Performance of Paracoccus pantotrophus MA3 in heterotrophic nitrification-anaerobic denitrification using formic acid as a carbon source.

Excess amount of nitrogen in wastewater has caused serious concerns, such as water eutrophication. Paracoccus pantotrophus MA3, a novel isolated strain of heterotrophic nitrification-anaerobic denitrification bacteria, was evaluated for nitrogen removal using formic acid as the sole carbon source. The results showed that the maximum ammonium removal efficiency was observed under the optimum conditions of 26.25 carbon to nitrogen ratio, 3.39% (v/v) inoculation amount, 34.64 °C temperature, and at 180 rpm shaking speed, respectively. In addition, quantitative real-time PCR technique analysis assured that the gene expression level of formate dehydrogenase, formate tetrahydrofolate ligase, 5,10-methylenetetrahydrofolate dehydrogenase, serine hydroxymethyltransferase, respiratory nitrate reductase beta subunit, L-glutamine synthetase, glutamate dehydrogenase, and glutamate synthase were up-regulated compared to the control group, and combined with nitrogen mass balance analysis to conclude that most of the ammonium was removed by assimilation. A small amount of nitrate and nearly no nitrite were accumulated during heterotrophic nitrification. MA3 exhibited significant denitrification potential under anaerobic conditions with a maximum nitrate removal rate of 4.39 mg/L/h, and the only gas produced was N2. Additionally, 11.50 ± 0.06 mg/L/h of NH4+-N removal rate from biogas slurry was achieved.

A novel application of Paracoccus pantotrophus for the decolorization of melanoidins from distillery effluent under static conditions.

Melanoidin is the hazardous byproduct formed during the production of ethanol in distilleries. In the present study, a highly effective melanoidin decolorizing bacterial isolate, SAG1, was isolated from the effluent enriched soil of a distillery. This strain, identified as Paracoccus pantotrophus, was highly efficient to decolorize melanoidins up to 81.2 ± 2.43% in the presence of glucose and NH4NO3. The effects of autoclaved as well as living cells and inoculums size on decolorization activity were investigated. The results indicated that only living cell showed the decolorization activity i.e. 78.6 ± 2.62%, while, no activity has been observed using autoclaved cells. The inoculums size of 8% v/v, showed maximum activity of 62.9 ± 3.00%. The isolate SAG1 was found to be more efficient in decolorizing the melanoidins from distillery effluent as compared to the reference culture Pseudomonas putida.

Kinetic enrichment of 34S during proteobacterial thiosulfate oxidation and the conserved role of SoxB in S-S bond breaking.

During chemolithoautotrophic thiosulfate oxidation, the phylogenetically diverged proteobacteria Paracoccus pantotrophus, Tetrathiobacter kashmirensis, and Thiomicrospira crunogena rendered steady enrichment of (34)S in the end product sulfate, with overall fractionation ranging between -4.6‰ and +5.8‰. The fractionation kinetics of T. crunogena was essentially similar to that of P. pantotrophus, albeit the former had a slightly higher magnitude and rate of (34)S enrichment. In the case of T. kashmirensis, the only significant departure of its fractionation curve from that of P. pantotrophus was observed during the first 36 h of thiosulfate-dependent growth, in the course of which tetrathionate intermediate formation is completed and sulfate production starts. The almost-identical (34)S enrichment rates observed during the peak sulfate-producing stage of all three processes indicated the potential involvement of identical S-S bond-breaking enzymes. Concurrent proteomic analyses detected the hydrolase SoxB (which is known to cleave terminal sulfone groups from SoxYZ-bound cysteine S-thiosulfonates, as well as cysteine S-sulfonates, in P. pantotrophus) in the actively sulfate-producing cells of all three species. The inducible expression of soxB during tetrathionate oxidation, as well as the second leg of thiosulfate oxidation, by T. kashmirensis is significant because the current Sox pathway does not accommodate tetrathionate as one of its substrates. Notably, however, no other Sox protein except SoxB could be detected upon matrix-assisted laser desorption ionization mass spectrometry analysis of all such T. kashmirensis proteins as appeared to be thiosulfate inducible in 2-dimensional gel electrophoresis. Instead, several other redox proteins were found to be at least 2-fold overexpressed during thiosulfate- or tetrathionate-dependent growth, thereby indicating that there is more to tetrathionate oxidation than SoxB alone.

Isolation, identification and removal of filamentous organism from SND based SBR degrading nitrophenols.

