Tn5-induced mutations affecting sulfur-oxidizing ability (Sox) of Thiosphaera pantotropha

Mutants of Thiosphaera pantotropha defective in chemolithoautotrophic growth were obtained by transpositional mutagenesis with Tn5 coding for kanamycin resistance. The suicide vehicle for introducing Tn5 to T. pantotropha was pSUP5011 harbored by Escherichia coli. Kanamycin-resistant isolates were screened for the inability to grow with reduced sulfur compounds (Sox-). Four classes of Sox- mutants were obtained. Three were of different pleiotropic phenotypes: (i) unable to grow with formate, nitrate, and xanthine; (this class strongly suggested the involvement of a molybdenum cofactor in inorganic sulfur-oxidizing ability); (ii) no growth with hydrogen; (iii) slight growth with hydrogen and formate. Two plasmids, pHG41 (about 450 kilobase pairs) and pHG42 (110 kilobases), were identified in lysates of T. pantotropha. In one Sox- mutant pHG41 could not be detected. Revertant analysis suggested that pHG41 and pHG42 were not involved in the Sox character.     

Bioenergetic examination of the heterotrophic nitrifier-denitrifier Thiosphaera pantotropha

The heterotrophic nitrifying-denitrifying bacterium Thiosphaera pantotropha is remarkable as it nitrifies and denitrifies simultaneously. With respect to nitrogenous compounds, whether nitrification or denitrification results in energy conservation is of interest. Proton translocation studies were performed to determine if energy was conserved by the bacterium during heterotrophic nitrification and denitrification. Hydrazine (N₂H _inf5 ^sup+ ) was employed as the heterotrophic nitrification substrate while nitrate, nitrite and nitrous oxide were used as denitrification substrates. Analysis of the data indicate that the bacterium does not conserve energy when hydrazine was the substrate. Conversely, energy was conserved when either nitrate, nitrite or nitrous oxide functioned as the oxidants during denitrification-dependent proton translocation experiments. Thiosphaera pantotropha thus is similar to other heterotrophic nitrifiers-denitrifiers in that it conserves energy while denitrifying but has not been observed to do so when heterotrophically nitrifying.     

Higher order structural organization of the nucleoid in the thiobacterium Thiosphaera pantotropha

Abstract Thiosphaera pantotropha cells treated with mitomycin C produced bacteriophages and showed cell lysis. Upon occurrence of cell lysis, samples were mounted for electron microscopy by negative staining. During mounting, the cell contents were spread at the surface of the support film. Besides polysomes, strands interpreted as DNA could be seen, most of them complexed with particles interpreted as DNA-binding proteins. Single and twisted strands were revealed, and complex structures with diameters around 35 nm were common. They exhibited an ordered arrangement of the proteins. Our findings suggest that bacterial chromosomal DNA complexed with DNA-binding proteins may be organized in higher order, similar to the compactation of nucleosome strands in eukaryotic chromosomal DNA.     

Simultaneous Nitrification and Denitrification in Aerobic Chemostat Cultures of Thiosphaera pantotropha

Thiosphaera pantotropha is capable of simultaneous heterotrophic nitrification and aerobic denitrification. Consequently, its nitrification potential could not be judged from nitrite accumulation, but was estimated from complete nitrogen balances. The maximum rate of nitrification obtained during these experiments was 93.9 nmol min⁻¹ mg of protein⁻¹. The nitrification rate could be reduced by the provision of nitrate, nitrite, or thiosulfate to the culture medium. Both nitrification and denitrification increased as the dissolved oxygen concentration fell, until a critical level was reached at approximately 25% of air saturation. At this point, the rate of (aerobic) denitrification was equivalent to the anaerobic rate. At this dissolved oxygen concentration, the combined nitrification and denitrification was such that cultures receiving ammonium as their sole source of nitrogen appeared to become oxygen limited and the nitrification rate fell. It appeared that, under carbon-and energy-limited conditions, a high nitrification rate was correlated with a reduced biomass yield. To facilitate experimental design, a working hypothesis for the mechanism behind nitrification and denitrification by T. pantotropha was formulated. This involved the basic assumption that this species has a “bottleneck” in its cytochrome chain to oxygen and that denitrification and nitrification are used to overcome this. The nitrification potential of other heterotrophic nitrifiers has been reconsidered. Several species considered to be “poor” nitrifiers also simultaneously nitrify and denitrify, thus giving a falsely low nitrification potential.     

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

Abstract 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.     

