The upper and lower limits of the mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Stoichiometry of oxidative phosphorylation

Determination of the intrinsic or mechanistic P/O ratio of oxidative phosphorylation is difficult because of the unknown magnitude of leak fluxes. Applying a new approach developed to overcome this problem (see our preceding paper in this journal), the relationships between the rate of O2 uptake [( Jo)3], the net rate of phosphorylation (Jp), the P/O ratio, and the respiratory control ratio (RCR) have been determined in rat liver mitochondria when the rate of phosphorylation was systematically varied by three specific means. (a) When phosphorylation is titrated with carboxyatractyloside, linear relationships are observed between Jp and (Jo)3. These data indicate that the upper limit of the mechanistic P/O ratio is 1.80 for succinate and 2.90 for 3-hydroxybutyrate oxidation. (b) Titration with malonate or antimycin yields linear relationships between Jp and (Jo)3. These data give the lower limit of the mechanistic P/O ratio of 1.63 for succinate and 2.66 for 3-hydroxybutyrate oxidation. (c) Titration with a protonophore yields linear relationships between Jp, (Jo)3, and (Jo)4 and between P/O and 1/RCR. Extrapolation of the P/O ratio to 1/RCR = 0 yields P/O ratios of 1.75 for succinate and 2.73 for 3-hydroxybutyrate oxidation which must be equal to or greater than the mechanistic stoichiometry. When published values for the H+/O and H+/ATP ejection ratios are taken into consideration, these measurements suggest that the mechanistic P/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation.     

NADH-Linked oxidative phosphorylation inNitrobacter agilis

A particulate cell-free fraction (144,000-X-g pellet) fromNitrobacter agilis catalyzes the acrobic or anaerobic oxidation of NADH. Phosphorylation coupled to the aerobic oxidation of NADH yields P/O ratios of 1.1. The net formation of ATP coupled to the anaerobic oxidation of NADH by nitrate yields P/NO₃ ratios of 0.7. Phosphate esterification is uncoupled by carbonylcyanide-m-chlorophenyl-hydrozone and is sensitive to inhibitors of the electron transport system.     

Interspecific variation in pollen–ovule ratio is negatively correlated with pollen transfer efficiency in a natural community

The pollination efficiency hypothesis has long been proposed as an explanation for interspecific variation in pollen–ovule (P:O) ratios. However, no empirical study on P:O ratios has directly and quantitatively measured pollen transfer efficiency (PE). Here, we use a PE index, defined as the proportion of pollen grains removed from anthers that are subsequently deposited on conspecific stigmas, as a direct and quantitative measure of PE. We investigated P:O ratios, pollen removal and pollen deposition in 26 plant species in an alpine meadow, over three consecutive years. Our community survey showed that nearly 5% of removed pollen was successfully deposited on conspecific stigmas. The PE index ranged from 0.01% up to 78.56% among species, and correlated negatively with the P:O ratio across years. This correlation was not changed by controlling for phylogenetic relationships among species, suggesting that the interspecific variation in P:O ratios can be attributed to the probability of pollen grains reaching a stigma. The results indicate that the pollination efficiency hypothesis can help to explain interspecific variation in P:O ratios.     

The energetic conversion competence of Escherichia coli during aerobic respiration studied by 31P NMR using a circulating fermentation system

