Atypical Features of Thermus thermophilus Succinate:Quinone Reductase

The Thermus thermophilus succinate:quinone reductase (SQR), serving as the respiratory complex II, has been homologously produced under the control of a constitutive promoter and subsequently purified. The detailed biochemical characterization of the resulting wild type (wt-rcII) and His-tagged (rcII-His₈-SdhB and rcII-SdhB-His₆) complex II variants showed the same properties as the native enzyme with respect to the subunit composition, redox cofactor content and sensitivity to the inhibitors malonate, oxaloacetate, 3-nitropropionic acid and nonyl-4-hydroxyquinoline-N-oxide (NQNO). The position of the His-tag determined whether the enzyme retained its native trimeric conformation or whether it was present in a monomeric form. Only the trimer exhibited positive cooperativity at high temperatures. The EPR signal of the [2Fe-2S] cluster was sensitive to the presence of substrate and showed an increased rhombicity in the presence of succinate in the native and in all recombinant forms of the enzyme. The detailed analysis of the shape of this signal as a function of pH, substrate concentration and in the presence of various inhibitors and quinones is presented, leading to a model for the molecular mechanism that underlies the influence of succinate on the rhombicity of the EPR signal of the proximal iron-sulfur cluster.     

The Nitric-oxide Reductase from Paracoccus denitrificans Uses a Single Specific Proton Pathway

The NO reductase from Paracoccus denitrificans reduces NO to N₂O (2NO + 2H⁺ + 2e⁻ → N₂O + H₂O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O₂-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O₂ reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O₂. We further show that electron transfer during single-turnover reduction of O₂ is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.     

The Dual-Functioning Fumarate Reductase Is the Sole Succinate:Quinone Reductase in Campylobacter jejuni and Is Required for Full Host Colonization

