Bacterial community composition in thermophilic microbial mats from five hot springs in central Tibet

Despite detailed study of selected thermophilic taxa, overall community diversity of bacteria in thermophilic mats remains relatively poorly understood. A sequence-based survey of bacterial communities from several hot spring locations in central Tibet was undertaken. Diversity and frequency of occurrence for 140 unique 16S rRNA gene phylotypes were identified in clone libraries constructed from environmental samples. A lineage-per-time plot revealed that individual locations have evolved to support relatively large numbers of phylogenetically closely related phylotypes. Application of the F _ST statistic and P test to community data was used to demonstrate that phylogenetic divergence between locations was significant, thus emphasizing the status of hot springs as isolated habitats. Among phylotypes, only the Chlorobi were ubiquitous to all mats, other phototrophs (Cyanobacteria and Chloroflexi) occurred in most but not all samples and generally accounted for a large number of recovered phylotypes. Phylogenetic analyses of phototrophic phylotypes revealed support for location-specific lineages. The alpha, beta and gamma proteobacteria were also frequently recovered phyla, suggesting they may be abundant phylotypes in mats, a hitherto unappreciated aspect of thermophilic mat biodiversity. Samples from one location indicated that where phototrophic bacteria were rare or absent due to niche disturbance, the relative frequency of proteobacterial phylotypes increased. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Close genetic relationship between Nitrobacter hamburgensis nitrite oxidoreductase and Escherichia coli nitrate reductases

The nitrite oxidoreductase (NOR) from the facultative nitrite-oxidizing bacterium Nitrobacter hamburgensis X14 was investigated genetically. In order to develop a probe for the gene norB, the N-terminal amino acid sequence of the NOR -subunit (NorB) was determined. Based on that amino acid sequence, an oligo-nucleotide was derived that was used for the identification and cloning of gene norB. Sequence analysis of DNA fragments revealed three adjacent open reading frames in the order norA, norX, norB. The DNA sequences of norX and norB represented complete genes while the open reading frame of norA was truncated by the cloning site. The deduced amino acid sequence of protein NorB contained four cysteine clusters with striking homology to those of iron-sulfur centers of bacterial ferredoxins. NorB shares significant sequence similarity to the -subunits (NarH, NarY) of the two dissimilatory nitrate reductases (NRA, NRZ) of Escherichia coli. Additionally, the derived amino acid sequence of the truncated open reading frame of norA showed striking resemblance to the -subunits (NarG, NarZ) of the E. coli nitrate reductases.     

Regulation of assimilatory nitrate reduction at the level of nitrite in Chlorella fusca

Batch cultures of Chlorella fusca excreted nitrite into the medium if gassed with air (0.03% CO₂), but they did not if supplied with air containing 5% CO₂. After a change from high to low CO₂ concentration in the gas stream, nitrite excretion started immediately. After an increase in CO₂ concentration to 5%, nitrite uptake started within only 30 min. Changes of in-vitro activities of nitrate reductase, nitrite reductase and glutamine synthetase did not correspond to changes of nitrite concentration in the medium and therefore could not explain these observations. A nitrite-binding site, whose activity corresponded with both nitrite excretion and uptake, was detected at the chloroplast envelope. From these data an additional regulatory step in the assimilatory nitrate-reduction sequence is suggested. This includes an envelopeprotein fraction probably regulating the availability of nitrite within the chloroplast.Abbreviations FMN riboflavin 5-phosphate - GS glutamine synthetase - NIR nitrite reductase - NR nitrate reductase     

Probing the archaeal diversity of a mixed thermophilic bioleaching culture by TGGE and FISH

