to study the mode, dynamics, and energetic component that drive the FMN binding process. As evidenced by MD simulation, FMN prefers to bind the cervix formed between the catalytic domain surrounded by strong conserved hydrogen bonding, electrostatic, and hydrophobic contacts. The slight movement and reorientation of FMN resulted in breakage of some crucial H-bonds and other nonbonded contacts, which were well compensated with newly formed H-bonds, electrostatic, and hydrophobic interactions. The critical residues aiding in tight anchoring of FMN within dimer were found to be strongly conserved in the bacterial system. The molecular mechanics combined with the Poisson-Boltzmann surface area binding free energy of the monomer portrayed that the van der Waals and electrostatic energy contribute significantly to the total free energy, where, the polar solvation energy opposes the binding of FMN. The proposed proximity relationships between enzyme and FMN binding site presented in this study will open up better avenues to engineer enzymes with optimized chromate reductase activity for sustainable bioremediation of heavy metals.