Phosphate transport system inParacoccus denitrificans

P_i uptake in cells or spheroplasts ofParacoccus denitrificans is biphasic; only the first rapid phase represents net P_i transport. The second phase is limited by the rate of P_i utilization inside the cell, i.e., mainly by its esterification, and as such it was inhibited by DCCD. The P_i/dicarboxylate antiporter does not seem to be operative, and its inhibitorn-butylmalonate did not exert specific inhibition. P_i transport is inhibited by SH reagents; the most potent inhibitor is PCMB, and mersalyl is much less effective. However, neither inhibitor affects efflux of accumulated P_i. The gradient of potassium ions may be involved in the P_i uptake, which is lowered in the presence of valinomycin. FCCP alone does not release accumulated P_i from spheroplasts unless they are preincubated with SCN^?. The results indicate that P_i enters the cell by symport with protons.     

Assimilation of ammonia inParacoccus denitrificans

Two pathways serve for assimilation of ammonia inParacoccus denitrificans. Glutamate dehydrogenase (NADP⁺) catalyzes the assimilation at a high NH₄ ⁺ concentration. If nitrate serves as the nitrogen source, glutamate is synthesized by glutamate-ammonia ligase and glutamate synthase (NADPH). At a very low NH₄ ⁺ concentration, all three enzymes are synthesized simultaneously. No direct relationship exists between glutamate dehydrogenase (NADP⁺) and glutamate-ammonia ligase inP. denitrificans, while the glutamate synthase (NADPH) activity changes in parallel with that of the latter enzyme. Ammonia does not influence the induction or repression of glutamate dehydrogenase (NADP⁺). The inner concentration of metabolites indicates a possible repression of glutamate dehydrogenase (NADP⁺) by the high concentration of glutamine or its metabolic products as in the case when NH₄ ⁺ is formed by assimilative nitrate reduction. No direct effect of the intermediates of nitrate assimilation on the synthesis of glutamate dehydrogenase (NADP⁺) was observed.     

C1 metabolism inParacoccus denitrificans: Genetics ofParacoccus denitrificans

Paracoccus denitrificans is able to grow on the C₁ compounds methanol and methylamine. These compounds are oxidized to formaldehyde which is subsequently oxidized via formate to carbon dioxide. Biomass is produced by carbon dioxide fixation via the ribulose biphosphate pathway. The first oxidation reaction is catalyzed by the enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively. Both enzymes contain two different subunits in an ₂₂ configuration. The genes encoding the subunits of methanol dehydrogenase (moxF andmoxI) have been isolated and sequenced. They are located in one operon together with two other genes (moxJ andmoxG) in the gene ordermoxFJGI. The function of themoxJ gene product is not yet known.MoxG codes for a cytochromec _551i , which functions as the electron acceptor of methanol dehydrogenase. Both methanol dehydrogenase and methylamine dehydrogenase contain PQQ as a cofactor. These so-called quinoproteins are able to catalyze redox reactions by one-electron steps. The reaction mechanism of this oxidation will be described. Electrons from the oxidation reaction are donated to the electron transport chain at the level of cytochromec. P. denitrificans is able to synthesize at least 10 differentc-type cytochromes. Five could be detected in the periplasm and five have been found in the cytoplasmic membrane. The membrane-bound cytochromec ₁ and cytochromec ₅₅₂ and the periplasmic-located cytochromec ₅₅₀ are present under all tested growth conditions. The cytochromesc _551i andc _553i , present in the periplasm, are only induced in cells grown on methanol, methylamine, or choline. The otherc-type cytochromes are mainly detected either under oxygen limited conditions or under anaerobic conditions with nitrate as electron acceptor or under both conditions. An overview including the induction pattern of allP. denitrificans c-type cytochromes will be given. The genes encoding cytochromec ₁, cytochromec ₅₅₀, cytochromec _551i , and cytochromec _553i have been isolated and sequenced. By using site-directed mutagenesis these genes were mutated in the genome. The mutants thus obtained were used to study electron transport during growth on C₁ compounds. This electron transport has also been studied by determining electron transfer rates inin vitro experiments. The exact pathways, however, are not yet fully understood. Electrons from methanol dehydrogenase are donated to cytochromec _551i . Further electron transport is either via cytochromec ₅₅₀ or cytochromec _553i to cytochromeaa ₃. However, direct electron transport from cytochromec _551i to the terminal oxidase might be possible as well. Electrons from methylamine dehydrogenase are donated to amicyanin and then via cytochromec ₅₅₀ to cytochromeaa ₃, but other routes are used also.P. denitrificans is studied by several groups by using a genetic approach. Several genes have already been cloned and sequenced and a lot of mutants have been isolated. The development of a host/vector system and several techniques for mutation induction that are used inP. denitrificans genetics will be described.     