The archaeal community present in a sample of Mixed Thermophilic Culture-B (MTC-B) from a laboratory-scale thermophilic bioleaching reactor was investigated by temperature gradient gel electrophoresis (TGGE) and fluorescence in situ hybridisation (FISH). Both techniques were specifically adapted for use on native state bioleaching samples, with a view to establishing convenient means for monitoring culture composition. Using the TGGE protocol developed, the relative species composition of the thermophilic bioleaching sample was analysed, and included four phylotypes belonging to the Sulfolobales, which were related to Stygiolobus azoricus, Metallosphaera sp. J1, Acidianus infernus and Sulfurisphaera ohwakuensis. However, the St. azoricus-like phylotype was difficult to resolve and some micro-heterogeneity was observed within this phylotype. Specific FISH probes were designed to qualitatively assess the presence of the phylotypes in MTC-B. The sample was dominated by Sf. ohwakuensis-like Archaea. In addition, the St. azoricus-like, Metallosphaera species-like and Acidianus species-like cells appeared in similar low abundance in the community. Most strikingly, FISH identified Sulfolobus shibatae-like cells present in low numbers in the sample even though these were not detected by PCR-dependent TGGE. These results highlight the importance of using more than one molecular technique when investigating the archaeal diversity of complex bioleaching reactor samples.     

Drought acclimation of two deciduous tree species of different layers in a temperate forest canopy

Water-use strategies of Populus tremula and Tilia cordata, and the role of abscisic acid in these strategies, were analysed. P. tremula dominated in the overstorey and T. cordata in the lower layer of the tree canopy of the temperate deciduous forest canopy. Shoot water potential (), bulk-leaf abscisic acid concentration ([ABA]_leaf), abscisic acid concentration in xylem sap ([ABA]_xyl), and rate of stomatal closure following the supply of exogenous ABA (v) decreased acropetally through the whole tree canopy, and foliar water content per area (w), concentration of the leaf osmoticum (c), maximum leaf-specific hydraulic conductance of shoot (L), stomatal conductance (g_s), and the threshold dose per leaf area of the exogenous ABA (d_a) required to reduce stomatal conductance increased acropetally through the tree canopy (from the base of the foliage of T. cordata to the top of the foliage of P. tremula) in non-stressed trees. The threshold dose per leaf dry mass of the exogenous ABA (d_w) required to reduce stomatal conductance, was similar through the tree canopy. After a drought period (3 weeks), the , w, L, g_s, d_a and d_w had decreased, and c and v had increased in both species. Yet, the effect of the drought period was more pronounced on L, g_s, d_a, d_w and v in T. cordata, and on , w and c in P. tremula. It was concluded that the water use of the species of the lower canopy layer—T. cordata, is more conservative than that of the species of the overstorey, P. tremula. [ABA]_leaf had not been significantly changed in these trees, and [ABA]_xyl had increased during the drought period only in P. tremula. The relations between [ABA]_leaf, [ABA]_xyl and the stomatal conductance, the osmotic adjustment and the shoot hydraulic conductance are also discussed.     

The electron transport system of Alcaligenes eutrophus H16

The electron transport system of autotrophically grown Alcaligenes eutrophus H16 has been investigated by spectroscopic and thermodynamic approaches. The results have been interpreted as evidence that isolated membranes contain a branched respiratory chain composed of three c-type haems (E _m,7=+160 mV, + 170 mV, and + 335 mV), five b-type haems (E _m,7=+ 5 mV, + 75 mV, + 205 mV, + 300 mV, and + 405 mV), two (possibly three) a-type haems [E _m,7= + 255 mV, + 350 mV, (+ 420 mV)], and nne d-type haem. EPR-analysis of the signals at g=1.93, g=2.02, and g=1.90 revealed the presence of iron-sulphur centres diagnostic of complexes I (NADH dehydrogenase), II (succinate dehydrogenase), and III (ubiquinol/cytochrome c oxidoreductase). The low potential b haems (+ 5 mV and + 75 mV) plus the Rieske protein (g=1.90, E _m,7=+ 280 mV), thought to be part of an orthodox bc ₁ complex, were present in low amounts as compared to their counterparts in membranes from Paracoccus denitrificans.CO-difference spectra in the presence of either succinate, NADH, hydrogen, ascorbate/TMPD, and/or dithionite as reductants, suggested the existance of four different oxidases composed by bo-, cb-, a-, and d-type haems.It is concluded that in contrast to other chemolithotrophes, e.g. P. denitrificans, autotrophic growth of Alcaligenes eutrophus utilizes a respiratory system in which the bc ₁ complex containing pathway is only partially involved in electron transport.Abbreviations Cytochrome c-551, number wavelength in nm - Cytochrome c ₂₇₀, number mid-point potential in mV - E _m,7 mid-point potential of an oxidation-reduction couple at pH 7.0 - KP buffer, potassium phosphate-buffer - OD optical density at 436 nm, 1 cm light path - TMPD N,N,N,N-tetramethyl-p-phenylenediamine     

