Thiosphaera Pantotropha Research Articles

Mutational analysis of mau genes involved in methylamine metabolism in Paracoccus denitrificans.

A chromosomal fragment containing DNA downstream from mauC was isolated from Paracoccus denitrificans. Sequence analysis of this fragment revealed the presence of four open reading frames, all transcribed in the same direction. The products of the putative genes were found to be highly similar to MauJ, MauG, MauM and MauN of Methylobacterium extorquens AM1. Using these four mau genes, 11 mau genes have been cloned from P. denitrificans to date. The gene order is mauRFBEDACJGMN, which is similar to that in M. extorquens AM1. mauL, present in M. extorquens AM1, seems to be absent in P. denitrificans. MauJ is predicted to be a cytoplasmic protein, and MauG a periplasmic protein. The latter protein contains two putative heme-binding sites, and has some sequence resemblance to the cytochrome c peroxidase from Pseudomonas aeruginosa. MauM is also predicted to be located in the periplasm, but MauN appears to be membrane associated. Both resemble ferredoxin-like proteins and contain four and two motifs, respectively, characteristic for [4Fe-4S] clusters. Inactivation of mauA, mauJ, mauG, mauM and mauN was carried out by introduction of unmarked mutations in the chromosomal copies of these genes. mauA and mauG mutant strains were unable to grow on methylamine. The mauJ mutant strain had an impaired growth rate and showed a lower dye-linked methylamine dehydrogenase (MADH) activity than the parent strain. Mutations in mauM and mauN had no effect on methylamine metabolism. The mauA mutant strain specifically lacked the beta subunit of MADH, but the alpha subunit and amicyanin, the natural electron acceptors of MADH, were still produced. The mauG mutant strain synthesized the alpha and beta subunits of MADH as well as amicyanin. However, no dye-linked MADH activity was found in this mutant strain. In addition, as the wild-type enzyme displays a characteristic fluorescence emission spectrum upon addition of methylamine, this property was lost in the mauG mutant strain. These results clearly show that MauG is essential for the maturation of the beta subunit of MADH, presumably via a step in the biosynthesis of tryptophan tryptophylquinone, the cofactor of MADH. The mau gene cluster mauRFBEDACJGMN was cloned on the broad-host vector pEG400. Transfer of this construct to mutant strains which were unable to grow on methylamine fully restored their ability to grow on this compound. A similar result was achieved for the closely related bacterium Thiosphaera pantotropha, which is unable to utilize methylamine as the sole sources of carbon and energy.

Mutational Analysis of Mau Genes Involved in Methylamine Metabolism in Paracoccus Denitrificans

A chromosomal fragment containing DNA downstream from mauC was isolated from Paracoccus denitrificans. Sequence analysis of this fragment revealed the presence of four open reading frames, all transcribed in the same direction. The products of the putative genes were found to be highly similar to MauJ, MauG, MauM and MauN of Methylobacterium extorquens AM1. Using these four mau genes, 11 mau genes have been cloned from P. denitrificans to date. The gene order is mauRFBEDACJGMN, which is similar to that in M. extorquens AM1. mauL, present in M. extorquens AM1, seems to be absent in P. denitrificans.MauJ is predicted to be a cytoplasmic protein, and MauG a periplasmic protein. The latter protein contains two putative heme‐binding sites, and has some sequence resemblance to the cytochrome c peroxidase from Pseudomonas aeruginosa. MauM is also predicted to be located in the periplasm, but MauN appears to be membrane associated. Both resemble ferredoxin‐like proteins and contain four and two motifs, respectively, characteristic for [4Fe‐4S] clusters.Inactivation of mauA, mauJ, mauG, mauM and mauN was carried out by introduction of unmarked mutations in the chromosomal copies of these genes. mauA and mauG mutant strains were unable to grow on methylamine. The mauJ mutant strain had an impaired growth rate and showed a lower dye‐linked methylamine dehydrogenase (MADH) activity than the parent strain. Mutations in mauM and mauN had no effect on methylamine metabolism. The mauA mutant strain specifically lacked the β subunit of MADH, but the α subunit and amicyanin, the natural electron acceptors of MADH, were still produced. The mauG mutant strain synthesized the α and β subunits of MADH as well as amicyanin. However, no dye‐linked MADH activity was found in this mutant strain. In addition, as the wild‐type enzyme displays a characteristic fluorescence emission spectrum upon addition of methylamine, this property was lost in the mauG mutant strain. These results clearly show that MauG is essential for the maturation of the β subunit of MADH, presumably via a step in the biosynthesis of tryptophan tryptophylquinone, the cofactor of MADH.The mau gene cluster mauRFBEDACJGMN was cloned on the broad‐host vector pEG400. Transfer of this construct to mutant strains which were unable to grow on methylamine fully restored their ability to grow on this compound. A similar result was achieved for the closely related bacterium Thiosphaera pantotropha, which is unable to utilize methylamine as the sole sources of carbon and energy.

