How denitrifiers defense ciprofloxacin: Insights from intracellular and extracellular stress response.

Overuse of antibiotics has led to their existence in nitrogen-containing water. The impacts of antibiotics on bio-denitrification and the metabolic response of denitrifiers to antibiotics are unclear. We systematically analyzed the effect of ciprofloxacin (CIP) on bio-denitrification and found that 5 mg/L CIP greatly inhibited denitrification with a model denitrifier (Paracoccus denitrificans). Nitrate reduction decreased by 32.89 % and nitrous oxide emission increased by 75.53 %. The balance analysis of carbon and nitrogen metabolism during denitrification showed that CIP exposure blocked electron transfer and reduced the flow of substrate metabolism used for denitrification. Proteomics results showed that CIP exposure induced denitrifiers to use the pentose phosphate pathway more for substrate metabolism. This caused a substrate preference to generate NADPH to prevent cellular damage rather than NADH for denitrification. Notably, despite denitrifiers having antioxidant defenses, they could not completely prevent oxidative damage caused by CIP exposure. The effect of CIP exposure on denitrifiers after removal of extracellular polymeric substances (EPS) demonstrated that EPS around denitrifiers formed a barrier against CIP. Fluorescence and infrared spectroscopy revealed that the binding effect of proteins in EPS to CIP prevented damage. This study shows that denitrifiers resist antibiotic stress through different intracellular and extracellular defense strategies.

Lactate oxidation in Paracoccus denitrificans.

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.

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.

Insights into the structure-function relationship of the NorQ/NorD chaperones from Paracoccus denitrificans reveal shared principles of interacting MoxR AAA+/VWA domain proteins.

BACKGROUND: NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families. RESULTS: We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that "plugs" the NorQ ring pore to the VWA domain of NorD with a protruding "finger" inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples. CONCLUSIONS: Based on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.

Structural Insight into Catalysis by the Flavin-Dependent NADH Oxidase (Pden_5119) of Paracoccus denitrificans.

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.

Proton-translocating NADH:ubiquinone oxidoreductase of Paracoccus denitrificans plasma membranes catalyzes FMN-independent reverse electron transfer to hexaammineruthenium (III).

NADH-OH, the specific inhibitor of NADH-binding site of the mammalian complex I, is shown to completely block FMN-dependent reactions of P. denitrificans enzyme in plasma membrane vesicles: NADH oxidation (in a competitive manner with Ki of 1 nM) as well as reduction of pyridine nucleotides, ferricyanide and oxygen in the reverse electron transfer. In contrast to these activities, the reverse electron transfer to hexaammineruthenium (III) catalyzed by plasma membrane vesicles is insensitive to NADH-OH. To explain these results, we hypothesize the existence of a non-FMN redox group of P. denitrificans complex I that is capable of reducing hexaammineruthenium (III), which is corroborated by the complex kinetics of NADH: hexaammineruthenium (III)-reductase activity, catalyzed by this enzyme. A new assay procedure for measuring succinate-driven reverse electron transfer catalyzed by P. denitrificans complex I to hexaammineruthenium (III) is proposed.

Preparation for Denitrification and Phenotypic Diversification at the Cusp of Anoxia: a Purpose for N2O Reductase Vis-à-Vis Multiple Roles of O2.