Isolation and characterization of a thermophilic sulfate-reducing bacterium Desulfotomaculum thermoacetoxidans sp. nov.

An obligately anaerobic thermophilic sporeforming sulfate-reducing bacterium, named strain CAMZ, was isolated from a benzoate enrichment from a 58°C thermophilic anaerobic bioreactor. The cells of strain CAMZ were 0.7 m by 2–5 m rods with pointed ends, forming single cells or pairs. Spores were central, spherical, and caused swelling of the cells. The Gram stain was negative. Electron donors used included lactate, pyruvate, acetate and other short chain fatty acids, short chain alcohols, alanine, and H₂/CO₂. Lactate and pyruvate were oxidized completely to CO₂ with sulfate as electron acceptor. Sulfate was required for growth on H₂/CO₂, and both acetate and sulfide were produced from H₂/CO₂-sulfate. Sulfate, thiosulfate, or elemental sulfur served as electron acceptors with lactate as the donor while sulfite, nitrate, nitrite, betaine, or a hydrogenotrophic methanogen did not. The optimum temperature for growth of strain CAMZ was 55–60°C and the optimum pH value was 6.5. The specific activities of carbon monoxide dehydrogenase of cells of strain CAMZ grown on lactate, H₂/CO₂, or acetate with sulfate were 7.2, 18.1, and 30.8 mol methyl viologen reduced min^–1 [mg protein]^–1, respectively, indicating the presence of the CO/Acetyl-CoA pathway in this organism. The mol%-G+C of strain CAMZ's DNA was 49.7. The new species name Desulfotomaculum thermoacetoxidans is proposed for strain CAMZ.     

Phylogenetic Evidence for the Existence of Novel Thermophilic Bacteria in Hot Spring Sulfur-Turf Microbial Mats in Japan