To determine the actual potential of the energetic conversion efficiency of Escherichia coli during aerobic respiration, apparent P/O ratios (P/O(app)) under either limited or standard glucose-feeding conditions were estimated. The previously reported circulating fermentation system (CFS) was used, and (31)P NMR saturation-transfer (ST) techniques were employed. By coupling with on-line NMR observations, CFS allowed us to evaluate cellular energetics directly, with both the dissolved oxygen tension and glucose feeding precisely controlled to prevent the effect of substrate-level phosphorylation based on aerobic or anaerobic acidogenesis in E. coli cells. Phosphate consumption rates under standard and limited glucose-conditions were estimated as 4.62 +/- 0.46 and 1.99 +/- 0.11 micromol/s g of dry cell weight (DCW), respectively. Using simultaneously assessed O(2) consumption rates, the P/O(app) values under these two conditions were estimated as 1.4 +/- 0.3 and 1.5 +/- 0.1, respectively. To correlate the obtained P/O(app) values with the potential efficiency of respiratory enzymes, we determined the activities of two NADH dehydrogenases (NDH 1 and 2) and two ubiquinol oxidases (bo- and bd-type) during the periods when ST was performed. NDH-1 activities in standard or limited glucose cultures were maintained at 57% or 58% of the total NADH oxidizing activity. The percentages of bo-type oxidase activity in relation to the total ubiqinol oxidizing activity under the standard and limited glucose conditions were 32% and 36%, respectively. These percentages of enzymatic activities represent the respiratory competence of E. coli cells, suggesting that, during the NMR observatory period, the enzymatic activity was not at a maximum, which could also explain the estimated P/O(app) values. If this is the case, enhancing the expression of the bo-type oxidase or disrupting of the bd-type oxidase gene could be effective approach to increasing both the P/O ratio and cellular yields.     

31P nuclear magnetic resonance and saturation transfer studies of the isolated perfused rat kidney

Previous studies using 31P nuclear magnetic resonance (NMR) saturation transfer techniques to quantitate the energy metabolism of the kidney have often resulted in estimates of adenosine triphosphate (ATP) turnover which are much lower than those predicted from the renal oxygen consumption and reasonable values of the P/O ratio. We measured the ATP turnover in isolated perfused kidneys of rats, using 31P NMR saturation transfer and a new procedure for quantitation of the intracellular Pi concentration. The estimated turnover rates of ATP were higher than previously reported. The P/O ratios calculated on the basis of these rates of ATP turnover and rates of renal oxygen consumption reported in the literature were within the range of theoretically possible values. Thus, 31P NMR saturation transfer can be used to quantitate the ATP turnover in the isolated perfused rat kidney.     

Characterization of null mutants of the glyoxylate cycle and gluconeogenic enzymes in S. cerevisiae through metabolic network modeling verified by chemostat cultivation.

Biomass yields for several null mutants in Saccharomyces cerevisiae were successfully predicted with a metabolic network model. Energetic parameters of the model were obtained from growth data in C-limited aerobic chemostat cultures of the corresponding wild-type strain, which exhibited a P/O ratio of 1.46, a non-growth-related maintenance of 56 mmol ATP/C-mol biomass/h, and a growth-related requirement of 655 mmol ATP/C-mol biomass. Biomass yields and carbon uptake rates were modeled for different mutants incapacitated in their glyoxylate cycle and their gluconeogenesis. Biomass yields were calculated for different feed ratios of glucose to ethanol, and decreases for higher ethanol fractions were correctly predicted for mutants with deletions of the malate synthase, the isocitrate lyase, or the phosphoenolpyruvate carboxykinase. The growth of the fructose- 1,6-bisphosphatase deletion mutant was anticipated less accurate, but the tendency was modeled correctly.     

An assessment of the role of proton leaks in the mechanistic stoichiometry of oxidative phosphorylation.

Rat liver mitochondria were incubated in the presence of varying concentrations of ATP, followed by ADP to initiate phosphorylation. Analysis of phosphorylation to oxygen ratios (P/O) was carried out with varied initial phosphorylation potentials (or ATP/ADP ratios). Rates of phosphorylation and respiration and magnitude of membrane potential (delta psi) were measured. The results are discussed in the framework of P/total O and P/"extra" O ratios in determination of the mechanistic P/O ratio. It is concluded that the former underestimates, and the latter overestimates the mechanistic P/O ratio.     