Four identical lab scale sequencing batch reactors R, R1, R2, and R3, were used to assess nitrophenol biodegradation using a single sludge biomass containing Thiosphaera pantotropha. Nitrophenols [4-Nitrophenol (4-NP), 2,4-dinitrophenol (2,4-DNP) and 2,4,6-trinitrophenol (2,4,6-TNP)] were biotransformed by heterotrophic nitrification and aerobic denitrification (SND). Reactor R was used as background control, whereas R1, R2, and R3 were fed with 4-NP, 2,4-DNP, and 2,4,6-TNP, respectively. The concentration of each nitrophenol was gradually increased from 2.5 to 200 mg/l along with increase in COD, during acclimation studies. The final COD maintained was 4,500 mg/l with each nitrophenolic loading of 200 mg/l. During late phase of acclimation and HRT study, a filamentous organism started appearing in 2,4-DNP and 2,4,6-TNP bioreactors. Filaments were never found in 4-NP and background control reactor. Biochemistry and physiology behind filamentous organism development, was studied to obtain permanent solution for its removal. The effect of different input parameters such as COD loading, DO levels, SVI etc. were analyzed. The morphology and development of filamentous organism were examined extensively using microscopic techniques involving ESEM, oil immersion, phase contrast, and dark field microscopy. The organism was grown and isolated on selective agar plates and was identified as member of Streptomyses species.

Electrochemical titrations and reaction time courses monitored in situ by magnetic circular dichroism spectroscopy.

Magnetic circular dichroism (MCD) spectra, at ultraviolet-visible or near-infrared wavelengths (185-2000 nm), contain the same transitions observed in conventional absorbance spectroscopy, but their bisignate nature and more stringent selection rules provide greatly enhanced resolution. Thus, they have proved to be invaluable in the study of many transition metal-containing proteins. For mainly technical reasons, MCD has been limited almost exclusively to the measurement of static samples. But the ability to employ the resolving power of MCD to follow changes at transition metal sites would be a potentially significant advance. We describe here the development of a cuvette holder that allows reagent injection and sample mixing within the 50-mm-diameter ambient temperature bore of an energized superconducting solenoid. This has allowed us, for the first time, to monitor time-resolved MCD resulting from in situ chemical manipulation of a metalloprotein sample. Furthermore, we report the parallel development of an electrochemical cell using a three-electrode configuration with physically separated working and counter electrodes, allowing true potentiometric titration to be performed within the bore of the MCD solenoid.

Sustainable biological system for the removal of high strength ammoniacal nitrogen and organic pollutants in poultry waste processing industrial effluent.

The effluent generated from poultry waste processing industries contains several organic compounds such as collagen, gelatin, bovine serum albumin, carbohydrates, essential fatty acids, and so forth. This enabled the establishment of poultry waste processing industries to produce value-added products such as animal feed and organic fertilizers. During poultry waste processing, huge amounts of ammoniacal nitrogen and organic pollutants such as proteins, various carbohydrates, and fatty materials are discharged into the effluent stream which contributes to several environmental issues. Because of the shortcomings of the current conventional treatment, the present study is about with the development of a sequential bioreactor system for the effective treatment of poultry waste processing industrial effluent. Facultative anaerobe Paracoccus pantotrophus FMR19 along with the indigenous isolate Bacillus albus MN527241 obtained from clarifying sludge was used as mixed consortia for the treatment of poultry waste processing industrial effluent. The mixed microbial consortia resulted in the maximum activity of enzymes such as protease (247 U/mL) and lipase (28.266 U/mL) thereby achieving 90% of ammoniacal nitrogen reduction and 98% of COD removal within five days. Further, the confirmatory analysis of poultry effluent treatment was carried out using gas chromatography-mass spectroscopy (GC-MS), High-Performance Liquid Chromatography (HPLC), Fourier Transform Infrared Spectroscopy (FT-IR), and SDS-PAGE. Hence, the sequential bioreactor-based treatment approach has proved to be highly effective in removal of organic pollutants in the poultry waste processing industrial effluent.Implications: The poultry waste processing industrial (PWPI) effluent contains huge ammoniacal nitrogen and COD and affects the environment. Aerobic moving bed biofilm reactor and up-flow anaerobic sludge blanket reactor are in current practice and shows considerable reduction in the ammoniacal nitrogen and COD in long retention time. Therefore, there is a need of sustainable treatment process that could effectively remove the organic pollutants from the effluent in short duration. Our study focused on the application of sequential bioreactor approach for the treatment of PWPI using aerobic followed by anaerobic treatment process and observed efficient organic pollutants removal in short duration.