So-called sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. This study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16S rRNA genes amplified from the bulk DNAs of the mats. Common clones with 16S rDNA sequences with similarity levels of 94.8 to 99% were isolated from sulfur-turf mat samples from two geographically remote hot springs. Phylogenetic analysis showed that the phylotypes of the common clones formed a major cluster with members of the Aquifex-Hydrogenobacter complex, which represents the most deeply branching lineage of the domain bacteria. Furthermore, the bacteria of the sulfur-turf mat phylotypes formed a clade distinguishable from that of other members of the Aquifex-Hydrogenobacter complex at the order or subclass level. In situ hybridization with clone-specific probes for 16S rRNA revealed that the common phylotype of sulfur-turf mat bacteria is that of the predominant sausage-shaped bacteria.Microbial mats develop in a wide variety of aquatic environments, including geothermal hot springs and hydrothermal vents. There are several types of thermophilic microbial mats, e.g., those of cyanobacteria, anoxygenic phototrophic bacteria, and chemotrophic sulfur bacteria, which differ according to the physical and chemical conditions they favor and other environmental factors (10, 38). These microbial mats in thermal habitats have been studied extensively as a peculiar microbial community of the ecosystem, in relation to the phylogeny and evolution of thermophilic prokaryotes, or as a source of new functional enzymes.So-called sulfur-turf microbial mats are macroscopic bundles of white filaments consisting of colorless sulfur bacteria and elemental sulfur particles that form in shallow streams of sulfide-containing high-temperature hot springs. Since first reported by Miyoshi in 1897 (33), this kind of microbial mat has been recorded for several geographically remote hot springs in Japan, although there have been only scattered reports of sulfur-turf microbial mats or chemotrophic sulfur streamers in geothermal springs in other countries (9, 13, 14). The sulfur-turf mats generally develop within a temperature range of 45 to 73°C, within a pH range of 6 to 9, and at discrete sulfide-oxygen interfaces in geothermal springs. These characteristics suggest that the major constituents of the sulfur-turf prokaryotic community are (hyper)thermophilic, neutrophilic, microaerophilic, and chemolithotrophic bacteria. Early studies of these sulfur-turf mats distinguished microscopically three morphotypes of bacteria, two of which were tentatively named Thiovibrio miyoshi and Thiothrix miyoshi (15). Moreover, in situ ecophysiological and microscopic studies have shown that one of these bacteria, the large sausage-shaped “Thiovibrio miyoshi,” predominates in sulfur-turf mats and oxidizes environmental sulfide to elemental sulfur and then to sulfate via thiosulfate (27–31). So far, however, it has not been possible to isolate and cultivate any thermophilic prokaryotes from the sulfur-turf mats predominated by these sausage-shaped bacteria with artificial media, and no attempt has been made to clarify their taxonomic and phylogenetic positions.Determination of 16S rRNA genes is a useful research strategy for identifying uncultivated prokaryotes and is now commonly performed in ecological studies. This technique, involving PCR amplification of 16S rRNA genes or synthesis of cDNAs from bulk 16S rRNAs of natural mixed microbial populations, has been used successfully for the phylogenetic characterization of prokaryotes in hydrothermal environments (6, 7, 34, 40, 41, 47, 48). In the present study, this approach was applied to characterize the sausage-shaped bacteria in sulfur-turf mats without isolating and cultivating them. Here we report that sulfur-turf mats contain novel thermophilic bacteria belonging to the earliest-branching lineage of the domain bacteria.     

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd 1-free background (NirS−) of Pseudomonas stutzeri

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of Achromobacter cycloclastes nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd ₁ nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d ₁, indicating that both heterologous hosts synthesized a cytochrome cd ₁ without the d ₁-group.Abbreviations Cu-NIR Cu-containing nitrite reductase - DDC diethyldithiocarbamate - EPR electron paramagnetic resonance - IPTG isopropyl--D-galactoside - SDS sodium dodecyl sulfate - LB medium Luria-Bertani medium     

Cytochrome aa 3 from Methylococcus capsulatus (Bath)

A cytochrome aa ₃-type oxidase was isolated with and without a c-type cytochrome (cytochrome c-557) from Methylococcus capsulatus Bath by ion-exchange and hydrophobic chromatography in the presence of Triton X-100. Although cytochrome c-557 was not a constitutive component of the terminal oxidase, the cytochrome c ascorbate-TMPD oxidase activity of the enzyme decreased dramatically when the ratio of cytochrome c-557 to heme a dropped below 1:3. On denaturing gels, the purified enzyme dissociated into three subunits with molecular weights of 46,000, 28,000 and 20,000. The enzyme contains two heme groups (a and a ₃), absorption maximum at 422 nm in the resting state, at 445 and 601 nm in the dithionite reduced form and at 434 and 598 nm in the dithionite reduced plus CO form. Denaturing gels of the cytochrome aa ₃-cytochrome c-557 complex showed the polypeptides associated with cytochrome aa ₃ plus a heme c-staining subunit with a molecular weight of 37,000. The complex contains approximately two heme a, one heme c, absorption maximum at 420 nm in the resting state and at 421, 445, 522, 557 and 601 nm in the dithionite reduced form. The specific activity of the purified enzyme was 130 mol O₂/min · mol heme a compared to 753 mol O₂/min · mol heme a when isolated with cytochrome c-557.Abbreviations MMO methan monooxygenase - sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - TMPD N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride - Na₂EDTA disodium ethylenediamine-tetraacetic acid     