Campylobacter jejuni encodes all the enzymes necessary for a complete oxidative tricarboxylic acid (TCA) cycle. Because of its inability to utilize glucose, C. jejuni relies exclusively on amino acids as the source of reduced carbon, and they are incorporated into central carbon metabolism. The oxidation of succinate to fumarate is a key step in the oxidative TCA cycle. C. jejuni encodes enzymes annotated as a fumarate reductase (Cj0408 to Cj0410) and a succinate dehydrogenase (Cj0437 to Cj0439). Null alleles in the genes encoding each enzyme were constructed. Both enzymes contributed to the total fumarate reductase activity in vitro. The frdA::cat⁺ strain was completely deficient in succinate dehydrogenase activity in vitro and was unable to perform whole-cell succinate-dependent respiration. The sdhA::cat⁺ strain exhibited wild-type levels of succinate dehydrogenase activity both in vivo and in vitro. These data indicate that Frd is the only succinate dehydrogenase in C. jejuni and that the protein annotated as a succinate dehydrogenase has been misannotated. The frdA::cat⁺ strain was also unable to grow with the characteristic wild-type biphasic growth pattern and exhibited only the first growth phase, which is marked by the consumption of aspartate, serine, and associated organic acids. Substrates consumed in the second growth phase (glutamate, proline, and associated organic acids) were not catabolized by the the frdA::cat⁺ strain, indicating that the oxidation of succinate is a crucial step in metabolism of these substrates. Chicken colonization trials confirmed the in vivo importance of succinate oxidation, as the frdA::cat⁺ strain colonized chickens at significantly lower levels than the wild type, while the sdhA::cat⁺ strain colonized chickens at wild-type levels.Campylobacter jejuni causes approximately two million cases of bacterial gastroenteritis in the United States annually (34). Humans are most often infected due to cross-contamination resulting from improper handling of poultry (27), which is the natural habitat of C. jejuni (28). The eradication of C. jejuni from poultry flocks is an important goal in reducing the number of campylobacteriosis cases.C. jejuni can rely solely on catabolism of small organic acids and amino acids as a carbon and energy source, and the products of this catabolism are used for glycolysis and the tricarboxylic acid (TCA) cycle (15, 29). Fumarate and succinate are key intermediates in the TCA cycle, and the interconversion of these compounds is a vital process in organisms that use the TCA cycle for central carbon metabolism. C. jejuni encodes a complete oxidative TCA cycle, which converts TCA intermediates (carboxylic acids) to CO₂, ATP, and reducing equivalents. One of the conversion steps, oxidation of succinate to fumarate, forms a reducing equivalent and is required for a complete cycle. Reduction of fumarate to succinate also occurs as part of the reductive TCA cycle, and this carbon fixation pathway has been proposed to be utilized by ɛ-proteobacteria found in deep-sea hydrothermal vents (3). C. jejuni encodes many of the reversible enzymes necessary for the reductive TCA cycle, including 2-oxoglutarate ferredoxin oxidoreductase (encoded by oorDABC) and pyruvate carboxylase (encoded by pycA and pycB) (29); however, C. jejuni does not encode an ATP citrate lyase, which is required for full cyclic reductive carboxylation (3). The fumarate-succinate interconversion is also involved in respiration (11), and fumarate has specifically been implicated as an electron acceptor that is an alternative to oxygen in other ɛ-proteobacteria (5, 17).C. jejuni encodes an enzyme which is annotated as a fumarate reductase (Cj0408 to Cj0410) and an enzyme which is annotated as a succinate dehydrogenase (Cj0437 to Cj0439) (29). Both of these enzymes are part of a large family of proteins called the succinate:quinone oxidoreductases (SQRs). These compounds are membrane-bound enzymes that either catalyze the two-electron oxidation of succinate to the two-electron reduction of quinone/quinol or, in the reverse direction, couple the oxidation of quinol/quinone to the reduction of fumarate to succinate. The amino acid sequence, however, does not dictate the in vivo function (18), and in characterized organisms like Escherichia coli both enzymes are able to reduce fumarate and oxidize succinate, albeit with a preference for one substrate (6, 21).The SQRs can be divided into three distinct classes based on function, all of which have similar subunit compositions and primary amino acid sequences. Class 1 SQRs couple the oxidation of succinate to the reduction of a high-redox-potential quinone like ubiquinone in vivo. Class 2 SQRs are the quinol:fumarate reductases, which couple the oxidation of menaquinol to the reduction of fumarate. And class 3 SQRs couple the oxidation of succinate to the reduction of a low-potential quinone, such as menaquinone, in vivo (11). Although each class has shared motifs, the in vivo function of an SQR enzyme cannot be resolved based on the primary sequence and must be determined experimentally. Fumarate reductase (Frd) activity has been reported to occur in the particulate fraction of C. jejuni cell lysates, and addition of formate to whole cells increased Frd activity (38), which implies that there is an active electron transport pathway. However, C. jejuni is unable to utilize fumarate as an alternative electron acceptor under anaerobic conditions (37, 41). C. jejuni can also use succinate as an electron donor to a respiratory quinone (12), which has been identified as either a menaquinone-6 or methylmenaquinone-6 (4). Yet succinate oxidation via menaquinone is an endergonic reaction; succinate has a redox midpoint potential (E_m) of 30 mV, and menaquinone is more electronegative (E_m = −80 mV). Although succinate oxidation coupled to menaquinone reduction would be an “uphill” reaction, class 3 SQRs can catalyze this reaction. Studies of gram-positive bacteria belonging to the genus Bacillus, as well as studies of sulfate-reducing bacteria, have shown that oxidation of succinate through menaquinone is driven by reverse transmembrane electron transport (18, 36, 45), and it is hypothesized that C. jejuni behaves similarly. The C. jejuni Frd enzyme contains three subunits, FrdC, FrdA, and FrdB, and the gene order in the operon is similar to that in Wolinella succinogenes (16, 19) and Helicobacter pylori (1, 9, 40). Based on Frd enzymes of other bacteria, FrdC (Cj0408) is the membrane anchor and diheme cytochrome b, FrdA (Cj0409) is a flavoprotein where the reduction of fumarate to succinate occurs, and FrdB (Cj0410) is an Fe-S protein (29). The succinate dehydrogenase of C. jejuni is also composed of three subunits, SdhABC encoded by Cj0437 to Cj0439 (29). SdhA is annotated as a succinate dehydrogenase flavoprotein subunit, SdhB is a putative succinate dehydrogenase Fe-S protein, and SdhC is a putative succinate dehydrogenase subunit C. According to ClustalW pairwise alignment, FrdA and SdhA of C. jejuni share 29% identity, FrdB and SdhB share 18% identity, and FrdC and SdhC share 13% identity.A better understanding of the C. jejuni TCA cycle may help identify metabolic pathways that are crucial to C. jejuni''s ability to thrive in poultry. The roles of the C. jejuni fumarate reductase and succinate dehydrogenase in the TCA cycle and respiration were investigated. Both enzymes contribute to the total fumarate reductase activity. We determined that the protein annotated as the fumarate reductase functions as the sole succinate dehydrogenase and that this enzyme is required for full colonization of chickens by C. jejuni. The sdh operon has been misannotated as the enzyme that it encodes exhibits no succinate dehydrogenase activity, as has recently been reported to be the case for the annotated succinate dehydrogenase of W. succinogenes (14).     