Voltammetric characterization of the aerobic energy-dissipating nitrate reductase of Paracoccus pantotrophus: exploring the activity of a redox-balancing enzyme as a function of electrochemical potential.

Paracoccus pantotrophus expresses two nitrate reductases associated with respiratory electron transport, termed NapABC and NarGHI. Both enzymes derive electrons from ubiquinol to reduce nitrate to nitrite. However, while NarGHI harnesses the energy of the quinol/nitrate couple to generate a transmembrane proton gradient, NapABC dissipates the energy associated with these reducing equivalents. In the present paper we explore the nitrate reductase activity of purified NapAB as a function of electrochemical potential, substrate concentration and pH using protein film voltammetry. Nitrate reduction by NapAB is shown to occur at potentials below approx. 0.1 V at pH 7. These are lower potentials than required for NarGH nitrate reduction. The potentials required for Nap nitrate reduction are also likely to require ubiquinol/ubiquinone ratios higher than are needed to activate the H(+)-pumping oxidases expressed during aerobic growth where Nap levels are maximal. Thus the operational potentials of P. pantotrophus NapAB are consistent with a productive role in redox balancing. A Michaelis constant (K(M)) of approx. 45 muM was determined for NapAB nitrate reduction at pH 7. This is in line with studies on intact cells where nitrate reduction by Nap was described by a Monod constant (K(S)) of less than 15 muM. The voltammetric studies also disclosed maximal NapAB activity in a narrow window of potential. This behaviour is resistant to change of pH, nitrate concentration and inhibitor concentration and its possible mechanistic origins are discussed.

Comparison of sulphide and nitrate removal from synthetic wastewater by pure and mixed cultures of nitrate-reducing, sulphide-oxidizing bacteria.

In this study, the activities of hydrogen sulphide (H2S) oxidation and nitrate (N-NO3-) reduction by three pure and mixed strains of nitrate-reducing, sulphide oxidizing bacteria (NR-SOB) were determined. Batch experiments were performed at 35 °C and pH 7.0-8.0 with initial H2S concentrations of 650-900 ppmv and N-NO3- concentrations of ∼120 mg/L. The strains MAL 1HM19, TPN 1HM1 and TPN 3HM1 were capable of removing 100% gas-phase H2S. The co-cultures showed better performance for H2S and N-NO3- removal. The mixed NR-SOB strains showed a higher H2S oxidation rate (143 ±â€¯18 ppmv/h), while the highest N-NO3- removal rate (5.5 ±â€¯0 and 5.1 ±â€¯0.6 N-NO3- mg/L·h) was obtained by a mixture of two NR-SOB strains. The 16S rDNA sequence analysis revealed that all strains belonged to the sub-class Alphaproteobacteria and are closely related to Paracoccus sp. (>99%).

Recombinant Sox Enzymes from Paracoccus pantotrophus Degrade Hydrogen Sulfide, a Major Component of Oral Malodor.

Hydrogen sulfide (H2S) is emitted from industrial activities, and several chemotrophs possessing Sox enzymes are used for its removal. Oral malodor is a common issue in the dental field and major malodorous components are volatile sulfur compounds (VSCs), including H2S and methyl mercaptan. Paracoccus pantotrophus is an aerobic, neutrophilic facultatively autotrophic bacterium that possesses sulfur-oxidizing (Sox) enzymes in order to use sulfur compounds as an energy source. In the present study, we cloned the Sox enzymes of P. pantotrophus GB17 and evaluated their VSC-degrading activities for the prevention of oral malodor. Six genes, soxX, soxY, soxZ, soxA, soxB, and soxCD, were amplified from P. pantotrophus GB17. Each fragment was cloned into a vector for the expression of 6×His-tagged fusion proteins in Escherichia coli. Recombinant Sox (rSox) proteins were purified from whole-cell extracts of E. coli using nickel affinity chromatography. The enzyme mixture was investigated for the degradation of VSCs using gas chromatography. Each of the rSox enzymes was purified to apparent homogeneity, as confirmed by SDS-PAGE. The rSox enzyme mixture degraded H2S in dose- and time-dependent manners. All rSox enzymes were necessary for degrading H2S. The H2S-degrading activities of rSox enzymes were stable at 25-80°C, and the optimum pH was 7.0. The amount of H2S produced by periodontopathic bacteria or oral bacteria collected from human subjects decreased after an incubation with rSox enzymes. These results suggest that the combination of rSox enzymes from P. pantotrophus GB17 is useful for the prevention of oral malodor.