Anaerobic growth and potential for amino acid production by nitrate respiration in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Oxygen limitation is a crucial problem in amino acid fermentation by Corynebacterium glutamicum. Toward this subject, our study was initiated by analysis of the oxygen-requiring properties of C. glutamicum, generally regarded as a strict aerobe. This organism formed colonies on agar plates up to relatively low oxygen concentrations (0.5% O₂), while no visible colonies were formed in the absence of O₂. However, in the presence of nitrate (), the organism exhibited limited growth anaerobically with production of nitrite (), indicating that C. glutamicum can use nitrate as a final electron acceptor. Assays of cell extracts from aerobic and hypoxic cultures yielded comparable nitrate reductase activities, irrespective of nitrate levels. Genome analysis revealed a narK2GHJI cluster potentially relevant to nitrate reductase and transport. Disruptions of narG and narJ abolished the nitrate-dependent anaerobic growth with the loss of nitrate reductase activity. Disruption of the putative nitrate/nitrite antiporter gene narK2 did not affect the enzyme activity but impaired the anaerobic growth. These indicate that this locus is responsible for nitrate respiration. Agar piece assays using l-lysine- and l-arginine-producing strains showed that production of both amino acids occurred anaerobically by nitrate respiration, indicating the potential of C. glutamicum for anaerobic amino acid production.     

The interaction of nitrogen assimilation with photosynthesis in nitrogen deficient cells of Chlorella

Nitrogen-limited chemostat cultures of Chlorella fusca var. vacuolata, when given nitrogen in the inorganic forms of nitrate, nitrite and ammonium divert photo-generated electrons, from CO₂ fixation to nitrogen assimilation. Addition of nitrate or nitrite, but not ammonium, stimulates rate of oxygen evolution. All but the most severely nitrogen-deficient culture have increased dark respiration rates after addition of inorganic nitrogen. The nitrite reduction step of nitrogen assimilation is the most light-dependent reaction.Abbreviation DCMU 3-(3, 4-dichloro)-1-1-dimethyl urea - CCCP carbonyl cyanide m-chlorophenylhydrazone     

Nitrite reduction in Rhizobium “hedysari” strain HCNT 1

Rhizobium hedysari strain HCNT 1 rapidly reduced nitrite to N₂O, only slowly reduced nitrate to nitrite and did not exhibit nitrous oxide reductase activity. Nitrite reduction in this rhizobium strain may be a detoxification mechanism for conversion of nitrite, which inhibits O₂ uptake, to non-toxic N₂O. Concentrations of nitrite as small as 3 M diminished O₂ uptake in whole cells. The bacterium did not couple energy conservation with nitrate or nitrite reduction. Cells neither grew anaerobically at the expense of these nitrogen oxides nor translocated protons during reduction of nitrite. Induction of nitrite reductase activity was not a response to the presence of nitrate or nitrite, but occurred instead when the O₂ concentration in culture atmospheres fell to <16.5% of air saturation. Sensitivity of cytochrome o, which is synthesized only in cells grown under O₂-limited conditions, may account for the toxicity of nitrite in strain HCNT 1.     