Competition between hydrogen peroxide and nitrate for electrons from the respiratory chains of Thiosphaera pantotropha and Rhodobacter capsulatus

Abstract Thiosphaera pantotropha and some strains of Rhodobacter capsulatus express both a periplasmic nitrate reductase and cytochrome c peroxidase when grown under aerobic conditions. Harvested cell suspensions of either species can respire nitrate in the presence of 200 μM O₂ (∼ 80% air saturation), at 70–80% of the anaerobic rate. Addition of hydrogen peroxide to such cells causes a 90% inhibition of nitrate reduction under anaerobic or aerobic conditions. The duration of the inhibition is proportional to the concentration of hydrogen peroxide added and can be ascribed to the expression of periplasmic peroxidases that compete with the nitrate reductase for electrons from the respiratory chain. The results reveal a hitherto unrecognised interaction between reactions of denitrification and the reduction of hydrogen peroxide by a periplasmic peroxidase that may have implications for the denitrification in microaerobic environments. The creation of aerobic conditions in bacterial cultures by addition of hydrogen peroxide, and relying on the generation of oxygen by endogenous catalase activity, is a commonly used technique for studying respiratory processes. The observations presented here demonstrate that results derived from such experiments should be interpreted with caution.     

The influence of carbon substrate on the activity of the periplasmic nitrate reductase in aerobically grown Thiosphaera pantotropha

The periplasmic nitrate reductase was assayed in intact cells of Thiosphaera pantotropha, after aerobic growth with either malate, succinate, acetate, butyrate or caproate present as sole carbon source. The level of enzyme activity was largely dependent upon carbon source and was lowest on malate and succinate, intermediate on acetate and highest on butyrate and caproate. The presence or absence of nitrate did not effect enzyme activity. The results indicate that, during aerobic growth, activity of the periplasmic nitrate reductase increases with the extent of reduction of the carbon substrate.Abbreviation MV⁺ reduced methylviologen     

The effect of electron acceptor variations on the behaviour of Thiosphaera pantotropha and Paracoccus denitrificans in pure and mixed cultures

Abstract The competitive advantages provided by a capacity for aerobic denitrification have been tested by comparing Thiosphaera pantotropha (which denitrifies aerobically and anaerobically), with a strain of Paracoccus denitrificans (which only denitrifies under anaerobic conditions) in acetate-limited chemostats. A comparison of μ -C_s curves based on K _s and μ _max measurements indicated that Pa. denitrificans could be expected to dominate mixtures of the two species at high growth rates when the dissolved oxygen was above 80% of air saturation and NH₃ was the sole source of nitrogen. The comparison also suggested t that at lower growth rates, lower dissolved oxygen tensions, and/or in the presence of nitrate, Tsa. pantotropha should have the competitive advantage. Chemostat experiments with mixtures of the two species showed that Tsa. pantotropha did, indeed, dominate the population when expected. However, when Pa. denitrificans was expected to dominate, only a small increase in the Pa. denitrificans numbers was possible before Tsa. pantotropha formed a biofilm on the walls of the chemostat instead of washing out, and was again able to out-compete Pa. denitrificans for acetate. Experiments with axenic chemostat cultures subjected to aerobic/anaerobic switches showed that Tsa. pantotropha , with its constitutive denitrifying system, was able to adjust smoothly to the changing environmental conditions and thus continued to grow. Pa. denitrificans does not have constitutive denitrifying enzymes, and could consequently not adjust its metabolism to the lack of oxygen rapidly enough. It therefore washed out at a rate equivalent to the dilution rate.     

The use of a metabolically structured model in the study of growth, nitrification, and denitrification by Thiosphaera pantotropha

Thiosphaera pantotropha is capable of aerobic heterotrophic nitrification and both aerobic and anaerobic denitrification. These phenomena have been studied in acetate-limited aerobic and anaerobic continuous cultures supplied with ammonia and nitrate. The internal reaction rates were defined, based on biochemical knowledge. The observable external conversion rates are related through a linear equation on the basis of the specified internal reaction rates. The linear equation is a Pirt relation extended for microbial systems with multiple electron donors (acetate and ammonia) and electron acceptors (oxygen and nitrate). The coefficients in this equation were estimated from the continuous culture measurements, and are composed of parameters involved in ATP production and consumption by the microorganism. It is shown that with realistic values for these parameters, the metabolically structured model describes the aerobic as well as the anaerobic experiments.     

