The Genetics of Reproductive Isolation in the Drosophila Simulans Clade: X Vs. Autosomal Effects and Male Vs. Female Effects

A strong effect of homozygous autosomal regions on reproductive isolation was found for crosses between the species in the Drosophila simulans clade. Second chromosome regions were introgressed from D. mauritiana and D. sechellia into D. simulans and tested for their homozygous effects on hybrid male and hybrid female sterility and inviability. Most introgressions are fertile as heterozygotes, yet produce sterile male offspring when made homozygous. The density of homozygous autosomal factors contributing to hybrid male sterility is comparable to the density of X chromosome factors for this level of resolution. Female sterility was also revealed, yet the disparity between male and female levels of sterility was great, with male sterility being up to 23 times greater than female sterility. Complete hybrid inviability was also associated with some regions of the second chromosome, yet there were no strong sex differences. In conclusion, we find no evidence to support a strong X chromosome bias in the evolution of hybrid sterility or inviability but do find a very strong sex bias in the evolution of hybrid sterility. In light of these findings, we reevaluate the current models proposed to explain the genetic pattern of reproductive isolation.     

Catalysis of nitrosyl transfer by denitrifying bacteria is facilitated by nitric oxide

Two denitrifying bacteria, Pseudomonas stutzeri and Achromobacter cycloclastes, were incubated with Na15NO2 and NaN3 under conditions that allowed catalysis of nitrosyl transfer from nitrite to azide. This transfer, which is presumed to be mediated by the heme- and copper-containing nitrite reductase of P. stutzeri and A. cycloclastes, respectively, leads to formation of isotopically mixed 14,15N2O, whereas denitrification leads to 15N2O. The conditions that emphasized nitrosyl transfer also partially inhibited the nitric oxide reductase system and led to accumulation of 15NO. Absorption of NO from the gas phase by acidic CrSO4 in a sidewell largely abolished nitrosyl transfer to azide. With these two organisms, which are thought to be representative of denitrifiers generally, catalysis of nitrosyl transfer seemed to depend on NO.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Nitric oxide reductase. Purification from Paracoccus denitrificans with use of a single column and some characteristics.

Nitric oxide reductase was purified from Paracoccus denitrificans very nearly to homogeneity by a simple method that involved the use of octyl glucoside to solubilize the enzyme from membranes and required a single hydroxyapatite column. The enzyme had specific activities of about 10 mumol NO reduced x min-1 x mg-1 at pH 6.5 in an amperometric assay system using phenazine methosulfate/ascorbate as the reducing agent and about 22 mumol NO reduced x min-1 x mg-1 at pH 5.0, which is the optimum pH. These values are based on average rates over kinetically complex progress curves and would be about three times greater if based on maximum rate values. The enzyme appeared to be reversibly inhibited by NOaq and to have a Km too low (probably less than or equal to 1 microM) to measure reliably by the amperometric method. The effective second-order rate constant of the enzyme lay within 1 to 2 orders of magnitude of the diffusion controlled limit. The enzyme was composed of a tight complex of two cytochromes: a cytochrome c (Mr = 17,500) and a cytochrome b (Mr = 38,000). The mole ratios of cytochrome c to cytochrome b and Mr 17,500 peptide to Mr 38,000 peptide were both about 1.7, and the heme content was about 3 mol/73,000 g (38,000 + 2(17,500)). Each subunit therefore contained only one heme group. The Mr 38,000 peptide aggregated when heated in the sample buffer used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to the ascorbate-based activity, the enzyme showed a little NADH-NO oxidoreductase activity which was not inhibited by antimycin A. The enzyme lost activity with a half-life of about 2 days at 4 degrees C but could be preserved at -20 degrees C and in liquid nitrogen. It seemed not to be inactivated by aerobic solutions. These observations, and the recent ones by Carr and Ferguson (Carr, G.J., and Ferguson, S.J. (1990) Biochem. J. 269, 423-429) with a partially purified preparation of nitric oxide reductase, establish that the enzyme from Pa. denitrificans is a cytochrome bc complex which resembles that from Pseudomonas stutzeri (Heiss, B., Frunzke, K., and Zumft, W.G. (1989) J. Bacteriol. 171, 3288-3297). There would appear to be no functional relationship between nitric oxide reductase and a Mr = 34,000 peptide of Pa. denitrificans membranes reported previously to be present in purified preparations of a nitric oxide reductase (Hoglen, J., and Hollocher, T.C. (1989) J. Biol. Chem. 264, 7556-7563).     

Electron paramagnetic resonance observations on the cytochrome c-containing nitrous oxide reductase from Wolinella succinogenes.

Nitrous oxide reductase from Wolinella succinogenes, an enzyme containing one heme c and four Cu atoms/subunit of Mr = 88,000, was studied by electron paramagnetic resonance (EPR) at 9.2 GHz from 6 to 80 K. In the oxidized state, low spin ferric cytochrome c was observed with gz = 3.10 and an axial Cu resonance was observed with g parallel = 2.17 and g perpendicular = 2.035. No signals were detected at g values greater than 3.10. For the Cu resonance, six hyperfine lines each were observed in the g parallel and g perpendicular regions with average separations of 45.2 and 26.2 gauss, respectively. The hyperfine components are attributed to Cu(I)-Cu(II) S = 1/2 (half-met) centers. Reduction of the enzyme with dithionite caused signals attributable to heme c and Cu to disappear; exposure of that sample to N2O for a few min caused the reappearance of the g = 3.10 component and a new Cu signal with g parallel = 2.17 and g perpendicular = 2.055 that lacked the simple hyperfine components attributed to a single species of half-met center. The enzyme lost no activity as the result of this cycle of reduction and reoxidation. EPR provided no evidence for a Cu-heme interaction. The EPR detectable Cu in the oxidized and reoxidized forms of the enzyme comprised about 23 and 20% of the total Cu, respectively, or about one spin/subunit. The enzyme offers the first example of a nitrous oxide reductase which can have two states of high activity that present very different EPR spectra of Cu. These two states may represent enzyme in two different stages of the catalytic cycle.     