Regulation of methylammonium transport inParacoccus denitrificans

Depending on the growth conditionsParacoccus denitrificans synthesizes two different carriers mediating uptake of methylamine. When used as a nitrogen source, methylamine is transported via a NH ₄ ⁺ carrier, and its transport is inhibited by NH ₄ ⁺ but not by ethylamine. When used as a carbon source, methylamine is transported by a specific alkylamine carrier, and its transport is inhibited by ethylamine but not by NH ₄ ⁺ . The NH ₄ ⁺ carrier is under nitrogen control, the alkylamine carrier under carbon control.Abbreviations MA Methylamine - FCCP p-trifluormethoxycarbonylcyanide-phenylhydrazone     

Regulation of methanol dehydrogenase synthesis inParacoccus denitrificans

The region downstream from the methanol dehydrogase (MDH) structural gene has been cloned and sequenced. MDH promoter activity have been studied by using a broad-host-range promoter probe vector.     

Genes coding for cytochromec oxidase inParacoccus denitrificans

Several loci on theParacoccus denitrificans chromosome are involved in the synthesis of cytochromec oxidase. So far three genetic loci have been isolated. One of them contains the structural genes of subunits II and III, as well as two regulatory genes which probably code for oxidase-specific assembly factors. In addition, two distinct genes for subunit I have been cloned, one of which is located adjacent to the cytochromec ₅₅₀ gene. An alignment of six promoter regions reveals only short common sequences.     

Metabolic regulation including anaerobic metabolism inParacoccus denitrificans

Under anaerobic circumstances in the presence of nitrateParacoccus denitrificans is able to denitrify. The properties of the reductases involved in nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase are described. For that purpose not only the properties of the enzymes ofP. denitrificans are considered but also those fromEscherichia coli, Pseudomonas aeruginosa, andPseudomonas stutzeri. Nitrate reductase consists of three subunits: the subunit contains the molybdenum cofactor, the subunit contains the iron sulfur clusters, and the subunit is a special cytochromeb. Nitrate is reduced at the cytoplasmic side of the membrane and evidence for the presence of a nitrate-nitrite antiporter is presented. Electron flow is from ubiquinol via the specific cytochromeb to the nitrate reductase. Nitrite reductase (which is identical to cytochromecd ₁) and nitrous oxide reductase are periplasmic proteins. Nitric oxide reductase is a membrane-bound enzyme. Thebc ₁ complex is involved in electron flow to these reductases and the whole reaction takes place at the periplasmic side of the membrane. It is now firmly established that NO is an obligatory intermediate between nitrite and nitrous oxide. Nitrous oxide reductase is a multi-copper protein. A large number of genes is involved in the acquisition of molybdenum and copper, the formation of the molybdenum cofactor, and the insertion of the metals. It is estimated that at least 40 genes are involved in the process of denitrification. The control of the expression of these genes inP. denitrificans is totally unknown. As an example of such complex regulatory systems the function of thefnr, narX, andnarL gene products in the expression of nitrate reductase inE. coli is described. The control of the effects of oxygen on the reduction of nitrate, nitrite, and nitrous oxide are discussed. Oxygen inhibits reduction of nitrate by prevention of nitrate uptake in the cell. In the case of nitrite and nitrous oxide a competition between reductases and oxidases for a limited supply of electrons from primary dehydrogenases seems to play an important role. Under some circumstances NO formed from nitrite may inhibit oxidases, resulting in a redistribution of electron flow from oxygen to nitrite.P. denitrificans contains three main oxidases: cytochromeaa ₃, cytochromeo, and cytochromeco. Cytochromeo is proton translocating and receives its electrons from ubiquinol. Some properties of cytochromeco, which receives its electrons from cytochromec, are reported. The control of the formation of these various oxidases is unknown, as well as the control of electron flow in the branched respiratory chain. Schemes for aerobic and anaerobic electron transport are given. Proton translocation and charge separation during electron transport from various electron donors and by various electron transfer pathways to oxygen and nitrogenous oxide are given. The extent of energy conservation during denitrification is about 70% of that during aerobic respiration. In sulfate-limited cultures (in which proton translocation in the NADH-ubiquinone segment of the respiratory chain is lost) the extent of energy conservation is about 60% of that under substrate-limited conditions. These conclusions are in accordance with measurements of molar growth yields.     