The cytochromes c-550 of Paracoccus denitrificans and Thiosphaera pantotropha: a need for re-evaluation of the history of Paracoccus cultures

Abstract The c -type cytochrome and protein profiles were compared for a number of cultures of Paracoccus denitrificans obtained from a range of culture collections. The cultures fell into two groups corresponding to the two original isolates of this bacterial species. One group, which included NCIMB 8944, ATCC 13543, ATCC 17741, ATCC 19367, Pd 1222 and DSM 413, were similar or identical to LMD 22.21. The second group, including DSM 65 and LMG 4218, were similar or identical to LMD 52.44. These groupings were not compatible with the recorded history of culture deposition. Mass spectrometry and amino acid sequence comparisons showed that the cytochrome c -550 of the LMD 52.44 culture group differed by 16% from that of the LMD 22.21 group, and yet was only 1% different from the cytochrome c -550 of Thiosphaera pantotropha . These results suggest that consideration should be given to creation of a new species of Paracoccus pantotropha , which would include Thiosphaera pantotropha and Paracoccus denitrificans LMD 52.44.     

Identification of a promoter region responsible for the senescence-specific expression of SAG12

SAG12, an Arabidopsis gene encoding a cysteine protease, is expressed only in senescent tissues. Studies of the expression patterns of a variety of genes showing senescence-specific or senescence-preferential expression indicate that plant senescence involves multiple regulatory pathways. In this study it is shown that the expression of SAG12 is specifically activated by developmentally controlled senescence pathways but not by stress- or hormone-controlled pathways. Using SAG12 as a molecular marker for the study of developmental senescence, we show that cytokinin, auxin, and sugars can repress developmental senescence at the molecular level. Studies using promoter deletions and recombination of promoter fragments indicate that a highly conserved region of the SAG12 promoter is responsible for senescence-specific regulation, while at least two other regions of the SAG12 promoter are important for full promoter activity. Extracts from young and senescent Arabidopsis leaves contain factors that exhibit differential binding to the senescence-responsive promoter element.     

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

Abstract 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.     

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

Abstract Most α-mannosidase activity (80%) in C. albicans was found in a soluble form. Addition of protease inhibitors to explore proteolytic release from a particulate cell component during enzyme preparation did not change this distribution. Molecular mass, calculated from gel filtration chromatography, was 417 kDa. Optimum pH was 6.0 with 50 mM Mes-Tris when p-nitrophenyl-α- d -mannopyranoside was used as substrate. Optimum temperature was 42°C with either 10 mM phosphate buffer (pH 6.8) or 50 mM Mes-Tris buffer (pH 6.0) and with 4-methylumbelliferyl-α- d -mannopyranoside as substrate. Apparent K _m values for p-nitrophenyl-α- d -mannopyranoside and 4-methylumbelliferyl-α- d -mannopyranoside were 3.3 mM and 0.1 mM, respectively. 1 mM 1-deoxymannojirimycin and 0.3 mM swainsonine inhibited the hydrolysis of 4-methylumbelliferyl-α- d -mannopyranoside by 67% and 83%, respectively, whereas that of p-nitrophenyl-α- d -mannopyranoside was only slightly diminished (10–15%).     

Determination of growth and coupled nitrification/denitrification by immobilized Thiosphaera pantotropha using measurement and modeling of oxygen profiles

An oxygen microsensor in combination with mathematical modeling was used to determine the behavior of immobilized Thiosphaera pantotropha. This organism can convert ammonia completely to nitrogen gas under aerobic conditions (coupled nitrification/denitrification) and denitrifies nitrate at highest rates under anaerobic conditions. Immobilization of T. pantotropha can result in aerobic and anaerobic zones inside the biocatalyst particle which will be advantageous for the conversion of ammonia and nitrate from wastewater. However, information of the effects of immobilization on the physiology of T. pantotropha is necessary for the development of such a system. This article gives the extension of a model developed to describe the behavior of chemostat cultures of T. pantotropha so that it can be used for immobilized cells. The original model was based on metabolic reaction equations. Kinetic and diffusion equations have now been added. Experimental verification was carried out using a stirred tank reactor and a Kluyver flask. After immobilization in agarose, the cells were grown in the particles under continuous culture conditions for 3 days. After 24 h the oxygen penetration depth showed a constant value of 100 mu, indicating that a steady state was reached. Scanning electron micrographs showed that large colonies of cells were present in this 100-mum aerobic layer.From the dynamics of the start-up phase, several parameters were determined from measurements of the oxygen concentration profiles made every few hours. The profiles simulated by the model were fitted to the measured data. The average value for the maximum specific growth rate was 0.52 h(-1), and the maximum oxygen conversion rate was 1.0 mol Cmol(-1) h(-1). The maximum specific acetate uptake rate was 2.0 mol Cmol(-1) h(-1), and the Monod constant for acetate was 2.9 x 10(-2) mol m(-3). The maximum specific nitrification rate was 0.58 x 10(-1) mol Cmol(-1) h(-1), and the amount of oxygen necessary for nitrification was 11% of the total oxygen uptake rate. Most of the kinetic parameters determined for the immobilized cells were in good agreement with those for the suspended cells. Only the maximum specific growth rate was significantly higher, and the maximum specific nitrification rate was some what lower than for suspended cells. The experimental results clearly show that an oxygen microsensor, in combination with mathematical modeling, can successfully be used to elucidate the kinetic behavior of immobilized, oxygen-consuming, cells.     

