ALBERT

Notes: Abstract Hydroxylamine is an intermediate in the oxidation of ammonia to nitrite, but until now it has not been possible to grow Nitrosomonas europaea on hydroxylamine. This study demonstrates that cells of N. europaea are capable of growing mixotrophically on ammonia and hydroxylamine. The molar growth yield on hydroxylamine (4.74 g mol−1 at a growth rate of 0.03 h−1) was higher than expected. Aerobically growing cells of N. europaea oxidized ammonia to nitrite with little loss of inorganic nitrogen, while significant inorganic nitrogen losses occurred when cells were growing mixotrophically on ammonia and hydroxylamine. In the absence of oxygen, hydroxylamine was oxidized with nitrite as electron acceptor, while nitrous oxide was produced. Anaerobic growth of N. europaea on ammonium, hydroxylamine and nitrite could not be observed at growth rates of 0.03 h−1 and 0.01 h−1.

Notes: Abstract The interconversion of adenine nucleotides during acetate fermentation was investigated with concentrated cell suspensions of Methanothrix soehngenii. Starved cells contained high levels of AMP (2.2 nmol/mg protein), but had hardly any ADP or ATP. The energy charge of these cells was 0.1. Immediately after the addition of the substrate acetate, the level of ATP increased, reaching a maximum of 1.4 nmol/mg protein, corresponding to an energy charge of 0.7 when half of the acetate was consumed. Once the acetate was depleted, the ATP concentration decreased to its original level of 0.1 nmol/mg protein. As M. soehngenii contained relatively high amounts of AMP, the luciferase system for the determination of ATP gave not always satisfactory results. Therefore a reliable method based on the separation of adenine nucleotides by anion exchange HPLC was used.

Notes: Abstract The methyl-CoM reductase from Methanothrix soehngenii was purified 18-fold to apparent homogeneity with 50% recovery in three steps. The native molecular mass of the enzyme estimated by gel-fitration was 280 kDa. SDS-polyacrylamide gel electrophoresis revealed three protein bands corresponding to Mr 63 900, 41 700 and 30 400 Da. The methyl-coenzyme M reductase constitutes up to 10% of the soluble cell protein. The enzyme has Km apparent values of 23 μM and 2 mM for N-7-mercaptoheptanoylthreonine phosphate (HS- HTP=componentB) and methyl-coenzyme M (CH3?CoM) respectively. At the optimum pH of 7.0 60 nmol of methane were formed per min per mg protein.

Notes: Abstract Phosphoenolpyruvate (PEP) carboxykinase is present in crude extracts of Corynebacterium glutamicum grown on both glucose and lactate. Preparation of PEP carboxykinase free from interfering PEP carboxylase and oxaloacetate decarboxylase showed an absolute dependence on divalent manganese and IDP for activity in the oxaloacetate (OAA) formation. Other diphosphate nucleotides could not substitute for IDP. The enzyme activity displayed Michaelis-Menten kinetics for the substrates PEP, IDP, KHCO3, OAA and ITP with a Km of 0.7 mM, 0.4 mM, 12 mM, 1.0 mM, and 0.5 mM, respectively. At the optimum pH of 6.6, 850 nmol of OAA were formed per min per mg of protein. ATP inhibited PEP carboxykinase in the OAA forming reaction for 60% at 0.1 mM, indicating that the enzyme mainly functions in gluconeogenesis.

Notes: Abstract The minimum threshold concentrations of acetate utilization and the enzymes responsible for acetate activation of several methanogenic bacteria were investigated and compared with literature data. The minimum acetate concentrations reached by hydrogenotrophic methane bacteria, which require acetate as carbon source, were between 0.4 and 0.6 mM. The acetoclastic Methanosarcina achieves acetate concentrations between 0.2 and 1.2 mM and Methanothrix between 7 and 70 μM. For the activation of acetate most of the hydrogenotrophic methane bacteria investigated use an acetyl-CoA synthetase with a relatively low Km (40–90 μM) for acetate. although the affinity for acetate was high, the hydrogenotrophic methane bacteria were not able to remove acetate to lower concentrations than the acetoclastic methane bacteria, neither in pure cultures nor in anaerobic granular sludge samples. Based on these observations, it is not likely that hydrogenotrophic methanogens compete strongly for acetate with the acetoclastic methane bacteria.

Notes: The chemolithoautotrophic anammox bacterium Candidatus“Kuenenia stuttgartiensis” grows anaerobically using ammonium as electron donor for nitrite reduction. More than 10% of the proteins in cell extracts of “K. stuttgartiensis” consist of c-type heme proteins. A 10 kDa soluble cytochrome c was purified from cell extracts using ultracentrifugation and anion exchange chromatography. The UV/Vis spectrum of the reduced cytochrome showed the γ, β and α absorption maxima at 419, 522 and 552 nm, respectively. The N-terminal amino acid sequence and peptide fragments of the tryptic digest of the protein were used to identify the corresponding gene. Analysis of the gene product showed that the protein was preceded by a 30 amino acids long leader sequence and that it belonged to the low-spin class ID cytochrome c. The CXXCH motive was located at the N-terminal site of the protein. The gene organization of the cytochrome showed some resemblance to cytochrome c clusters of unknown function in the genome of Nitrosomonas europaea and Geobacter sulfurreducens PCA.