Function of terminal acceptors in the biosynthesis of denitrification pathway components inParacoccus denitrificans

Limited aeration of cell suspension in growth medium was used to study the kinetics of formation of nitrite reductase and nitrous-oxide reductase and their physiological electron donor, cytochromec-550, during the anaerobic adaptation ofParacoccus denitrificans. The crucial step in the regulation of synthesis of these components is the repressive effect of oxygen while nitrogenous acceptors (NO₃ ⁻, NO₂ ⁻, N₂O) probably play no role as inducers. The time course of the enzyme activites was analogous (after a lag phase a sharp increase with a maximum after 3 h) and differed from the kinetics of synthesis of cytochromec-550 (gradual rise throughout the 8-h experiment).     

Loss of penicillin resistance inParacoccus denitrificans induced by mitomycin C and acridine orange

In an attempt to eliminate the penicillin resistance gene ofP. denitrificans by curing agents, such as acridine orange (AO) and mitomycin C, it was observed that AO treatment caused temporary phenotypic curing where development of sensitivity was a function of concentration of both the curing agent and benzylpenicillin. However, curing with mitomycin produced sensitive clones at a frequency of 6×10⁻³ and two permanently cured clones were isolated. Heavy metal resistance and resistance to other drugs, however, remain unchanged in the mitomycin-cured isolate.     

Cytochromec oxidase inParacoccus denitrificans. Protein,chemical, structural,and evolutionary aspects

Preparations and protein chemical characterizations performed with cytochromec oxidase (E.C. 1.9.3.1) from the purple bacteriumParacoccus denitrificans are reviewed. The simplest catalytically competent complex of the enzyme consists of two subunits of 62012 and 27999 Da. The theoretical hemea/protein ratio of the purified enzyme is 22.0 nmol/mg. The amino acid sequences of both proteins are compared with examples of subunits I and II of mitochondrial terminal oxidases from the main kingdoms of eukaryotes. The significance of the emerging conserved features such as membrane penetration patterns, invariant residues, stoichiometry, and sites of prosthetic groups are discussed. TheParacoccus enzyme represents the only prokaryotic oxidase detailed so far, which is directly related to the mitochondrial oxidases by common ancestry in the growing O₂ atmosphere.     

Metabolic pathways inParacoccus denitrificans and closely related bacteria in relation to the phylogeny of prokaryotes

Denitrification and methylotrophy inParacoccus denitrificans are discussed. The properties of the enzymes of denitrification: the nitrate-nitrite antiporter, nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase are described. The genes for none of these proteins have yet been cloned and sequenced fromP. denitrificans. A number of sequences are available for enzymes fromEscherichia coli, Pseudomonas stutzeri andPseudomonas aeruginosa. It is concluded that pathway specificc-type cytochromes are involved in denitrification. At least 40 genes are involved in denitrification.In methanol oxidation at least 20 genes are involved. In this case too pathway specificc-type cytochromes are involved. The sequence homology between the quinoproteins methanol dehydrogenase, alcoholdehydrogenase and glucose dehydrogenase is discussed. This superfamily of proteins is believed to be derived from a common ancestor. ThemoxFJGI operon determines the structural components of methanol dehydrogenase and the associatedc-type cytochrome. Upstream of this operon 3 regulatory proteins were found. The mox Y protein shows the general features of a sensor protein and the moxX protein those of a regulatory protein. Thus a two component regulatory system is involved in both denitrification and methylotrophy.The phylogeny of prokaryotes based on 16S rRNA sequence is discussed. It is remarkable that the 16S rRNA ofThiosphaera pantotropha is identical to that ofP. denitrificans. Still these bacteria show a number of differences.T. pantotropha is able to denitrify under aerobic circumstances and it shows heterotrophic nitrification. Nitrification and heterotrophic nitrification are found in species belonging to the -and -subdivisions of purple non-sulfur bacteria. Thus the occurrence of heterotrophic nitrification inT. pantotropha which belongs to the -subdivision of purple non-sulfur bacteria is a remarkable property. FurthermoreT. pantotropha contains two nitrate reductases of which the periplasmic one is supposed to be involved in aerobic denitrification. The nitrite reductase is of the Cu-type and not of the cytochromecd ₁ type as inP. denitrificans. Also the cytochromeb of theQbc complex ofT. pantotropha is highly similar to its counterpart inP. denitrificans. It is hypothesized that the differences between these two organisms which both contain large megaplasmids is due to a combination of loss of genetic information and plasmid-coded properties. The distribution of a number of complex metabolic systems in eubacteria and in a number of species belonging to the -group of purple non sulphur bacteria is reviewed. Two possibilities to explain this haphazard distribution are considered: 1. Lateral gene transfer between distantly related micro organisms occurs frequently. 2. The eubacterial ancestors must have possessed already these properties. The distribution of these properties is due to sporadic loss during evolutionary divergence.With respect to the occurrence and frequency of lateral gene transfer two opposing views exist. According to molecular biologists lateral gene transfer occurs frequently and is very easy. Bacteria are supposed to form one large gene pool. On the other hand population geneticists have provided evidence that strong systems operate that establish reproductive isolation between diverged species and even between closely related cell lines.Data on amino acid sequences of nitrogenase proteins, cytochromesc, cytochrome oxidases, -subunits of ATP synthase and tryptophan biosynthetic enzymes of various micro organisms were reviewed. In all these cases phylogenetic trees could be constructed based on the amino acid sequence data. In all cases this phylogenetic tree was similar to the one based on 16S rRNA homology. Only in one case evidence for the occurrence of lateral gene transfer was obtained. Therefore it is concluded that lateral gene transfer played a minor role in the distribution of complex metabolic systems among prokaryotes. It must be stressed that this does not exclude the possibility that lateral gene transfer occurred frequently in the initial stage of bacterial evolution. It is hypothesized that the appearance of nitrogen fixation, denitrification and cytochrome oxidase formation were early events in the evolution of micro organisms. Both systems are supposed to have evolved only once. Subsequently the capacity to fix nitrogen or to denitrifymust have been lost many times, just as photosynthetic capacity is supposed to have been lost many times. During evolution many systems have been lost leading to a haphazard distribution of metabolic characters among bacteria. As an example it is suggested that organisms with a respiratory chain similar to that ofEscherichia coli arose by loss of the capacity to form the Qbc complex andc-type cytochromes. The remaining systems could be controlled much better however than in the ancestral organisms.     