The terminal oxidases of Paracoccus denitrificans

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding sub unit I of the aa₃-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (ActaDI, ActaDII), indicating that they are isoforms of subunit I of the aa₃-type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. Thisprotohaem-containing oxidase, called cytochrome bb₃, is the oniy quinoi oxidase expressed under the conditions used, in a triple oxidase mutant (ActaDI, ActaDII, cyoB::Km^R) an alternative cyto-chrome c oxidase has been characterized; this cbb₃-type oxidase has been partially purified. Both cytochrome aa₃ and cytochrome bb₃ are redox-driven proton pumps. The proton-pumping capacity of cytochrome cbb₃ has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.     

Structure of Escherichia coli Succinate:Quinone Oxidoreductase with an Occupied and Empty Quinone-binding Site

Three new structures of Escherichia coli succinate-quinone oxidoreductase (SQR) have been solved. One with the specific quinone-binding site (Q-site) inhibitor carboxin present has been solved at 2.4 Å resolution and reveals how carboxin inhibits the Q-site. The other new structures are with the Q-site inhibitor pentachlorophenol and with an empty Q-site. These structures reveal important details unresolved in earlier structures. Comparison of the new SQR structures shows how subtle rearrangements of the quinone-binding site accommodate the different inhibitors. The position of conserved water molecules near the quinone binding pocket leads to a reassessment of possible water-mediated proton uptake networks that complete reduction of ubiquinone. The dicarboxylate-binding site in the soluble domain of SQR is highly similar to that seen in high resolution structures of avian SQR (PDB 2H88) and soluble flavocytochrome c (PDB 1QJD) showing mechanistically significant structural features conserved across prokaryotic and eukaryotic SQRs.Succinate:quinone oxidoreductase (SQR,⁴ succinate dehydrogenase) and menaquinol:fumarate oxidoreductase (QFR, fumarate reductase), members of the Complex II family, are homologous integral membrane proteins which couple the interconversion of succinate and fumarate with quinone and quinol (1–4). SQR is a key enzyme in the Krebs cycle, oxidizing succinate to fumarate during aerobic growth and reducing quinone to quinol and, thus, acts as a direct link between the Krebs cycle and the respiratory chain. QFR is found in anaerobic or facultative bacteria and lower eukaryotes, where it couples the oxidation of reduced quinones to the reduction of fumarate (1, 4). Escherichia coli SQR has four subunits, two hydrophilic subunits exposed to the cytoplasm (SdhA and SdhB), which interact with two hydrophobic membrane-intrinsic subunits (SdhC and SdhD) (5). SdhA contains the dicarboxylate-binding site and a covalently bound FAD cofactor which cycles between FAD and FADH₂ redox states during succinate oxidation (6). The electrons from succinate oxidation are sequentially transferred via a [2Fe-2S], a [4Fe-4S], and a [3Fe-4S] iron-sulfur cluster relay system in SdhB to a quinone-binding site (Q_P) located at the interface of the SdhB, SdhC, and SdhD subunits. SdhC and SdhD are both composed of three transmembrane helices and coordinate a low spin b-type heme via His residues contributed by each subunit (7, 8).The first structural information about members of the Complex II family came from x-ray structures of the QFR enzymes from E. coli at 3.3 Å resolution (9) and Wolinella succinogenes at 2.2 Å resolution (10). These structures revealed details of the overall architecture of the subunits, the position of key redox cofactors, the electron transfer pathway, and the quinone-binding sites. At around the same time, the structures of soluble fumarate reductases found in anaerobic and microaerophilic bacteria and structurally homologous to the flavoprotein subunit of Complex II were solved by x-ray crystallography (1). Analysis of these soluble fumarate reductases has proven particularly informative in describing the mechanism of fumarate reduction and succinate oxidation at the dicarboxylate-binding site (11–14).Structures of SQRs lagged behind those of the QFRs until the structure of the E. coli enzyme was solved at 2.6 Å (15). This structure, solved in space group R32, revealed that the E. coli enzyme is packed as a trimer. The structures of the SdhA and SdhB subunits were highly similar to those of E. coli and W. succinogenes QFRs, but the transmembrane SdhC