Archaeal diversity and geochemical energy yields in a geothermal well on Vulcano Island, Italy

The archaeal diversity in a shallow geothermal well on Vulcano Island, Italy was characterized using culture‐independent 16S rDNA sequence analysis. Environmental DNA was extracted from 56 °C well water, and the 16S ribosomal RNA gene was amplified with archaea‐specific primers. Restriction fragment length polymorphism (RFLP) analysis of ～250 clones revealed 35 unique patterns, which were sequenced and analyzed. These yielded 17 operational taxonomic units, of which 13, 3, and 1 were unique cren‐, eury‐, and korarchaeotal sequences, respectively. The majority of the crenarchaeotal phylotypes formed a novel, deeply‐branching clade that includes sequences from other hydrothermal environments, but no cultured representatives. Three phylotypes represent novel lineages in the Thermoproteales and two phylotypes represent a novel genus of Euryarchaeota. One euryarchaeotal phylotype was nearly identical (99%) to Palaeococcus helgesonii, an aerotolerant, hyperthermophilic fermenter previously isolated from the same well. To place this diverse archaeal community in the geochemical framework of this ecosystem, we calculate values of Gibbs free energy of 145 organic and inorganic redox reactions at in situ conditions. Energy yields ranged from 0 to 125 kJ per mole of electrons transferred. The most exergonic organic reactions were organic carbon oxidation with O₂ (>100 kJ/mol e^?), followed by oxidation with (61–93 kJ/mol e^?), Fe(III) (43–60 kJ/mol e^?), and S⁰/ (6–27 kJ/mol e^?) as terminal electron acceptors. Overall, energy yields from inorganic reactions were similar to those of the organic reactions considered, but were less systematic with respect to terminal electron acceptor. The oxidation of methane coupled with Fe(III) reduction yielded the most energy (123 kJ/mol e^?). However, the most exergonic inorganic reactions were predominantly O₂, , or reduction. Reduction of , S⁰, CO₂, and CO yielded significantly less energy (0–18 kJ/mol e^?). Metabolisms of the cultured organisms identified in the Pozzo Istmo archaeal clone library were exergonic. However, most of the archaeal diversity remains uncultured and energetic calculations reveal an extensive suite of potential lithotrophic and heterotrophic metabolisms that could be exploited by these novel organisms.     

Cytochromes in Thiobacillus tepidarius and the respiratory chain involved in the oxidation of thiosulphate and tetrathionate

Thiobacillus tepidarius was shown to contain cytochrome(s) c with absorption maxima at 421, 522 and 552 nm in room temperature reduced minus oxidized difference spectra, present at 1.1–1.2 nmol per mg dry wt and present in both membrane and soluble fractions of the cell. The membrane-bound cytochrome c (1.75 nmol per mg membrane protein) had a midpoint potential (E_m, pH 7.0) of 337 mV, while the soluble fractions appeared to contain cytochrome(s) c with E_m (pH 7.0) values of about 270 and 360 mV. The organism also contained three distinct membrane-bound b-type cytochromes (totalling 0.33 nmol per mg membrane protein), each with absorption maxima in reduced minus oxidized difference spectra at about 428, 532 and 561 nm. The E_m (pH 7.0) values for the three cytochromes b were 8 mV (47.8% of total), 182 mV (13.7%) and 322 mV (38.5%). No a- or d-type cytochromes were detectable spectrophotometrically in the intact organism or its membrane and soluble fractions. Evidence is presented for both CO-binding and CO-unreactive cytochromes b or o, and CO-binding cytochrome(s) c. From redox effects observed with CO it is proposed that a cytochrome c donates electrons to a cytochrome b, and that a high potential cytochrome b or o may be acting as the terminal oxidase in substrate oxidation. This may be the 445 nm pigment, a photodissociable CO-binding membrane haemoprotein. Substrate oxidation was relatively insensitive to CO-inhibition, but strongly inhibited by cyanide and azide. Thiosulphate oxidation couples directly to cytochrome c reduction, but tetrathionate oxidation is linked (probably via ubiquinone Q-8) to reduction of a cytochrome b of lower potential than the cytochrome c. The nature of possible electron transport pathways in Thiobacillus tepidarius is discussed. One speculative sequence is: c^– b₈ b₁₈₂ c₂₇₀ c₃₃₇ b₃₂₂/c₃₆₀ O₂ Abbreviations E_m midpoint electrode potential - E _inf0 ^sup pH 7, standard electrode potential at pH 7.0 - Q-8 coenzyme Q-8 (ubiquinone-40)     