Dynamics of dissolved oxygen isotopic ratios: a transient model to quantify primary production,community respiration,and air–water exchange in aquatic ecosystems

Dissolved O(2) is an important aquatic ecosystem health indicator. Metabolic and gas exchange (G) rates, which control O(2) concentration, are affected by nutrient loading and other environmental factors. Traditionally, aquatic metabolism has been reported as primary production:community respiration (P:R) ratios using diel measurements and interpretations of dissolved O(2) and/or CO(2) concentrations, and recently using stable isotopes (delta(18)O, Delta(17)O) and steady state assumptions. Aquatic ecosystems, such as rivers and ponds, are not at steady state and exhibit diel changes, so steady state approaches are often inappropriate. A dynamic O(2) stable isotope model (photosynthesis-respiration-gas exchange; PoRGy) is presented here, requiring a minimum of parameters to quantify daily averaged P, R, and G rates under transient field conditions. Unlike steady state approaches, PoRGy can address scenarios with 100% O(2) saturation but with delta(18)O-O(2) values that are not at air equilibrium. PoRGy successfully accounts for isotopic G when applied to an oxygen isotope equilibration laboratory experiment. PoRGy model results closely matched the diel O(2) and delta(18)O-O(2) data from three field sites with different P:R:G ratios and various P, R and G rates. PoRGy provides a new research tool to assess ecosystem health and to pose environmental impact-driven questions. Using daily averaged rates was successful and thus they can be used to compare ecosystems across seasons and landscapes.     

Comparative genomics of two ‘Candidatus Accumulibacter' clades performing biological phosphorus removal

Members of the genus Candidatus Accumulibacter are important in many wastewater treatment systems performing enhanced biological phosphorus removal (EBPR). The Accumulibacter lineage can be subdivided phylogenetically into multiple clades, and previous work showed that these clades are ecologically distinct. The complete genome of Candidatus Accumulibacter phosphatis strain UW-1, a member of Clade IIA, was previously sequenced. Here, we report a draft genome sequence of Candidatus Accumulibacter spp. strain UW-2, a member of Clade IA, assembled following shotgun metagenomic sequencing of laboratory-scale bioreactor sludge. We estimate the genome to be 80–90% complete. Although the two clades share 16S rRNA sequence identity of >98.0%, we observed a remarkable lack of synteny between the two genomes. We identified 2317 genes shared between the two genomes, with an average nucleotide identity (ANI) of 78.3%, and accounting for 49% of genes in the UW-1 genome. Unlike UW-1, the UW-2 genome seemed to lack genes for nitrogen fixation and carbon fixation. Despite these differences, metabolic genes essential for denitrification and EBPR, including carbon storage polymer and polyphosphate metabolism, were conserved in both genomes. The ANI from genes associated with EBPR was statistically higher than that from genes not associated with EBPR, indicating a high selective pressure in EBPR systems. Further, we identified genomic islands of foreign origins including a near-complete lysogenic phage in the Clade IA genome. Interestingly, Clade IA appeared to be more phage susceptible based on it containing only a single Clustered Regularly Interspaced Short Palindromic Repeats locus as compared with the two found in Clade IIA. Overall, the comparative analysis provided a genetic basis to understand physiological differences and ecological niches of Accumulibacter populations, and highlights the importance of diversity in maintaining system functional resilience.     

Oxidative phosphorylation in Micrococcus denitrificans

P/O ratios were measured in membrane particles obtained from cells of Micrococcus denitrificans, while growing on different carbon sources. The membrane particles obtained from cells growing actively on glucose, succinate, ethanol and propanol as the carbon and energy sources catalyzed oxidative phosphorylation and yielded respective P/O ratios of 1.4, 1.2, 0.8, and 0.5 with NADH, and 0.8, 0.6, 0.6, and 0.5 with succinate as the electron donors. Not such a difference in P/O ratio is observed in intact resting cells grown with different carbon sources. It is concluded that the influence of the carbon source is probably directed towards the efficiency of oxidative phosphorylation in membrane particles and not in the growing cells.For the aerobic carbon source-limited chemostat cultures the following maximum growth yields were determined: 40.2 and 34.2 for succinate and oxgen, 41.7 and 36.5 for malate and oxygen, 81.4 and 39.4 for mannitol and oxygen, and 77.8 and 43.4 for gluconate and oxygen respectively. With a mathematical model (de K waadsteniet et al., in press) the P/O ratio was valued at 1.4–1.7. Y _ATP at =0.2 was valued at 8.7–10.9; Y _ATP ^max at 9.6–13.2 and m _e at 0.6–4.5 for the most precise experiment (gluconate-limited). The calculation of these growth parameters has been discussed.     

