Detrital Controls on Soil Solution N and Dissolved Organic Matter in Soils: A Field Experiment

We established a long-term field study in an old growth coniferous forest at the H.J. Andrews Experimental Forest, OR, USA, to address how detrital quality and quantity control soil organic matter accumulation and stabilization. The Detritus Input and Removal Treatments (DIRT) plots consist of treatments that double leaf litter, double woody debris inputs, exclude litter inputs, or remove root inputs via trenching. We measured changes in soil solution chemistry with depth, and conducted long-term incubations of bulk soils from different treatments in order to elucidate effects of detrital inputs on the relative amounts and lability of different soil C pools. In the field, the addition of woody debris increased dissolved organic carbon (DOC) concentrations in O-horizon leachate and at 30 cm, but not at 100 cm, compared to control plots, suggesting increased rates of DOC retention with added woody debris. DOC concentrations decreased through the soil profile in all plots to a greater degree than did dissolved organic nitrogen (DON), most likely due to preferential sorption of high C:N hydrophobic dissolved organic matter (DOM) in upper horizons; percent hydrophobic DOM decreased significantly with depth, and hydrophilic DOM had a much lower and less variable C:N ratio. Although laboratory extracts of different litter types showed differences in DOM chemistry, percent hydrophobic DOM did not differ among soil solutions from different detrital treatments in the field, suggesting that microbial processing of DOM leachate in the field consumed easily degradable components, thus equalizing leachate chemistry among treatments. Total dissolved N leaching from plots with intact roots was very low (0.17 g m⁻² year⁻¹), slightly less than measured deposition to this very unpolluted forest (~s 0.2 g m⁻² year⁻¹). Total dissolved N losses showed significant increases in the two treatments without roots whereas concentrations of DOC decreased. In these plots, N losses were less than half of estimated plant uptake, suggesting that other mechanisms, such as increased microbial immobilization of N, accounted for retention of N in deep soils. In long-term laboratory incubations, soils from plots that had both above- and below-ground litter inputs excluded for 5 years showed a trend towards lower DOC loss rates, but not lower respiration rates. Soils from plots with added wood had similar respiration and DOC loss rates as control soils, suggesting that the additional DOC sorption observed in the field in these soils was stabilized in the soil and not readily lost upon incubation.     

Ionic mechanism for contractile response to hyposmotic challenge in canine basilar arteries

A hyposmotic challenge elicited contraction of isolated canine basilar arteries. The contractile response was nearly abolished by the removal of extracellular Ca²⁺ and by the voltage-dependent Ca²⁺ channel (VDCC) blocker nicardipine, but it was unaffected by thapsigargin, which depletes intracellular Ca²⁺ stores. The contraction was also inhibited by Gd³⁺ and ruthenium red, cation channel blockers, and Cl^– channel blockers DIDS and niflumic acid. The reduction of extracellular Cl^– concentrations enhanced the hypotonically induced contraction. Patch-clamp analysis showed that a hyposmotic challenge activated outwardly rectifying whole cell currents in isolated canine basilar artery myocytes. The reversal potential of the current was shifted toward negative potentials by reductions in intracellular Cl^– concentration, indicating that the currents were carried by Cl^–. Moreover, the currents were abolished by 10 mM BAPTA in the pipette solution and by the removal of extracellular Ca²⁺. Taken together, these results suggest that a hyposmotic challenge activates cation channels, which presumably cause Ca²⁺ influx, thereby activating Ca²⁺-activated Cl^– channels. The subsequent membrane depolarization is likely to increase Ca²⁺ influx through VDCC and elicit contraction. stretch-activated cation channels; Ca²⁺-activated Cl^– channels; voltage-dependent Ca²⁺ channels; large-conductance Ca²⁺-activated K⁺ channels; gadolinium     

sacB-5-Fluoroorotic acid-pyrE-based bidirectional selection for integration of unmarked alleles into the chromosome of Rhodobacter capsulatus