The impact of organic matter on nitric oxide formation by Nitrosomonas europaea

Chemolithoautotrophically growing cells of Nitrosomonas europaea quantitatively oxidized ammonia to nitrite under aerobic conditions with no loss of inorganic nitrogen. Significant inorganic nitrogen losses occurred when cells were growing mixotrophically with ammonium, pyruvate, yeast extract and peptone. Under oxygen limitation the nitrogen losses were even higher. In the absence of oxygen pyruvate was metabolized slowly while nitrite was consumed concomitantly. Nitrogen losses were due to the production of nitric oxide and nitrous oxide. In mixed cultures of Nitrosomonas and Nitrobacter, strong inhibition of nitrite oxidation was reproducibly measured. NO and ammonium were not inhibitory to Nitrobacter. First evidence is given that hydroxylamine, the intermediate of the Nitrosomonas monooxygenase-reaction, is formed. 0.2 to 1.7 M NH₂OH were produced by mixotrophically growing cells of Nitrosomonas and Nitrosovibrio. Hydroxylamine was both a selective inhibitory agent to Nitrobacter cells and a strong reductant which reduced nitrite to NO and N₂O. It is discussed whether chemodenitrification or denitrification is the most abundant process for NO and N₂O production of Nitrosomonas.     

Thermophilic anaerobic fermentation of hemicellulose and hemicellulose-derived aldose sugars by Thermoanaerobacter strain B6A

The thermophilic, anaerobic fermentation of hemicellulosic subtrates by Thermoanaerobacter strain B6A was investigated using a variety of commercially-available hemicelluloses which had been characterized by physical and chemical analysis. Products of hemicellulose fermentation included ethanol, acetic acid, lactic acid, H₂, and CO₂ in ratios which varied depending on the hemicellulose used. Both the rate and extent of substrate utilization (as estimated from product formation) varied in the order: unidentified mannan/xyloglucan > xylan > 4-O-methylglucuronoxylan > arabinoxylan > type II arabinogalactan=0. Rates of product formation were enhanced up to twofold by autoclaving of substrates,which partially depolymerized the substrates and in some cases altered their composition. Total extent of product formation, however, was similar in autoclaved and non-autoclaved substrates. Hemicellulose fermentations were mediated by one or more constitutive extracellular enzyme activities. Component monosaccharides of hemicelluloses in their natural isomeric configurations supported rapid growth (_max = 0.3–1.0 h^–1), while unnatural isomers were not utilized.The wide carbohydrate utilization spectrum of this strain apparently reflects its role as a versatile primary consumer in the hot spring bacterial-algal mat from which it was isolated.Abbreviations GC Gas chromatography - HPLC High-performance liquid chromatography - MS Mass spectroscopy - TLC Thinlayer chromatography - TMS Trimethylsilyl Contribution No. 3696 of the Central Research and Development Department     

Electron transport-linked proton translocation at nitrite reduction inCampylobacter sputorum subspeciesbubulus