Extended x-ray absorption fine structure and electron paramagnetic resonance of nitrous oxide reductase from Pseudomonas aeruginosa strain P2.

The copper centers of nitrous oxide reductase from Pseudomonas aeruginosa strain P2 were studied by x-ray and electron paramagnetic resonance (EPR) spectroscopy. The enzyme is dimeric and contains four Cu atoms and about seven cysteine residues/subunit of Mr = 73,000. The extended x-ray absorption fine structure (EX-AFS) spectrum was analyzed for enzyme as isolated (oxidized or slightly reduced), enzyme exposed briefly to air, reduced enzyme, and enzyme at pH 7 after having been activated by standing at pH 10. The average Cu ligand environment in the first shell was best modeled for all forms of the enzyme by a combination of N/O and S atoms at a total coordination number between 3 and 4 and bond distances ranging from 1.96-2.03 A for Cu-N/O and 2.20-2.25 A for Cu-S. The data could be fit without using Cu-Cu interactions. Overall the results are similar to those reported for the enzyme for Pseudomonas stutzeri (Scott, R. A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086). The first derivative EPR spectra of the Cu(II) centers at 15 and 45 K were qualitatively similar among enzyme as isolated and enzyme exposed to N2O or air. These three nominally oxidized samples showed an axial signal with g perpendicular = 2.03 and g parallel = 2.15-2.16. Hyperfine structure was observed in both the g parallel and g perpendicular regions with splittings of 43 and 25 gauss, respectively. These hyperfine components are attributed to exchange coupled Cu(I)-Cu(II) S = 1/2 (half-met) centers. In the enzyme as isolated and after exposure to N2O, about 3/4 of the Cu was EPR silent, whereas after exposure to air the signal integrated to about half the Cu concentration. The EPR spectrum of enzyme activated at pH 10 but frozen at pH 7 was a composite of spectra from activated and inactive species. The activated species presented a complex set of narrow hyperfine components which may arise from contributions from more than one species of half-met center.     

Nitrous oxide reductase of Flexibacter canadensis: a unique membrane-bound enzyme

Abstract The subcellular distribution of nitrous oxide reductase was studied in the gliding soil bacterium Flexibacter canadensis . Nitrous oxide reductase activity, as measured by the methyl viologen-nitrous oxide oxidoreductase assay, was associated entirely with the membrane fraction of cell-free extracts. The enzyme was liberated from the membranes with use of detergents but not by high-salt concentrations, thus implying that nitrous oxide reductase is an integral membrane protein. The nitrous oxide reductase of F. canadensis is the first reported example of a membrane-bound form of this respiratory enzyme.     

15N tracer studies on the reduction of nitrite by the purified dissimilatory nitrite reductase of Pseudomonas aeruginosa. Evidence for direct production of N2O without free NO as an intermediate

Nitrite reductase (cytochrome c,d1) was purified from Pseudomonas aeruginosa. In the presence of the reducing system, ascorbate-N,N,N',N'-tetramethylphenyl-enediamine, which alone had no ability to reduce nitrite or NO at pH 7.5, the enzyme catalyzed the reduction of nitrite to NO and N2O as major and minor products, respectively, as determined by gas chromatography-mass spectrometry. The rate of reduction of NO to N2O was considerably lower than the rate of reduction of nitrite to N2O and might be zero. The N2O produced in a system containing [15N]nitrite and natural NO was more highly enriched in 15N than was the NO pool and, in this regard, closely resembled the enrichment of the nitrite pool. The amount of 14N in the NO pool changed little, if any, as the result of enzymatic processes. For the enzyme, free NO seems not to be an intermediate between nitrite and N2O, just as was found by this laboratory for certain intact denitrifying bacteria. The results are consistent with reduction of nitrite to enzyme-bound NO, which can partition between release and further reduction.     

Studies on the differential inhibition by azide on the nitrite/nitrous oxide level of denitrification.

A gas chromatographic method was used to demonstrate that nitrite can counteract the inhibition by azide of nitrous oxide reductase activity in denitrifiers. This effect explains why azide (and cyanide) can inhibit nitrogen production from nitrous oxide in these organisms but have little effect on nitrogen production from nitrite. Although the physiological basis by which nitrite opposes the action of azide remains unknown, extensive destruction of azide by nitrite can be ruled out as an explanation.     

Oxidation of dithiothreitol during turnover of nitric oxide reductase: evidence for generation of nitroxyl with the enzyme from Paracoccus denitrificans.

The stoichiometric relationship between thiol oxidized and NO reduced was studied for the reaction catalyzed by nitric oxide reductase from Paracoccus denitrificans. The reaction systems consisted of dithiothreitol, ascorbate, phenazine methosulfate, enzyme and NO, or that system minus ascorbate. The mole ratio of thiol groups oxidized to NO reduced was observed to be 2.3 to 1.5 over a range of NO from 0.09 to 0.35 mumol. A ratio of 1.0 was expected for the simple reduction of NO by 1-electron to N2O. The oxidation of additional thiol is attributed to the trapping of nitrosyl hydride (nitroxyl, NO/NOH) by thiol.