The flavoprotein SoxF functions in chemotrophic thiosulfate oxidation of Paracoccus pantotrophus in vivo and in vitro.

Paracoccus pantotrophus strain GBsoxFDelta carries a deletion in the soxF gene that inactivates flavoprotein SoxF-sulfide dehydrogenase. This strain grew with thiosulfate slower than the wild type. GBsoxFDelta cells oxidized thiosulfate at a rate of 40% and hydrogen sulfide at a rate of 45% of the wild type. Complementation of GBsoxFDelta with plasmid pRIsoxF carrying the soxF gene increased these rates to 83% and 70%, respectively. However, GBsoxFDelta and GBsoxFDelta (pRIsoxF) oxidized thiosulfate and hydrogen sulfide to sulfate as evident from the yield of electrons. The thiosulfate oxidation rate of cell-free extracts of strain GBsoxFDelta was increased when supplemented with SoxF isolated from the wild type. However, SoxF did not affect the thiosulfate-oxidizing activity of the Sox enzyme system as reconstituted from the 'as-isolated' four Sox proteins. These data demonstrated that SoxF enhanced chemotrophic thiosulfate oxidation in vivo and acted on some component or condition present in whole cells and cell-free extracts but not present in the reconstituted system.

Evaluation of nitrate removal by continuous culturing of an aerobic denitrifying bacterium, Paracoccus pantotrophus.

Nitrate removal under aerobic conditions was investigated using pure cultures of Paracoccus pantotrophus, which is a well-known aerobic-denitrifying (AD) bacterium. When a high concentration of cultures with a high carbon/nitrogen (C/N) ratio was preserved at the beginning of batch experiments, subsequently added nitrate was completely removed. When continuous culturing was perpetuated, a high nitrate removal rate (66.5%) was observed on day 4 post-culture, although gradual decreases in AD ability with time were observed. The attenuation in AD ability was probably caused by carbon limitation, because when carbon concentration of inflow water was doubled, nitrate removal efficiency improved from 18.1% to 59.6%. Bacterial community analysis using the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method showed that P. pantotrophus disappeared in the suspended medium on day 8 post-culture, whereas other bacterial communities dominated by Acidovorax sp. appeared. Interestingly, this replaced bacterial community also showed AD ability. As P. pantotrophus was detected as attached colonies around the membrane and bottom of the reactor, this bacterium can therefore be introduced in a fixed form for treatment of wastewater containing nitrate with a high C/N ratio.

Start-up of sequencing batch reactor with Thiosphaera pantotropha for treatment of high-strength nitrogenous wastewater and sludge characterization.

Biological treatment of high-strength nitrogenous wastewater is challenging due to low growth rate of autotrophic nitrifiers. This study reports bioaugmentation of Thiosphaera pantotropha capable of simultaneously performing heterotrophic nitrification and aerobic denitrification (SND) in sequencing batch reactors (SBRs). SBRs fed with 1:1 organic-nitrogen (N) and NH4+-N were started up with activated sludge and T. pantotropha by gradual increase in N concentration. Sludge bulking problems initially observed could be overcome through improved aeration and mixing and change in carbon source. N removal decreased with increase in initial nitrogen concentration, and only 50-60 % removal could be achieved at the highest N concentration of 1000 mg L-1 at 12-h cycle time. SND accounted for 28 % nitrogen loss. Reducing the settling time to 5-10 min and addition of divalent metal ions gradually improved the settling characteristics of sludge. Sludge aggregates of 0.05-0.2 mm diameter, much smaller than typical aerobic granules, were formed and progressive increase in settling velocity, specific gravity, Ca2+, Mg2+, protein, and polysaccharides was observed over time. Granulation facilitated total nitrogen (TN) removal at a constant rate over the entire 12-h cycle and thus increased TN removal up to 70 %. Concentrations of NO2--N and NO3--N were consistently low indicating effective denitrification. Nitrogen removal was possibly limited by urea hydrolysis/nitrification. Presence of T. pantotropha in the SBRs was confirmed through biochemical tests and 16S rDNA analysis.

Sulphate production by Paracoccus pantotrophus ATCC 35512 from different sulphur substrates: sodium thiosulphate, sulphite and sulphide.