Campylobacter sputorum subspeciesbubulus contains a membrane-bound nitrite reductase which catalyses the six-electron reduction of nitrite to ammonia. Formate andL-lactate are used as hydrogen donors. Cells ofC. sputorum grown with nitrate or nitrite contain cytochromes of theb-andc-type and a carbon monoxide-binding cytochromec. In addition, a special membrane-bound carbon monoxide-binding pigment is found. Nitrite reduction with formate orL-lactate as a hydrogen donor is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Nitrite reduction by bacterial suspensions with lactate as a hydrogen donor is strongly inhibited by carbonylcyanide-m-chlorophenyl-hydrazone (CCCP) whereas nitrite reduction with formate as a hydrogen donor is not inhibited at all. H⁺/O values and H⁺/NO ₂ ^- values were measured with ascorbate + N,N,N,N-tetramethyl-p-phenylenediamine (TMPD), formate (in the absence and presence of carbonic anhydrase) andL-lactate as a hydrogen donor. The results are summarized in a scheme for electron transport from formate or lactate to oxygen or nitrite which shows a periplasmic orientation of formate dehydrogenase and nitrite reductase and a cytoplasmic orientation of lactate dehydrogenase and oxygen reduction, and which shows proton translocation with a H⁺/2e value of 2.0. The H⁺/O and H⁺/NO ₂ ^- values predicted by this scheme are in good agreement with the experimental values.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - MTPP⁺ methyltriphenylphosphonium cation - TMPD N,N,N,N-tetramethyl-p-phenylenediamine; H⁺/O (H⁺/NO ₂ ^- ), number of protons liberated in the outer bulk phase at the reduction of one atom O (one ion NO ₂ ^- ); H⁺/2e (q⁺/2e), number of protons (charges) translocated across the cytoplasmic membrane during flow of two electrons to an acceptor     

Photosynthetic electrogenic events in native membranes ofChloroflexus aurantiacus. Flash-induced charge displacements within the reaction center-cytochromec 554 complex

The thermophilic phototrophChloroflexus aurantiacus possesses a photosynthetic reaction center (RC) containing a pair of menaquinones as primary (Q_A) and secondary (Q_B) electron acceptors and a bacteriochlorophyll dimer (P) as a primary donor. A tetraheme cytochromec ₅₅₄ with two high(H)- and two low(L)-potential hemes operates as an immediate electron donor for P. The following equilibrium E_m,7 values were determined by ESR for the hemes in whole membrane preparations: 280 mV (H1), 150 mV (H2), 95 mV (L1) and 0 mV (L2) (Van Vliet et al. (1991) Eur. J. Biochem. 199: 317–323). Partial electrogenic reactions induced by a laser flash inChl. aurantiacus chromatophores adsorbed to a phospholipid-impregnated collodion film were studied electrometrically at pH 8.3. The photoelectric response included a fast phase of generation ( < 10 ns, phase A). It was ascribed to the charge separation between P⁺ and Q_A ^– as its amplitude decreased both at high and low E_h values (E_m,high=360±10 mV, estimated E_m,low\s-160 mV) in good agreement with E_m values for P/P⁺ and Q_A/Q_A ^– redox couples. A slower kinetic component appeared upon reduction of the cytochromec ₅₅₄ hemes (phase C). With H1 reduced before the flash the amplitude of phase C was equal to 15–20% of that of phase A and its rise time was 1.2–1.3 s: we attribute this phase to the electrogenic electron transfer from H1 to P⁺. Pre-reduction of H2 decreased the value to about 700–800 ns and increased the amplitude of phase C to 30–35% of that of phase A. Pre-reduction of L1 further accelerated phase C (up to of 500 ns) and induced a reverse electrogenic phase with of 12 s and amplitude equal to 10% of phase A. Upon pre-reduction of L2 the rise time of phase C was decreased to about 300 ns and its amplitude decreased by 30%. The acceleration in the onset of phase C is explained by the acceleration of the rate-limiting H1 P electrogenic reaction after reduction of the other hemes due to their electrostatic influence; a P-H1-(L1-L2)-H2 alignment of redox centers with an approximately rhombic arrangement of the cytochromec ₅₅₄ hemes is proposed. The observed reverse phase is ascribed to the post-flash charge redistribution between the hemes. Redox titration of the amplitude of phase C yielded the E_m,8.3 values of H1, H2 and L2 hemes: 340±10 mV for H1, 160±20 mV for H2 and –40±40 mV for L2.