Adaptation to anoxia by synthesizing a denitrification proteome costs metabolic energy, and the anaerobic respiration conserves less energy per electron than aerobic respiration. This implies a selective advantage of the stringent O2 repression of denitrification gene transcription, which is found in most denitrifying bacteria. In some bacteria, the metabolic burden of adaptation can be minimized further by phenotypic diversification, colloquially termed "bet-hedging," where all cells synthesize the N2O reductase (NosZ) but only a minority synthesize nitrite reductase (NirS), as demonstrated for the model strain Paracoccus denitrificans. We hypothesized that the cells lacking NirS would be entrapped in anoxia but with the possibility of escape if supplied with O2 or N2O. To test this, cells were exposed to gradual O2 depletion or sudden anoxia and subsequent spikes of O2 and N2O. The synthesis of NirS in single cells was monitored by using an mCherry-nirS fusion replacing the native nirS, and their growth was detected as dilution of green, fluorescent fluorescein isothiocyanate (FITC) stain. We demonstrate anoxic entrapment due to e--acceptor deprivation and show that O2 spiking leads to bet-hedging, while N2O spiking promotes NirS synthesis and growth in all cells carrying NosZ. The cells rescued by the N2O spike had much lower respiration rates than those rescued by the O2 spike, however, which could indicate that the well-known autocatalytic synthesis of NirS via NO production requires O2. Our results bring into relief a fitness advantage of pairing restrictive nirS expression with universal NosZ synthesis in energy-limited systems. IMPORTANCE Denitrifying bacteria have evolved elaborate regulatory networks securing their respiratory metabolism in environments with fluctuating oxygen concentrations. Here, we provide new insight regarding their bet-hedging in response to hypoxia, which minimizes their N2O emissions because all cells express NosZ, reducing N2O to N2, while a minority express NirS + Nor, reducing NO2- to N2O. We hypothesized that the cells without Nir were entrapped in anoxia, without energy to synthesize Nir, and that they could be rescued by short spikes of O2 or N2O. We confirm such entrapment and the rescue of all cells by an N2O spike but only a fraction by an O2 spike. The results shed light on the role of O2 repression in bet-hedging and generated a novel hypothesis regarding the autocatalytic nirS expression via NO production. Insight into the regulation of denitrification, including bet-hedging, holds a clue to understanding, and ultimately curbing, the escalating emissions of N2O, which contribute to anthropogenic climate forcing.

Enhancement of biological denitrification by the addition of novel sRNA Pda200 under antibiotic pressure.

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.

Interaction of Venturicidin and Fo·F1-ATPase/ATP Synthase of Tightly Coupled Subbacterial Particles of Paracoccus denitrificans in Energized Membranes.

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.

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.

Different responses of representative denitrifying bacterial strains to gatifloxacin exposure in simulated groundwater denitrification environment.

The impact of antibiotics on denitrification in the ecological environment has attracted widespread attention. However, the concentration threshold and inhibitory effect of the same antibiotic on denitrification mediated by mixed denitrifying microbes were conflicting in some studies. In this study, Paracoccus denitrificans, Acidovorax sp., and Pseudomonas aeruginosa were selected as representative denitrifying bacterial strains to explore the response of a single strain to gatifloxacin (GAT) exposure in groundwater denitrification. The results showed that the nitrate and nitrite removal efficiencies of Pseudomonas aeruginosa decreased by 34.87-36.25 % and 18.27-23.31 %, respectively, with exposure to 10 µg/L GAT, accompanied by a significant decline in denitrifying enzyme activity and gene expression. In contrast, the elevated denitrifying enzyme activity and gene expression of Paracoccus denitrificans promoted its nitrate and nitrite reduction by 2.09-10.00 % and 0-8.44 %, respectively. Additionally, there were no obvious effects on the removal of nitrate and nitrite by Acidovorax sp. in the presence of 10 µg/L GAT, which was consistent with the variation in denitrifying enzyme activity and total gene expression levels. The fit results of the Monod equation and its modification further elucidated the nitrate degradation characteristics from the perspective of denitrification kinetics. Furthermore, antibiotic resistance gene (ARG) analysis showed that the addition of 10 µg/L GAT (approximately 30 days) did not observably increase the relative abundance of ARGs. This study provides some preliminary understanding of the response differences of representative denitrifying bacterial strains to antibiotic exposure in groundwater denitrification.

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.

Simultaneous removal of high concentrations of ammonia nitrogen and calcium by the novel strain Paracoccus denitrificans AC-3 with good environmental adaptability.

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.

Phage Genes Induce Quorum Sensing Signal Release through Membrane Vesicle Formation.