One of the problems in waste water treatment plants (WWTPs) is the increase in emissions of hydrogen sulphide (H2S), which can cause damage to the health of human populations and ecosystems. To control emissions of this gas, sulphur-oxidizing bacteria can be used to convert H2S to sulphate. In this work, sulphate detection was performed by spectrophotometry, ion chromatography and atomic absorption spectrometry, using Paracoccus pantotrophus ATCC 35512 as a reference strain growing in an inorganic broth supplemented with sodium thiosulphate (Na2S2O3·5H2O), sodium sulphide (Na2S) or sodium sulphite (Na2SO3), separately. The strain was metabolically competent in sulphate production. However, it was only possible to observe significant differences in sulphate production compared to abiotic control when the inorganic medium was supplemented with sodium thiosulphate. The three methods for sulphate detection showed similar patterns, although the chromatographic method was the most sensitive for this study. This strain can be used as a reference for sulphate production in studies with sulphur-oxidizing bacteria originating from environmental samples of WWTPs.

Paracoccus pantotrophus NapC can reductively activate cytochrome cd1 nitrite reductase.

The oxidized "as isolated" form of Paracoccus pantotrophus cytochrome cd1 nitrite reductase has a bis-histidinyl coordinated c heme and a histidine/tyrosine coordinated d1 heme. This form of the enzyme has previously been shown to be kinetically incompetent. Upon reduction, the coordination of both hemes changes and the enzyme is kinetically activated. Here, we show that P. pantotrophus NapC, a tetraheme c-type cytochrome belonging to a large family of such proteins, is capable of reducing, and hence activating, "as isolated" cytochrome cd1. NapC is the first protein from P. pantotrophus identified as being capable of this activation step and, given the periplasmic co-location and co-expression of the two proteins, is a strong candidate to be a physiological activation partner.

Biotinylated polypyrrole films: an easy electrochemical approach for the reagentless immobilization of bacteria on electrode surfaces.

Biotinylated bacteria were immobilized onto biotin/avidin modified electrode surfaces. Firstly, an electrospotting deposition method, followed by fluorescence microscopy, showed that bacteria were specifically grafted onto a gold surface. Fluorescence intensity versus the quantity of bacteria deposited on the surface was correlated, allowing determination of the microbial saturation point. Secondly, biotinylated bacteria were immobilized onto a glassy carbon macro-electrode in order to assess immobilized bacterial denitrification activity. During a 7-day trial, the modified electrode completely denitrified 5 mM nitrate, with a rate of 1.66 mM/day over the first 3 days. When the same electrode was placed in fresh nitrate solution, the denitrification rate dropped to 0.80 mM/day. Crucially, the immobilized bacteria did not become detached from the electrode during the study.

Comparison of aerobic denitrifying activity among three cultural species with various carbon sources.

Abilities of three aerobic denitrifiers such as Alcaligenes faecalis, Microvirgula aerodenitrificans and Paracoccus pantotrophus were compared from the viewpoints of nitrate removal efficiency and organic matter utilization. First, the effect of carbon source was investigated. Although nitrate reduction was observed in all strains under aerobic conditions, a change of carbon source considerably affected the denitrification ability. In the case of P. pantotrophus, nitrate and nitrite were completely removed in three days under sodium acetate or leucine as a carbon source. In the case of A. faecalis, sufficient nitrate removal was observed only when sodium acetate or ethanol was added. P. pantotrophus and A. faecalis showed a higher ability of nitrate removal than that of M. aerodenitrificans. Therefore, P. pantotrophus was selected in order to investigate the effects of concentration and repetitive addition of carbon. Sodium acetate was used as a sole carbon source. Nitrate was not reduced when the carbon concentration was below 500 mgC/L. However, when carbon source was added repeatedly, nitrate was reduced under 100 mgC/L after the optical density of the bacterium reached above 1.0. This result indicated that a high enough level of bacterial density was necessary to express aerobic denitrification activity.

Nitrophenol removal by simultaneous nitrification denitrification (SND) using T. pantotropha in sequencing batch reactors (SBR).

Nitrophenol removal was assessed using four identical lab scale sequencing batch reactors R (background control), R1 (4-nitrophenol i.e. 4-NP), R2 (2,4-dinitrophenol i.e. 2,4-DNP), and R3 (2,4,6-trinitrophenol i.e. 2,4,6-TNP). In the present study, the SND based SBR system was used to carry out total nitrogen removal at reduced aeration (DO=2mg/L) using a specifically designed single sludge biomass containing Thiosphaera pantotropha. The concentration of each of the nitrophenols was gradually increased from 2.5 to 200mg/L during acclimation. The nitrophenols were used as the sole source of nitrogen during study. A synthetic feed was designed to direct SND in the bioreactors. It was observed that overall removal for 4-NP was 98% and for 2,4-DNP and 2,4,6 TNP, removals varied between 83% and 84%. The COD removal for 4-NP was 99% and for 2,4-DNP and 2,4,6-TNP was 97-98% during acclimation. Total nitrogen and nitrophenol removals were achieved via SND.