Methanol oxidation in a spontaneous mutant of Thiosphaera pantotropha with a methanol-positive phenotype is catalysed by a dye-linked ethanol dehydrogenase

A spontaneous Thiosphaera pantotropha mutant (Tp9002) that is able to grow on methanol has been isolated. With hybridization experiments it has been demonstrated that mxaF, the gene encoding the large subunit of methanol dehydrogenase, is absent from T. pantotropha. In Tp9002, a dye-linked enzyme activity was found with a substrate specificity similar to that of the dye-linked ethanol dehydrogenase from Pseudomonas aeruginosa. The N-terminus of a 26-kDa cytochrome c, exclusively synthesized in Tp9002, is homologous to the N-terminus of the electron acceptor of ethanol dehydrogenase. These results suggest that in Tp9002 a dye-linked ethanol dehydrogenase is responsible for methanol oxidation, using a 26-kDa cytochrome c as electron acceptor.

Methanol oxidation in a spontaneous mutant ofThiosphaera pantotrophawith a methanol-positive phenotype is catalysed by a dye-linked ethanol dehydrogenase

A spontaneous Thiosphaera pantotropha mutant (Tp9002) that is able to grow on methanol has been isolated. With hybridization experiments it has been demonstrated that mxaF, the gene encoding the large subunit of methanol dehydrogenase, is absent from T. pantotropha. In Tp9002, a dye-linked enzyme activity was found with a substrate specificity similar to that of the dye-linked ethanol dehydrogenase from Pseudomonas aeruginosa. The N-terminus of a 26-kDa cytochrome c, exclusively synthesized in Tp9002, is homologous to the N-terminus of the electron acceptor of ethanol dehydrogenase. These results suggest that in Tp9002 a dye-linked ethanol dehydrogenase is responsible for methanol oxidation, using a 26-kDa cytochrome c as electron acceptor.

Aerobic Denitrification Studies on Activated Sludge Mixed with Thiosphaera Pantotropha

Nitrate nitrogen levels upto 425 mg l−1 (D.O = 2.5 mg l−1) were treated under aerobic conditions by activated sludge mixed with Thiosphaera pantotropha. Efficiency of nitrate nitrogen removal ranged from 75–85% for different reactors operated at HRT between 0.5 to 1 day and SRT of 2–8 days.

Characterization of the paramagnetic iron-containing redox centres of Thiosphaera pantotropha periplasmic nitrate reductase

Electron paramagnetic resonance spectroscopy signals attributable to low-spin haem c in the oxidised protein and [4Fe4S] 1+ in the dithionite-reduced protein were identified, at low temperature, in Thiosphaera pantotropha periplasmic nitrate reductase. Spin integration of these signals as well as elemental analysis suggest a stoichiometry of 1.3–1.6 c-haem and 1 [4Fe4S] cluster per enzyme molecule. The E m (at pH 7.4) of the [4F4S] 2+,1+ couple, −160 mV, means that it is unlikely to be physiologically reducible. Peptide sequences from the 90 kDa subunit indicate that the enzyme is a member of the family of molybdopterin guanine dinucleotide-binding polypeptides, the majority of which possess a putative [4Fe4S] cluster binding sequence and thus may also bind a (low potential) iron—sulphur cluster.

Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha.