Notes: Degenerate primers to detect nrfA were designed by aligning six nrfA sequences including Escherichia coli K-12, Sulfurospirillum deleyianum and Wolinella succinogenes. These primers amplified a 490 bp fragment of nrfA. The ability of these primers to detect nrfA was tested with chromosomal DNA isolated from a variety of bacteria: they could distinguish between bacteria in which the gene is known to be present or absent. The positive reference organisms spanned the various classes of Proteobacteria, suggesting that these primers are probably generic. The primer pair F1 and R1 was also used successfully to analyse nrfA diversity from community DNA isolated from a sulphate reducing bioreactor, and from two established Anammox reactors (for an aerobic amm onia ox idation, in which nitrite is reduced by ammonia to dinitrogen gas). The nrfA clones isolated from these three sources grouped with the Bacteroidetes phylum. The nrfA primers also amplified 570 bp fragments from the Anammox community DNA. These fragments encoded a protein with four haem-binding motifs typical of a c-type cytochrome, but were unrelated to the NrfA nitrite reductase. A BLAST search failed to reveal similarity to any known proteins. However, similarity was found to one sequence, which was annotated as rapC (response regulator aspartate phosphatase), in the genome of the planctomycete Rhodopirellula baltica. These sequences possibly belong to a new class of c-type cytochrome that might be specific to members of the order Planctomycetales. The data are consistent with the proposal that cytochrome c nitrite reductases, present in the periplasm of Gram-negative bacteria, are widely distributed in many different environments where they provide a short circuit in the biological nitrogen cycle by reducing nitrite directly to ammonia.

Notes: In the Completely Autotrophic Nitrogen removal Over Nitrite (CANON) process, aerobic and anaerobic ammonia oxidizing bacteria cooperate to remove ammonia in one oxygen-limited reactor. Kinetic studies, microsensor analysis, and fluorescence in situ hybridization on CANON biomass showed a partial differentiation of processes and organisms within and among aggregates. Under normal oxygen-limited conditions (∼5 μM O2), aerobic ammonia oxidation (nitrification) was restricted to an outer shell (〈100 μm) while anaerobic ammonia oxidation (anammox) was found in the central anoxic parts. Larger type aggregates (〉500 μm) accounted for 68% of the anammox potential whereas 65% of the nitrification potential was found in the smaller aggregates (〈500 μm). Analysis with ∼5 μM O2 microsensors showed that the thickness of the activity zones varied as a function of bulk O2 and NO−2 concentrations and flow rate.

Notes: Abstract Acetate is the precursor of approximately two-thirds of the methane produced in anaerobic bioreactors. Only two genera of methanogenic archae are known to use acetate as sole energy source: Methanosarcina and Methanothrix. Methanosarcina appears to be a generalist with a high growth rate, but low affinity for acetate. Methanothrix is a specialist having a high affinity for acetate, but low growth rate. Methanothrix shows a much lower minimum threshold for acetate utilization (7–70 μM) than Methanosarcina (0.2–1.2 mM). This is consistent with the evidence that Methanothrix is found in environments with low acetate concentrations.The acetate degradation by acetotrophic methanogens starts with an activation of acetate to acetyl-coenzyme A. In Methanosarcina spp. this activation is catalysed by an acetate kinase/phosphotransacetylase system at the expense of one ATP. Acetyl-coenzyme A synthetase activates acetate in Methanothrix, with concomitant hydrolysis of one ATP to AMP and PPi. Both enzyme systems have been purified and comparison of the kinetic properties confirmed the hypothesis that low acetate concentrations favour Methanothrix. The gene encoding for acetyl-CoA synthetase of Methanothrix was isolated from a genomic library and actively expressed in Escherichia coli. The deduced amino acid sequence showed homology to proteins with similar function and contained two putative ATP binding sites.The most characteristic and complex enzyme involved in the acetate degradation by acetotrophic methanogens is carbon monoxide dehydrogenase. The enzyme has been purified from both Methanothrix and Methanosarcina, and represents 5–10% of the soluble protein of these microorganisms. CO dehydrogenase is proposed to catalyse both the cleavage of acetyl-CoA in a methyl-, carbonyl- and CoA-moiety, and the oxidation of the carbonyl group to CO2. This multifunctional redox enzyme contains several iron, acid-labile sulfur and nickel atoms. These atoms are arranged into several paramagnetic complexes, which have been studied by EPR spectroscopy. The low spin recovery of the different paramagnetic centers makes statements about structure and functions difficult. There are good spectroscopic and genetic indications that the CO dehydrogenase of Methanothrix contains at least one ferrodoxin-like [4Fe-4S] cluster, which could play a role in the electron transfer of the CO oxidation. Further, in EPR spectra of concentrated samples of CO dehydrogenase from Methanothrix a very unusual signal was observed, which showed great similarity to putative [6Fe-6S] prismane clusters.The final step in the methanogenesis from acetate, the reduction of methyl-coenzyme M, is catalysed by methyl-coenzyme M methylreductase. The enzyme purified from Methanothrix and Methanosarcina showed great homology with the methyl-CoM methylreductase of other methanogenic archae, although the specific activity was rather low (60–125 nmol min−1 mg−1).The reduction of the heterodisulfide between coenzyme M and component B is proposed to be the common site for energy conservation in all methanogens. Acetoclastic methanogens, however, need additional sites of energy conservation to compensate for their high energy input in acetate activation. The oxidation of CO to CO2 could form one possible site. The partially membrane-associated pyrophosphatase of Methanothrix could form another site of energy conservation.