Effect of shock and mixed loading on the performance of SND based sequencing batch reactors (SBR) degrading nitrophenols.

The effect of nitrophenolic shock loads on the performance of three lab scale SBRs was studied using a synthetic feed. Nitrophenols were biotransformed by Simultaneous heterotrophic Nitrification and aerobic Denitrification (SND) using a specially designed single sludge biomass containing Thiosphaera pantotropha. Reactors R1, R2 and R3 were fed with 200mg/L concentration of 4-nitrophenol (4-NP), 2,4-dinitrophenol (2,4-DNP), and 2,4,6-trinitrophenol (2,4,6-TNP) whereas reactor R was used as a background control. Three nitrophenolic shock loadings of 400, 600 and 800 mg/Ld were administrated by increasing the influent nitrophenolic concentration while keeping the hydraulic retention time as 48 h. The shocks were given continuously for a period of 4 days before switching back to normal nitrophenolic loading (200mg/Ld). The reactors were allowed to recover to normal performance level before administrating the next nitrophenolic shock load. The study showed that a nitrophenolic shock load, as high as 600 mg/Ld was completely degraded by the 4-NP & 2,4-DNP bioreactors while almost half degraded by the 2,4,6-TNP bioreactor without affecting the reactor's performance irreversibly. After resuming the normal nitrophenolic loading, it took almost 8-10 days for the reactors to recover from the shock effect. The study was further extended to evaluate the maximum possible mixed nitrophenolic loading (4-NP:2,4-DNP:2,4,6-TNP 1:1:1) to which a reactor (R3) containing 2,4,6-TNP acclimated single sludge biomass can be exposed without hampering the reactor performance irreversibly. The reactor was able to achieve pseudo-steady-state at a mixed nitrophenolic loading of 300 mg/Ld with more than 90% removal of all the three nitrophenols, but could remove half of the mixed nitrophenolic loading of 600 mg/Ld.

NirF is a periplasmic protein that binds d1 heme as part of its essential role in d1 heme biogenesis.

The cytochrome cd1 nitrite reductase from Paracoccus pantotrophus catalyses the one electron reduction of nitrite to nitric oxide using two heme cofactors. The site of nitrite reduction is the d1 heme, which is synthesized under anaerobic conditions by using nirECFD-LGHJN gene products. In vivo studies with an unmarked deletion strain, ΔnirF, showed that this gene is essential for cd1 assembly and consequently for denitrification, which was restored when the ΔnirF strain was complemented with wild-type, plasmid-borne, nirF. Removal of a signal sequence and deletion of a conserved N-terminal Gly-rich motif from the NirF coded on a plasmid resulted in loss of in vivo NirF activity. We demonstrate here that the product of the nirF gene is a periplasmic protein and, hence, must be involved in a late stage of the cofactor biosynthesis. In vitro studies with purified NirF established that it could bind d1 heme. It is concluded that His41 of NirF, which aligns with His200 of the d1 heme domain of cd1, is essential both for this binding and for the production of d1 heme; replacement of His41 by Ala, Cys, Lys and Met all gave nonfunctional proteins. Potential functions of NirF are discussed.

Interaction between Sox proteins of two physiologically distinct bacteria and a new protein involved in thiosulfate oxidation.

Organisms using the thiosulfate-oxidizing Sox enzyme system fall into two groups: group 1 forms sulfur globules as intermediates (Allochromatium vinosum), group 2 does not (Paracoccus pantotrophus). While several components of their Sox systems are quite similar, i.e. the proteins SoxXA, SoxYZ and SoxB, they differ by Sox(CD)(2) which is absent in sulfur globule-forming organisms. Still, the respective enzymes are partly exchangeable in vitro: P. pantotrophus Sox enzymes work productively with A. vinosum SoxYZ whereas A. vinosum SoxB does not cooperate with the P. pantotrophus enzymes. Furthermore, A. vinosum SoxL, a rhodanese-like protein encoded immediately downstream of soxXAK, appears to play an important role in recycling SoxYZ as it increases thiosulfate depletion velocity in vitro without increasing the electron yield.