The periplasmic nitrate reductase of Thiosphaera pantotropha has been purified from a mutant strain (M-6) that overproduces the enzyme activity under anaerobic growth conditions. The enzyme is a complex of a 93-kDa polypeptide and a 16-kDa nitrate-oxidizable cytochrome c552. The complex contains molybdenum; a fluorescent compound with spectral features of a pterin derivative can be extracted. In contrast to the dissimilatory membrane-bound nitrate reductases, the periplasmic nitrate reductase shows high specificity for nitrate as a substrate and is insensitive to inhibition by azide. The 93-kDa subunit exhibits immunological cross-reactivity with the catalytic subunit of Rhodobacter capsulatus N22DNAR+ periplasmic nitrate reductase. Mass spectrometric comparisons of holo-cytochrome c552 and apo-cytochrome c552 demonstrated that the polypeptide bound two haem groups. Mediated redox potentiometry of the cytochrome indicated that the haem groups have reduction potentials (pH = 7.0) of approximately -15 mV and + 80 mV. The functional significance of these potentials is discussed in relation to the proposed physiological role of the enzyme as a redox valve.

Simultaneous nitrification‐denitrification in a rotating biological contactor

Abstract Simultaneous nitrification\aerobic denitrification studies of a high strength synthetic nitrogenous fertilizer wastewater in a single stage were carried out using a rotating biological contactor (RBC). Mixed culture biofilm consisting of Nitrifiers and Thiosphaera pantotropha (a heterotrophic nitrifier and aerobic denitrifier) was acclimated to 1386 mg l‐1 of TKN & 400 mg l‐1 of nitrate nitrogen. The effect of different nitrogen loadings and hydraulic retention times (HRTs) on percentage removal of TKN and nitrate nitrogen simultaneously, was investigated. A maximum TKN removal of 95.6 percent could be achieved at a nitrogen loading of 9.36 gN m‐2 d‐1 and HRT of 2 days. The results showed that the process is zero order with respect to nitrogen removal. The HRT studied were in the range of 0.5–3 days. The corresponding percentage TKN removal ranged from 43.9–93.7. Nitrate removal of 97–98% could also be achieved simultaneously. Nitrite accumulated significantly throughout these studies. Nitrogen l...

Properties of a Paracoccus denitrificans mutant deleted in cytochrome c550 indicate that a copper protein can substitute for this cytochrome in electron transport to nitrite, nitric oxide and nitrous oxide

Summary: Disruption of the gene for the periplasmic cytochrome c550 in Paracoccus denitrificans did not result in substantial alteration of rates of electron flow from physiological substrates to the reductases for nitrite, nitric oxide and nitrous oxide, thus confirming and extending earlier findings. In wild-type cells the chelating agent diethyldithiocarbamate (DDC) caused a partial inhibition of these electron transport processes. When the same concentrations of this inhibitor were added to a mutant of P. denitrificans in which the gene for cytochrome c550 was specifically disrupted an almost complete inhibition of these reactions was observed. It is known from previous work with the closely related organism Thiosphaera pantotropha that DDC effectively and rapidly removes copper from the periplasmic pseudoazurin that is also found in P. denitrificans. Therefore, it is concluded that in the absence of cytochrome c550, a copper protein, probably pseudoazurin, acts as the electron carrier between the cytochrome bc 1 complex and the reductases for nitrite, nitric oxide and nitrous oxide. Cells with the gene for cytochrome c550 deleted continued to give a positive Nadi test.

Purification of hydroxylamine oxidase from Thiosphaera pantotropha: Identification of electron acceptors that couple heterotrophic nitrification to aerobic denitrification

Thiosphaera pantotropha, a Gram-negative heterotrophic nitrifying bacterium, expresses a soluble 20 kDa monomeric periplasmic hydroxylamine oxidase that differs markedly from the hydroxylamine oxidase found in autotrophic bacteria. This enzyme can use the periplasmic redox proteins, cytochrome C 551 and pseudoazurin as electron acceptors, both of which can also donate electrons to denitrification enzymes. A model of electron transfer is proposed, that suggests a coupling of nitrification to denitrification and provides a mechanism by which nitrification can play a role in dissipating reductant.

Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity.

Chlorate-resistant mutants of the denitrifying bacterium Thiosphaera pantotropha were generated by transposon Tn5 mutagenesis. One class was deficient in membrane-bound nitrate reductase activity but retained a periplasmic nitrate reductase activity. Using transposon marker rescue it was shown that in one such mutant, M-6, the transposon was inserted in the membrane-bound nitrate reductase beta subunit structural gene (termed narH in order to be consistent with the nomenclature of the Escherichia coli major nitrate reductase operon). The translated sequence (total of 106 amino acids) from around the point of transposon insertion showed approximately 90% amino acid identity with the beta subunits of the E. coli nitrate reductases. Under anaerobic growth conditions M-6 overproduced the periplasmic nitrate reductase activity allowing anaerobic growth with nitrate as electron acceptor. A regulatory link was inferred between the presence of the membrane-bound nitrate reductase and expression of the periplasmic nitrate reductase. This is the first demonstration of full denitrification in an organism possessing only a periplasmic nitrate reductase.