and SdhD subunits showed differences compared with their QFR counterparts. The structure revealed the position of the redox sites and the dicarboxylate- and quinone-binding (Q) sites. The heme b molecule was shown to lie away from the electron transfer pathway, suggesting electrons are preferentially transferred from the [3Fe-4S] cluster to ubiquinone, on the grounds of the edge-to-edge distances and redox potentials of the relevant groups. The structure revealed density in the Q-site that was interpreted as ubiquinone, and the position of the binding site was confirmed by the structure of the E. coli enzyme co-crystallized with the Q-site inhibitor 2-(1-methyl-hexyl)-4,6-dinitrophenol (DNP-17, PDB code 1NEN (15)). The E. coli enzyme was subsequently co-crystallized with the Q-site inhibitor Atpenin A5 (AA5) (PDB code 2ACZ (16)). This inhibitor was bound deeper into the quinone-binding site than ubiquinone or DNP-17, suggesting that there are two binding positions for ubiquinone in its binding site. The structure also identified a water-mediated proton pathway, proposed to deliver protons to the quinone-binding site. The first structure of a mitochondrial SQR was from porcine heart at 2.4 Å resolution (PDB code 1ZOY (17). This structure revealed a monomer in the asymmetric unit, suggesting that mitochondrial SQRs were likely to function as monomers. Superposition of the porcine and E. coli SQR structures revealed the high structural similarity of the SdhA and SdhB subunits and the conservation in position of the redox cofactors. Larger divergences were observed in the transmembrane subunits.Further structural information about SQRs was obtained by analysis of structures of avian SQR crystallized with oxaloacetate (2.2 Å resolution, PDB code 1YQ3), with 3-nitropropionate (2.4 Å resolution, PDB code 1YQ4), and with the Q-site inhibitor carboxin (2.1 Å resolution, PDB code 2FBW) (18). These structures revealed important differences in the position of key residues in the dicarboxylate-binding site compared with the E. coli and porcine structures. Arg-297 (equivalent to Arg-298 in porcine and Arg-286 in E. coli SQRs) was ideally located to act as a general base catalyst, accepting a proton during dehydrogenation of succinate, as in the soluble Shewanella flavocytochrome c3 (PDB code 1QJD) (11), suggesting conservation of mechanism between these distantly related enzymes. An unusual cis-serine peptide bond was proposed to position another arginine residue for binding dicarboxylates. Density for the dicarboxylate in 1YQ3 and 2FBW was shown to be distinctly non-planar and could be modeled by the “malate-like intermediate” seen in 1QJD. The nature of the ligand in the dicarboxylate site was further analyzed in a 1.74 Å resolution structure of avian SQR (PDB code 2H88), confirming the high structural similarity of the ligand and binding site residues in the SQR and flavocytochrome c3 structures (11, 12, 14).Despite the structural information described above, there are still unresolved issues regarding the structure and function of SQRs and QFRs. These include the location of conserved waters, which may form a channel involved in protonation of quinone, and the ability of the Q-site to accommodate different quinones and inhibitors. To further address these issues, we pursued structure-function studies of E. coli SQR. We developed alternative crystallization conditions that provided crystals more reproducibly and diffracting to higher resolution. By exchanging the enzyme into decyl-β-d-maltoside (DM) during purification, it was possible to crystallize the enzyme in the orthorhombic P2₁2₁2₁ space group. These crystals routinely diffracted in the 3–3.5 Å resolution range. Co-crystallization with the biochemically well characterized Q-site inhibitor carboxin improved diffraction to 2.1–2.8 Å. This structure shows new features related to the dicarboxylate-binding site of E. coli SQR including a rare cis-peptide bond in SdhA, as found in avian SQR (14), which helps shape the geometry of the active site. Comparisons of the structure with those of SQR binding PCP and SQR with an empty Q-site show how subtle rearrangements of the Q-site accommodate the different inhibitors. The orientation of carboxin in the Q-site differs with computational predictions (16) and with that seen in avian SQR (2FBW). The position of conserved water molecules around the Q-site suggests a new water-mediated proton uptake pathway consistent with recent mutational and biophysical studies (19).     