Isolation,sequencing and mutational analysis of a gene cluster involved in nitrite reduction inParacoccus denitrificans

By using the gene encoding the C-terminal part of thecd ₁-type nitrite reductase ofPseudomonas stutzeri JM300 as a heterologous probe, the corresponding gene fromParacoccus denitrificans was isolated. This gene,nirS, codes for a mature protein of 63144 Da having high homology withcd ₁-type nitrite reductases from other bacteria. Directly downstream fromnirS, three othernir genes were found in the ordernirECF. The organization of thenir gene cluster inPa. denitrificans is different from the organization ofnir clusters in some Pseudomonads.nirE has high homology with a S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (uro'gen III methylase). This methylase is most likely involved in the hemed ₁ biosynthesis inPa. denitrificans. The third gene,nirC, codes for a small cytochromec of 9.3 kDa having high homology with cytochromec _55X ofPs. stutzeri ZoBell. The 4th gene,nirF, has no homology with other genes in the sequence databases and has no relevant motifs. Inactivation of either of these 4 genes resulted in the loss of nitrite and nitric oxide reductase activities but not of nitrous oxide reductase activity.nirS mutants lack thecd ₁-type nitrite reductase whilenirE, nirC andnirF mutants produce a small amount ofcd ₁-type nitrite reductase, inactive due to the absence of hemed ₁. Upstream from thenirS gene the start of a gene was identified which has limited homology withnosR, a putative regulatory gene involved in nitrous oxide reduction. A potential FNR box was identified between this gene andnirS.Abbreviations SDS sodium dodecyl sulfate - NBT nitroblue tetrazolium - PAGE polyacrylamide gel electrophoresis     

Uncouplers can shuttle between localized energy-coupling sites during photophosphorylation by chromatophores of Rhodopseudomonas capsulata N22.

Two models of the action of uncoupler molecules in inhibiting photophosphorylation in bacterial chromatophores are considered: either uncoupler molecules shuttle rapidly between energy-coupling sites, or uncoupler molecules that are bound to particular sites in the chromatophores for a time that is comparable with the turnover time of the photophosphorylation apparatus may uncouple by a co-operative "substoichiometric' mechanism. It is found that the titre of uncoupler necessary to cause complete uncoupling is lowered if the rate of photophosphorylation is initially decreased by partially restricting electron flow with an appropriate titre of antimycin A. This result indicates that uncoupler molecules shuttle rapidly between energy coupling in which the energized intermediate between electron transport and phosphorylation is delocalized over the entire chromatophore membrane and those in which it is not. If the rate of photophosphorylation is partially restricted with the covalent H+-translocating ATP synthase inhibitor dicyclohexylcarbodi-imide, the titre of uncoupler necessary to effect complete inhibition of photophosphorylation is also decreased relative to that in which the covalent H+-ATP synthase inhibitor is absent. This important result appears to be inconsistent with models of electron-transport phosphorylation in which the "energized state' of the chromatophore membrane that is set up by electron transport and utilized in photophosphorylation is delocalized over the entire chromatophore membrane.     