INTRA-INFLORESCENCE VARIABILITY IN POLLEN/OVULE RATIOS IN THE CLEISTOGAMOUS SPECIES LAMIUM AMPLEXICAULE (LABIATAE)

A comparative study was made of the various floral forms in the main shoot inflorescence of the cleistogamous species Lamium amplexicaule L. (Labiatae). This study revealed significant differences in pollen/ovule (P/O) ratios between successively produced flowers. The variation in P/O ratio is due to a change in pollen count/flower and not in ovule number. The study included two plant populations, one grown in November when only closed flowers are produced, and the other grown in April when open flower production occurs at the upper nodes. The intra-inflorescence variation in P/O ratio among the spring population showed a correlation between P/O ratio and breeding system. The cleistogamous flowers had low P/O ratios of about 200 while the chasmogamous flowers had higher P/O ratios of about 600.     

Growth of Pseudomonas aeruginosa on nitrous oxide.

Three strains of Pseudomonas aeruginosa were grown anaerobically on exogenous N2O in a defined medium under conditions that assured the maintenance of highly anaerobic conditions for periods of 1 week or more. The bacteria were observed reproducibly to increase their cell density by factors of 3 to 9, but not more, depending on the initial amount of N2O. Growth on N2O was cleanly blocked by acetylene. Cell yields, CO2 production, and N2O uptake all increased with initial PN2O at PN2O less than or equal to 0.1 atm. Growth curves were atypical in the sense that growth rates decreased with time. This is the first observation of growth of P. aeruginosa on N2O as the sole oxidant. N2O was shown to be an obligatory, freely diffusible intermediate during growth of strains PAO1 and P1 on nitrate. All three strains used this endogenous N2O efficiently for growth. For strains PAO1 and P1, it was confirmed that exogenous N2O had little effect on the cell yields of cultures growing with nitrate; thus, for these strains exogenous N2O neither directly inhibited growth nor was used significantly for growth. On the other hand, strain P2 grew abundantly on exogenous N2O when small and growth-limiting concentrations of nitrate or nitrate (2 to 10 mM) were included in the medium. The dramatic effect of these N-anions was realized in large part even when the exogenous N2O was introduced immediately after the quantitative conversion of anion-nitrogen to N2. No evidence was found for a factor in filter-sterilized spent medium that stimulated fresh inocula to grow abundantly on N2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth of Pseudomonas aeruginosa on nitrous oxide

Three strains of Pseudomonas aeruginosa were grown anaerobically on exogenous N2O in a defined medium under conditions that assured the maintenance of highly anaerobic conditions for periods of 1 week or more. The bacteria were observed reproducibly to increase their cell density by factors of 3 to 9, but not more, depending on the initial amount of N2O. Growth on N2O was cleanly blocked by acetylene. Cell yields, CO2 production, and N2O uptake all increased with initial PN2O at PN2O less than or equal to 0.1 atm. Growth curves were atypical in the sense that growth rates decreased with time. This is the first observation of growth of P. aeruginosa on N2O as the sole oxidant. N2O was shown to be an obligatory, freely diffusible intermediate during growth of strains PAO1 and P1 on nitrate. All three strains used this endogenous N2O efficiently for growth. For strains PAO1 and P1, it was confirmed that exogenous N2O had little effect on the cell yields of cultures growing with nitrate; thus, for these strains exogenous N2O neither directly inhibited growth nor was used significantly for growth. On the other hand, strain P2 grew abundantly on exogenous N2O when small and growth-limiting concentrations of nitrate or nitrate (2 to 10 mM) were included in the medium. The dramatic effect of these N-anions was realized in large part even when the exogenous N2O was introduced immediately after the quantitative conversion of anion-nitrogen to N2. No evidence was found for a factor in filter-sterilized spent medium that stimulated fresh inocula to grow abundantly on N2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pollen and ovule production in wind-pollinated species with special reference to Juncus