Spontaneous mutation of Thiosphaera pantotropha enabling growth on methanol correlates with synthesis of a 26 kDa c-type cytochrome

Thiosphaera pantotropha grows on methanol as carbon and energy source following spontaneous mutation to a Mox+ phenotype after incubation in media containing methanol. Acquisition of ability to grow on methanol correlates with the appearance of a c-type cytochrome, molecular mass 26 kDa, which is suggested for the product of the moxG gene, which is the electron acceptor from methanol in related bacteria, but which is absent from T. pantotropha. Mutation leading to growth on methylamine as carbon and energy source was not observed despite the presence of in vitro methylamine dehydrogenase activity in cells grown on choline. Lack of growth on methylamine may correlate with the absence of amicyanin, which is the obligatory electron acceptor from methylamine dehydrogenase in other organisms.

Spontaneous mutation ofThiosphaera pantotrophaenabling growth on methanol correlates with synthesis of a 26 kDac-type cytochrome

Thiosphaera pantotropha grows on methanol as carbon and energy source following spontaneous mutation to a Mox+ phenotype after incubation in media containing methanol. Acquisition of ability to grow on methanol correlates with the appearance of a c-type cytochrome, molecular mass 26 kDa, which is suggested to substitute for the product of the moxG gene, which is the electron acceptor from methanol in related bacteria, but which is absent from T. pantotropha. Mutation leading to growth on methylamine as carbon and energy source was not observed despite the presence of in vitro methylamine dehydrogenase activity in cells grown on choline. Lack of growth on methylamine may correlate with the absence of amicyanin, which is the obligatory electron acceptor from methylamine dehydrogenase in other organisms.

Sequence and expression of the gene encoding the respiratory nitrous-oxide reductase from Paracoccus denitrificans. New and conserved structural and regulatory motifs.

The structural gene for the respiratory nitrous-oxide reductase from Paracoccus denitrificans has been cloned using a probe derived from the structural gene, nosZ, for this enzyme from Pseudomonas stutzeri. The cloned gene could be expressed surprisingly well (presumably yielding an apo-protein) using an expression vector in Escherichia coli. Sequencing the nosZ gene from P. denitrificans has shown that the periplasmic nitrous-oxide reductase of this organism is highly similar in sequence to previously derived primary sequences for the enzyme from three other organisms. As with the other reductases, an unusually long signal sequence is deduced and a common motif of GXXRRXXLG near the beginning of this sequence is present. The results of N-terminal sequencing of the mature nitrous-oxide reductase from the closely related organism Thiosphaera pantotropha indicate that processing of the P. denitrificans precursor occurs between amino acids at positions 57 and 58. The predicted signal peptide is therefore of the same length and of similar overall structure to that previously described for the P. denitrificans methylamine dehydrogenase small subunit (MauA). The P. denitrificans sequence for the mature nitrous-oxide reductase reduces from 14 to 11 and 6 to 4, respectively, the number of conserved histidine and methionine residues compared to previous sequences. Three cysteine and four tryptophan residues, previously identified as conserved amongst nitrous-oxide reductases, are found in the Paracoccus enzyme. A comparison of the sequence of the C-terminal region of the nitrous-oxide-reductase sequence with that for the CuA region of subunit II of the cytochrome aa3 from P. denitrificans reveals considerable sequence similarities. Upstream of the structural gene for nosZ are sequences TTGAAGCTTAACCAG (centred at position -21 with respect to the start codon) and CCCGGTGGTCATCAAG (centred at position -126). Although both could be FNR (ANR) boxes, the latter is far more probable to have this role because only it is likely to be upstream of a promoter site. This is the first indication at the DNA sequence level for the existence of this regulatory system in P. denitrificans. Analysis of the flanking DNA sequences revealed reading frames upstream and downstream of the nosZ gene showing similarity to the nosR and nosD genes, respectively, of Pseudomonas species. An S30 in vitro transcription/translation system was developed for P. denitrificans which permitted the expression of the cloned gene for nitrous-oxide reductase and which will be of general value in other studies of this organism.