Membrane vesicles (MVs) released from the bacterium Paracoccus denitrificans Pd1222 are enriched with the quorum sensing (QS) signaling molecule N-hexadecanoyl-l-homoserine lactone (C16-HSL). However, the biogenesis of MVs in Pd1222 remains unclear. Investigations on MV formation are crucial for obtaining a more detailed understanding of the dynamics of MV-assisted signaling. In the present study, live-cell imaging showed that P. denitrificans Pd1222 produced MVs through cell lysis under DNA-damaging conditions. DNA sequencing of MVs and a transcriptome ana-lysis of cells indicated that the expression of a prophage region was up-regulated at the onset of MV formation under DNA-damaging conditions. A further sequence ana-lysis identified a putative endolysin (Pden_0381) and holin (Pden_0382) in the prophage region. The expression of these genes was regulated by RecA. Using gene knockout mutants, we showed that prophage-encoded endolysin was critical for MV formation by P. denitrificans Pd1222 under DNA-damaging conditions. MV triggering by endolysin was dependent on the putative holin, which presumably transported endolysin to the periplasmic space. C16-HSL quantification revealed that more signals were released into the milieu as a consequence of the effects of endolysin. Using a QS reporter strain, we found that the QS response in P. denitrificans was stimulated by inducing the expression of endolysin. Collectively, these results provide novel insights into the mechanisms by which a bacterial cell-to-cell communication system is manipulated by phage genes.

Energy homeostasis is a conserved process: Evidence from Paracoccus denitrificans' response to acute changes in energy demand.

Paracoccus denitrificans is a model organism for the study of oxidative phosphorylation. We demonstrate a very high respiratory capacity compared to mitochondria when normalizing to cytochrome aa3 content even in the absence of alternative terminal oxidases. To gain insight into conserved mechanisms of energy homeostasis, we characterized the metabolic response to K+ reintroduction. A rapid 3-4-fold increase in respiration occurred before substantial cellular K+ accumulation followed by a sustained increase of up to 6-fold that persisted after net K+ uptake stopped. Proton motive force (Δp) was slightly higher upon addition of K+ with ΔpH increasing and compensating for membrane potential (ΔΨ) depolarization. Blocking the F0F1-ATP synthase (Complex V) with venturicidin revealed that the initial K+-dependent respiratory activation was primarily due to K+ influx. However, the ability to sustain an increased respiration rate was partially dependent on Complex V activity. The 6-fold stimulation of respiration by K+ resulted in a small net reduction of most cytochromes, different from the pattern observed with chemical uncoupling and consistent with balanced input and utilization of reducing equivalents. Metabolomics showed increases in glycolytic and TCA cycle intermediates together with a decrease in basic amino acids, suggesting an increased nitrogen mobilization upon K+ replenishment. ATP and GTP concentrations increased after K+ addition, indicating a net increase in cellular potential energy. Thus, K+ stimulates energy generation and utilization resulting in an almost constant Δp and increased high-energy phosphates during large acute and steady state changes in respiration. The specific energy consuming processes and signaling events associated with this simultaneous activation of work and metabolism in P. denitrificans remain unknown. Nevertheless, this homeostatic behavior is very similar to that observed in mitochondria in tissues when cellular energy requirements increase. We conclude that the regulation of energy generation and utilization to maintain homeostasis is conserved across the prokaryote/eukaryote boundary.

Cryo-EM structures of intermediates suggest an alternative catalytic reaction cycle for cytochrome c oxidase.

Cytochrome c oxidases are among the most important and fundamental enzymes of life. Integrated into membranes they use four electrons from cytochrome c molecules to reduce molecular oxygen (dioxygen) to water. Their catalytic cycle has been considered to start with the oxidized form. Subsequent electron transfers lead to the E-state, the R-state (which binds oxygen), the P-state (with an already split dioxygen bond), the F-state and the O-state again. Here, we determined structures of up to 1.9 Å resolution of these intermediates by single particle cryo-EM. Our results suggest that in the O-state the active site contains a peroxide dianion and in the P-state possibly an intact dioxygen molecule, the F-state may contain a superoxide anion. Thus, the enzyme's catalytic cycle may have to be turned by 180 degrees.