The reproductive biology of wind-pollinated species in terms of pollen and ovule production is rarely studied compared with zoophilous species, despite available hypotheses on the effect of growth form and life-history traits on reproductive investment. Here, we use published data and new data for species of Juncus and Luzula (Juncaceae) to test the hypotheses that, in wind-pollinated species, woody perennials should exhibit larger pollen–ovule (P/O) ratios than herbaceous species and that species with separate sexes have larger P/O ratios than homoecious species. In total, we report pollen and ovule production for 291 wind-pollinated species, including 19 Juncus and 5 Luzula species. Compared with other wind-pollinated species, Juncus exhibits unusually low P/O ratios (log P/O = 2.06 ± 0.46) because of high ovule production. We argue that the high ovule and seed production in Juncus, associated with frequent self-fertilization, may be beneficial in habitats preferred by the genus. In general, we found higher P/O ratios in woody perennials (log P/O = 4.37 ± 1.18) or in species with separate sexes (log P/O = 4.28 ± 1.12) than in herbaceous (log P/O = 3.51 ± 0.77) or homoecious (log P/O = 3.52 ± 0.80) species, respectively. However, when we analyzed woody perennials separately, we found no significant difference in P/O ratios between homoecious and nonhomoecious species. We argue that woody perennials, independent of dicliny, may be preferentially outcrossed and therefore exhibit decreased variation in mating systems compared with herbs. Because the degree of outcrossing correlates with P/O ratios, differences between homoecious and nonhomoecious woody perennials could be less pronounced.     

Pollen–ovule ratios are strongly correlated with floral reciprocity,in addition to sexual system,in Mussaenda (Rubiaceae)

Heterostyly is a floral polymorphism that increase inter‐morph pollen transfer and promote disassortative mating. Breakdown of heterostyly has happened many times, either leading to dioecy or monomorphism. Mussaenda is a genus with diverse sexual systems including distyly, dioecy, floral monomorphism, and homostyly, making it an ideal system to study the evolution of floral traits and their relationship with sexual system shifts. Here, floral traits and pollen–ovule (P/O) ratios were estimated and used to test hypotheses about the relationships among P/O ratios, floral trait evolution, and sexual system shifts. Our results revealed that there is no significant difference of reciprocity in upper level sexual organs between species with different sexual systems. The reciprocity indices of the lower level sexual organs, however, are smaller in species with functional dioecy than in those with distyly. P/O ratios in dimorphic Mussaenda species were relatively lower than in monomorphic outcrossing species, but did not differ significantly between species with distyly and functional dioecy. Populational P/O ratios were negatively correlated with reciprocity index in the dimorphic species. We suggest that the loss of function in lower level organs in species with functional dioecy has resulted from less strict reciprocity. The relationship between P/O ratios and reciprocity indices strongly support that efficient disassortative pollination of distylus flowers may have promoted evolution towards low P/O ratios.     

Prebiotic synthesis in atmospheres containing CH4, CO,and CO2

The prebiotic synthesis of organic compounds using a spark discharge on various simulated primitive earth atmospheres at 25 degrees C has been studied. Methane mixtures contained H2 + CH4 + H2O + N2 + NH3 with H2/CH4 molar ratios from 0 to 4 and pNH3 = 0.1 torr. A similar set of experiments without added NH3 was performed. The yields of amino acids (1.2 to 4.7% based on the carbon) are approximately independent of the H2/CH4 ratio and whether NH3 was present, and a wide variety of amino acids are obtained. Mixtures of H2 + CO + H2O + N2 and H2 + CO2 + H2O + N2, with and without added NH3, all gave about 2% yields of amino acids at H2/CO and H2/CO2 ratios of 2 to 4. For a H2/CO2 ratio of 0, the yield of amino acids is extremely low (10(-3)%). Glycine is almost the only amino acid produced from CO and CO2 model atmospheres. These results show that the maximum yield is about the same for the three carbon sources at high H2/carbon ratios, but that CH4 is superior at low H2/carbon ratios. In addition, CH4 gives a much greater variety of amino acids than either CO or CO2. If it is assumed that an abundance of amino acids more complex than glycine was required for the origin of life, then these results indicate the requirement for CH4 in the primitive atmosphere.     