Separate binding sites for antimycin and mucidin in the respiratory chain of the bacterium Paracoccus denitrificans and their occurrence in other denitrificans bacteria.

By means of the method of fluorimetric titration it has been shown that mucidin does not affect the attachment of antimycin to membranes from anaerobically grown Paracoccus denitrificans. The fluorimetric titration with antimycin can be used in the determination of the amount of the cytochrome bc1 complex in the membrane. In cells inhibited with antimycin, the oxidation of cytochromes c was accompanied by the reduction of cytochrome b; in the presence of mucidin this effect did not take place. The results, which indicated a difference in binding sites, were interpreted in terms of the Q-cycle [Mitchell (1976) J. Theor. Biol. 62, 327-367; Trumpower (1981) Biochim. Biophys. Acta 639, 129-155]. Comparable sensitivity towards antimycin and mucidin was shown by other typical denitrifying bacteria: Pseudomonas stutzeri and Alcaligenes xylosoidans, subspecies denitrificans.     

Surface potential and energy-coupling in bioenergy-conserving membrane systems

In order to understand the localization of dyes and the nature of their responses in membranes and particularly in those involved in energy-conservation processes, the influence of micelles of neutral and ionic surfactants on the pK _a of solubilized fluorophoric (umbelliferone) and chromophoric (bromthymol blue and methyl red) indicator dyes is studied. It is shown that the pK _a of the indicator adsorbed onto micelles shifted towards the acid extreme with cationic micelles, to the alkaline side with anionic micelles while it was not significantly modified by the neutral ones. Maximal displacements were observed with Methyl Red where the difference in pK _a between anionic and cationic micelles was as large as 3 pH units. Phospholipid liquid crystals (Liposomes) of phosphatidylcholine with and without adsorbed long-chain ions introduced in order to confer to it a net surface charge induced displacements of the pK _a of UBF analogous to those detected in the presence of detergent micelles. It was demonstrated that UBF can monitor reversal of charge phenomena such as that obtained by the interaction of phosphatidylcholine + dicetyl phosphate liposomes (anionic colloid) with poly-L-lysine (cationic colloid). The partition of the indicator dyes between micellar and aqueous phases was determined by gel filtration revealing thequasi exclusive presence of the dyes in the micellar phase. Fluorescence polarization measurement of solubilized UBF in either ionic micelles or submitochondrial particles indicate that the dye tumbling rate is as rapid as in pure water suggesting that the dye is mobile in an interfacial environment where it can experience modifications due to changes in surface potential. The use of UBF as a probe of respiration-dependent energy-linked reactions in submitochondrial particles is presented. The available data on the use of indicator dyes in mitochondrial, chloroplast and bacterial chromatophore membranes is reevaluated, on the basis of the evidence of the extreme sensitivity of these probes to surface charge. The implications of these results and considerations are discussed in terms of the importance of the surface potential in the primary event of the energy-coupling process in oxidative and photosynthetic phosphorylation.A preliminary account of this research has been presented elsewhere (IV International Biophysics Congress of the International Union of Pure and Applied Biophysics, Moscow, August 1972).     

Assessment of the addition of Thiobacillus denitrificans and Thiomicrospira denitrificans to chemolithoautotrophic denitrifying bioreactors

The aim of the present study was to assess the impact of adding cultures of Thiobacillus denitrificans and Thiomicrospira denitrificans to two upflow anaerobic sludge bed (UASB) reactors: one inoculated with granular sludge and the other filled only with activated carbon (AC). The performances of the bioreactors and the changes in biomass were compared with a non-bioaugmented control UASB reactor inoculated with granular sludge. The reactors inoculated with granular sludge achieved efficiencies close to 90% in nitrate and thiosulfate removal for loading rates as high as 107 mmol-NO3 -/l per day and 68 mmol-S2O3 2-/l per day. Bioaugmentation with Tb. denitrificans and Tm. denitrificans did not enhance the efficiency compared to that achieved with non-bioaugmented granular sludge. The loading rates and efficiencies were 30-40% lower in the AC reactor. In all the reactors tested, Tb. denitrificans became the predominant species. The results strongly suggest that this bacterium was responsible for denitrification and sulfoxidation within the reactors. We additionally observed that granules partially lost their integrity during operation under chemolithoautotrophic conditions, suggesting limitations for long-term operation if bioaugmentation is applied in practice.