Urea production by Thiosphaera pantotropha and by anaerobic enrichment cultures from marine sediments

The production of urea by Thiosphaera pantotropha was studied. Batch cultures were grown on acetate as energy source and with NO3− or O2 as terminal electron acceptor. Urea accumulated in the media during exponential growth in aerobic and anaerobic cultures of T. pantotropha. Urea production continued after the cells had entered the stationary growth phase. Bacterial ability to produce urea was supported by studies of cultures enriched for denitrifying, sulphate-reducing and fermenting bacteria. The results implied that urea production was common among bacteria normally considered to be important in marine sediments.

Urea production by Thiosphaera pantotropha and by anaerobic enrichment cultures from marine sediments

Urea production by Thiosphaera pantotropha and by anaerobic enrichment cultures from marine sediments

Crystallization and Preliminary Crystallographic Study of Cytochrome cd 1 Nitrite Reductase from Thiosphaera pantotropha

Single crystals of cytochrome cd 1 nitrite reductase from Thiosphaera pantotropha have been grown in the presence of ammonium sulphate by vapour diffusion techniques. Crystals are suitable for X-ray analysis and diffract to d min = 1·5 Å. They belong to space group P2 1 with unit cell dimensions of a = 107·8 Å, b = 61·9 Å, c = 101·2 Å, β = 112·5°. The crystallographic asymmetric unit contains a dimer.

Interactions between respiration and denitrification during growth of Thiosphaera pantotropha in continuous culture

The effects of oxygen on the use of nitrate as an electron acceptor by the denitrifying bacterium Thiosphaera pantotropha were investigated during growth on acetate. In batch cultures under aerobic conditions nitrate was not utilised and the growth rate constant was 0.55 h−1. The corresponding value for growth on nitrate under anoxic conditions was 0.37 h−1. In acetate-limited continuous cultures with feedback control of the dissolved oxygen concentration, nitrate utilisation was totally inhibited by the lowest concentration of oxygen tested (22 μM). Carbon conversion efficiencies with acetate increased from 0.28 with nitrate to 0.44 with oxygen. The rates of nitrification calculated from nitrogen balance studies were not greater than 1.5% of the rate of anoxic denitrification.

Interactions between respiration and denitrification during growth ofThiosphaera pantotrophain continuous culture

The effects of oxygen on the use of nitrate as an electron acceptor by the denitrifying bacterium Thiosphaera pantotropha were investigated during growth on acetate. In batch cultures under aerobic conditions nitrate was not utilised and the growth rate constant was 0.55 h−1. The corresponding value for growth on nitrate under anoxic conditions was 0.37 h−1. In acetate-limited continuous cultures with feedback control of the dissolved oxygen concentration, nitrate utilisation was totally inhibited by the lowest concentration of oxygen tested (22 μM). Carbon conversion efficiencies with acetate increased from 0.28 with nitrate to 0.44 with oxygen. The rates of nitrification calculated from nitrogen balance studies were not greater than 1.5% of the rate of anoxic denitrification.

Methanol and methylamine utilization result from mutational events in Thiosphaera pantotropha

With choline as carbon source Thiosphaera pantotropha GB17 grew with a doubling time (td) of 6 h. The cellular yield was 55.8 g dry cell weight per mol of choline, indicating that its methyl moieties were used for growth. However, T. pantotropha was unable to grow with methanol or with methylamine as carbon source. Mutants were isolated from liquid or from solid media able to grow with methanol (Mox+) as carbon or methylamine as nitrogen source (Mam+). The Mox+ mutant GB17M grew with a mean td of 11.7h and a growth yield of 8.9 g dry cell weight per mol of methanol. Diauxic growth of strain GB17M was observed with mixtures of pyruvate and methanol as substrates in batch culture. Methanol led to the formation of methanol dehydrogenase, formate dehydrogenase, ribulosebisphosphate carboxylase and of a soluble cytochrome c-551.5. Tn5-insertional mutants defective in the thiosulfate oxidizing enzyme system or in hydrogenase acquired the Mox+ phenotype. However, Tn5-insertional mutants defective in either a c-type cytochrome or the molybdenum cofactor did not mutate to the Mox+ phenotype, indicating common functions in thiosulfate and in methanol metabolism.