Differences in infectivity and induction of infertility: a comparative study of Chlamydia trachomatis strains in the murine model

Chlamydia trachomatis, although commonly asymptomatic in women, can result in chronic sequelae, such as pelvic inflammatory disease, ectopic pregnancy and infertility. However, a clear relationship has not been determined between specific serovars and the ability to lead to upper genital tract infection or infertility. Thus, in order to investigate differences in pathogenicity, C3H/HeN mice were infected in the ovarian bursa with the C. trachomatis strains D (UW-3/Cx), F (N.I.1), F (IC-Cal-3) and E (Bour). Differences both in the amount of vaginal shedding as well as subsequent fertility rates were observed between D (UW-3/Cx) and F (N.I.1) compared to F (IC-Cal-3) and E (Bour). Approximately 50% of the mice infected with the D (UW-3/Cx) and F (N.I.1) strains had vaginal shedding for up to 3–4 weeks after infection and fertility rates of less than 25%. Furthermore, mice inoculated with D (UW-3/Cx) and F (N.I.1) showed infertility even in the absence of medroxy progesterone acetate (MPA) treatment. In contrast, both MPA and non-MPA treated mice infected with F (IC-Cal-3) or E (Bour) did not show vaginal shedding and had fertility rates between 45 and 88%. Mutations in the CT135 open reading frame have been associated with virulence. However, no nucleotide differences were found among the four isolates for CT135. This murine model of infection with C. trachomatis may help with the understanding of disease pathology in humans and ultimately vaccine development.     

A high-yield preparative method for isolation of rat liver mitochondria

A differential centrifugation method for the preparation of rat liver mitochondria is described, which results in final mitochondrial yields of at least 35 to 40 mg of mitochondrial protein per gram wet weight of liver. These mitochondria are shown to be functionally and ultrastructurally intact. They exhibit acceptor control ratios of 6 to 8 and ADP/O ratios near 2.0 with succinate as a substrate. In addition, they appear homogeneous by electron microscopy criteria. This method can be used to prepare rat liver mitochondria in high yield on either a large- or a small-scale basis.     

不同海拔藜芦种群繁殖特征的初步研究

 在北京东灵山地区,沿一定海拔梯度,对藜芦（Veratrum nigrum）这一雄花两性花同株的多年生草本植物的繁殖特征进行了初步研究。结果表明：藜芦开花植株的生物量显著大于未开花植株的生物量。藜芦开花植株大小存在最小临界值,总种群的最小临界值为2.61 g,不同种群的繁殖临界值存在一定的差异。根据花粉/胚珠比（Pollen / ovule ratio,P/O比）推测,藜芦应属于以异交为主的混合交配系统。雄花的大小与生物量都比两性花小,而且其开放时间也晚于两性花,但二者的花粉生产量却没有显著差异。这种现象与人们针对雄花两性花同株植物的雄花功能所提出的最优资源分配假说（Optimal resource allocation hypothesis）是一致的。     

Oxidative phosphorylation and respiratory control in mitochondria from Aspergillus niger

1. Tightly coupled mitochondria were isolated from Aspergillus niger by using an all-glass homogenizer followed by differential centrifugation. 2. The mitochondria oxidized the common intermediates of the tricarboxylic acid cycle, NADH(2) and the ascorbate-tetramethyl-p-phenylenediamine system. 3. High P/O ratios and control of respiration by ADP were obtained with all substrates tested. The average P/O ratios observed were: 1.5-1.8 with succinate as substrate [respiratory control ratio (RC) 2-4]; 0.8-1.0 with ascorbate-tetramethyl-p-phenylenediamine (RC 1.2-1.5); 1.4-1.8 with NADH(2) (RC 2-3); 2.4-2.8 with alpha-oxoglutarate (RC 3-5). 4. Bovine serum albumin (0.05-0.2%) was essential for tightly coupled respiration to be observed. 5. Coupled oxidation of exogenous NADH(2) was relatively insensitive to rotenone and Amytal. 6. The mitochondria responded to specific inhibitors and uncoupling agents in a manner similar to that of mammalian mitochondria. 7. It was concluded that the isolated mitochondria from A. niger show respiratory properties similar to those reported for intact yeast and mammalian mitochondria.