Effect of oxygen on ubiquinone-10 production by Paracoccus denitrificans

Summary Ubiquinone-10 (Q₁₀) production was measured in batch cultures of Paracoccus denitrificans grown for 8 h at increasing oxygen concentrations (0–21 % O₂ in the sparging gas). Whereas the cellular level of Q₁₀ decreased monotonically from 1.2 to 0.5 mol/g d.w., the total yield of Q₁₀ was maximal at 2.5 % O₂ and amounted to 350 nmol (0.3 mg) per L of culture.     

The nitric oxide reductase of Paracoccus denitrificans.

The nitric oxide (NO) reductase activity of the cytoplasmic membrane of Paracoccus denitrificans can be solubilized in dodecyl maltoside with good retention of activity. The solubilized enzyme lacks NADH-dependent activity, but can be assayed with isoascorbate plus 2,3,5,6-tetramethylphenylene-1,4-diamine as electron donor and with horse heart cytochrome c as mediator. Reduction of NO was measured with an amperomeric electrode. The solubilized enzyme could be separated from other electron-transport components, including the cytochrome bc1 complex and nitrite reductase, by several steps of chromatography. The purified enzyme had a specific activity of 11 mumols.min-1.mg of protein-1 and the Km(NO) was estimated as less than 10 microM. The enzyme formed N2O from NO with the expected stoichiometry. These observations support the view that NO reductase is a discrete enzyme that participates in the denitrification process. The enzyme contained both b- and c-type haems. The former was associated with a polypeptide of apparent molecular mass 37 kDa and the latter with a polypeptide of 18 kDa. Polypeptides of 29 and 45 kDa were also identified in the purified protein which showed variable behaviour on electrophoresis in polyacrylamide gels.     

Proton-Translocating ATP-Synthase of Paracoccus denitrificans: ATP-Hydrolytic Activity

Tightly coupled membranes of Paracoccus denitrificans catalyze oxidative phosphorylation but are incapable of ATP hydrolysis. The conditions for observation and registration of the venturicidin-sensitive ATPase activity of subbacterial particles derived from this organism are described. The ATP hydrolytic activity does not appear after prolonged incubation in the presence of pyruvate kinase and phosphoenol pyruvate (to remove ADP), EDTA (to remove Mg²⁺) and/or inorganic phosphate, whereas the activity dramatically increases after energization of the membranes. ATP hydrolysis by activated ATPase is coupled with electric potential formation. Inorganic phosphate prevents and azide promotes a decline of the enzyme activity during ATP hydrolysis. The addition of uncouplers results in rapid and complete inactivation of ATPase. The dependent ATPase activity increases upon dilution of the membranes. The results are discussed as evidence for the presence of distinct ATP-synthase and ATP-hydrolase states of F_oF₁ complex in the coupling membranes (Vinogradov, A. D. (1999) Biochemistry (Moscow), 64, 1219-1229). The proposal is made that part of the free energy released from oxidoreduction in the respiratory chain is used to maintain active conformation of the energy-transducing proteins.     

Atypical Polyphosphate Accumulation by the Denitrifying Bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-β-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.     

The cytochrome c peroxidase of Paracoccus denitrificans.

The size, visible absorption spectra, nature of haem and haem content suggest that the cytochrome c peroxidase of Paracoccus denitrificans is related to that of Pseudomonas aeruginosa. However, the Paracoccus enzyme shows a preference for cytochrome c donors with a positively charged 'front surface' and in this respect resembles the cytochrome c peroxidase from Saccharomyces cerevisiae. Paracoccus cytochrome c-550 is the best electron donor tested and, in spite of an acidic isoelectric point, has a markedly asymmetric charge distribution with a strongly positive 'front face'. Mitochondrial cytochromes c have a much less pronounced charge asymmetry but are basic overall. This difference between cytochrome c-550 and mitochondrial cytochrome c may reflect subtle differences in their electron transport roles. A dendrogram of cytochrome c1 sequences shows that Rhodopseudomonas viridis is a closer relative of mitochondria than is Pa. denitrificans. Perhaps a mitochondrial-type cytochrome c peroxidase may be found in such an organism.     

Biodegradation of Isopropanol by a Solvent-Tolerant Paracoccus denitrificans Strain

The biodegradation of high concentration isopropanol (2-propanol, IPA) at 16 g/L was investigated by a solvent-tolerant strain of bacteria identified as Paracoccus denitrificans for the first time by 16S rDNA gene sequencing. The strain P. denitrificans GH3 was able to utilize the high concentration of IPA as the sole carbon source within a minimal salts medium with a cell density of 1.5 × 10⁸ cells/mL. The optimal conditions were found as follows: initial pH 7.0, incubation temperature 30°C, with IPA concentration 8 g/L. Under the optimal conditions, strain GH3 utilized 90.3% of IPA in 7 days. Acetone, the major intermediate of aerobic IPA biodegradation, was also monitored as an indicator of microbial IPA utilization. Both IPA and acetone were completely removed from the medium following 216 hr and 240 hr, respectively. The growth of strain GH3 on IPA as a sole carbon and energy source was well described by the Andrews model with a maximum growth rate (μ _max ) = 0.0277/hr, a saturation constant (K _S ) = 0.7333 g/L, and an inhibition concentration (Ki) = 8.9887 g/L. Paracoccus denitrificans GH3 is considered to be well used in degrading IPA in wastewater.     

Atypical polyphosphate accumulation by the denitrifying bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-beta-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.