Limited proteolysis of synapsin I. Identification of the region of the molecule responsible for its association with microtubules

Synapsin I is a highly asymmetric neuronal structural phosphoprotein implicated in the regulation of neurotransmitter release probably by the multiple interactions it can contract with membranous and cytoskeletal elements of the neuronal cell. In order to locate the region(s) of synapsin I responsible for its association with microtubules, we have first studied synapsin I limited digestion by trypsin. The resulting polypeptides were localized in the synapsin I molecule by using three different criteria: their kinetics of appearance, their collagenase sensitivity, and the presence of the synapsin phosphorylation site 1 (cyclic AMP dependent). Synapsin I digestion kinetics are not affected by phosphorylation at this site. Analysis of the ability of various synapsin I tryptic fragments in mixture to cosediment with microtubules shows that a 44-kDa fragment corresponding to the NH2-terminal hydrophobic head of the molecule contains a binding site for polymerized tubulin. This fragment competes with native synapsin I for binding on microtubules. None of the polypeptides belonging to the tail region of synapsin I (COOH-terminal half of the molecule) were found to cosediment with microtubules.     

Identification of the region responsible for fibril formation in the CAD domain of caspase-activated DNase

Caspase-activated DNase (CAD) has a compact domain at its N-terminus (CAD domain, 87 amino acid residues), which comprises one alpha-helix and five beta-strands forming a single sheet. The CAD domain of CAD (CAD-CD) forms amyloid fibrils containing alpha-helix at low pH in the presence of salt. To obtain insights into the mechanism of amyloid fibril formation, we identified the peptide region essential for fibril formation of CAD-CD and the region responsible for the salt requirement. We searched for these regions by constructing a series of deletion and point mutants of CAD-CD. Fibril formation by these CAD-CD mutants was examined by fluorescence analysis of thioflavin T and transmission electron microscopy. C-Terminal deletion and point mutation studies revealed that an aromatic residue near the C-terminus (Trp81) is critical for fibril formation. In addition, the main chain conformation of the beta5 strand, which forms a hydrophobic core with Trp81, was found to be important for the fibril formation by CAD-CD. The N-terminal 30 amino acid region containing two beta-strands was not essential for fibril formation. Rather, the N-terminal region was found to be responsible for the requirement of salt for fibril formation.     

Identification of the chromosomal region responsible for high-temperature stress tolerance during the grain-filling period in rice

An unusually high temperature during the grain-filling period, such as that caused by global warming, impairs the quality of rice (Oryza sativa L.) grains. This sensitivity to high-temperature stress is different among cultivars, suggesting the possibility of developing a high-temperature-tolerant cultivar. Since marker-assisted selection would reduce time and labor in breeding for such a quantitative trait, we determined the chromosomal region responsible for high-temperature tolerance during the grain-filling period. A high-temperature-sensitive japonica cultivar Tohoku 168 and a tolerant japonica cultivar Kokoromachi were selected as the parental lines of recombinant inbred lines (RILs) by high-temperature stress treatment from 5 to 10 days after anthesis, which was found to be the period most critical for grain quality. Using the RILs, whose genotypes were determined by analysis with 131 DNA markers which were selected as polymorphic markers between these two cultivars from 2,648 DNA markers tested, the quantitative trait locus (QTL) for the percentage of white-back grains was mapped on chromosome 6. The Kokoromachi allele of the QTL, which had a positive additive effect on the high-temperature tolerance, was introduced into the Tohoku 168 genome by repeated backcrossings with marker-assisted selection. Using high-temperature stress treatment of the near isogenic lines developed, the QTL on chromosome 6 was localized within a 1.9-Mb region between two DNA markers, ktIndel001 and RFT1. These DNA markers would be useful not only for breeding high-temperature-tolerant cultivars but also for map-based cloning of the QTL.