The gram-negative, purple nonsulfur, facultative photosynthetic bacterium Rhodobacter capsulatus is a widely used model organism and has well-developed molecular genetics. In particular, interposon mutagenesis using selectable gene cartridges is frequently employed for construction of a variety of chromosomal knockout mutants. However, as the gene cartridges are often derived from antibiotic resistance-conferring genes, their numbers are limited, which restricts the construction of multiple knockout mutants. In this report, sacB-5-fluoroorotic acid (5FOA)--pyrE-based bidirectional selection that facilitates construction of unmarked chromosomal knockout mutations is described. The R. capsulatus pyrE gene encoding orotate phosphoribosyl transferase, a key enzyme of the de novo pyrimidine nucleotide biosynthesis pathway, was used as an interposon in a genetic background that is auxotrophic for uracil (Ura-) and hence resistant to 5FOA (5FOA(r)). Although Ura+ selection readily yielded chromosomal allele replacements via homologous recombination, selection for 5FOA(r) to replace pyrE with unmarked alleles was inefficient. To improve the latter step, 5FOA(r) selection was combined with sucrose tolerance selection using a suicide plasmid carrying the Bacillus subtilis sacB gene encoding levansucrase that induces lethality upon exposure to 5% (wt/vol) sucrose in the growth medium. Sucrose-tolerant, 5FOA(r) colonies that were obtained carried chromosomal unmarked mutant alleles of the target gene via double crossovers between the resident pyrE-marked and incoming unmarked alleles. The effectiveness of this double selection was proven by seeking insertion and deletion alleles of helC involved in R. capsulatus cytochrome c biogenesis, which illustrated the usefulness of this system as a genetic means for facile construction of R. capsulatus unmarked chromosomal mutants.     

Nitrile pathway involving acyl-CoA synthetase: overall metabolic gene organization and purification and characterization of the enzyme

Two open reading frames (nhpS and acsA) were identified immediately downstream of the previously described Pseudomonas chlororaphis B23 nitrile hydratase (NHase) gene cluster (encoding aldoxime dehydratase, amidase, the two NHase subunits, and an uncharacterized protein). The amino acid sequence deduced from acsA shows similarity to that of acyl-CoA synthetase (AcsA). The acsA gene product expressed in Escherichia coli showed acyl-CoA synthetase activity toward butyric acid and CoA as substrates, with butyryl-CoA being synthesized. From the E. coli transformant, AcsA was purified to homogeneity and characterized. The quality of the recombinant protein was verified by the NH2-terminal amino acid sequence and the results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The apparent Km values for butyric acid, CoA, and ATP were 0.32 +/- 0.04, 0.37 +/- 0.02, and 0.22 +/- 0.02 mm, respectively. AcsA was shown to be a short-chain acyl-CoA synthetase, according to the catalytic efficiencies (kcat/Km) for various acids. The substrate specificity of AcsA was similar to those of aldoxime dehydratase, NHase, and amidase, the genes of which coexist in the same orientation in the gene cluster. P. chlororaphis B23 grew when cultured in a medium containing butyraldoxime as the sole carbon and nitrogen source. The activities of aldoxime dehydratase, NHase, and amidase were detected together with that of acyl-CoA synthetase under the culture conditions used. Moreover, on culture in a medium containing butyric acid as the sole carbon source, acyl-CoA synthetase activity was also detected. Together with the adjacent locations of the aldoxime dehydratase, NHase, amidase, and acyl-CoA synthetase genes, these findings suggest that the four enzymes are sequentially correlated with one another in vivo to utilize butyraldoxime as a carbon and nitrogen source. This is the first report of an overall "nitrile pathway" (aldoxime-->nitrile-->amide-->acid-->acyl-CoA) comprising these enzymes.     

Amino acid residues associated with cluster N3 in the NuoF subunit of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli

The NuoF subunit, which harbors NADH-binding site, of Escherichia coli NADH-quinone oxidoreductase (NDH-1) contains five conserved cysteine residues, four of which are predicted to ligate cluster N3. To determine this coordination, we overexpressed and purified the NuoF subunit and NuoF+E subcomplex in E. coli. We detected two distinct EPR spectra, arising from a [4Fe-4S] cluster (g(x,y,z)=1.90, 1.95, and 2.05) in NuoF, and a [2Fe-2S] cluster (g(x,y,z)=1.92, 1.95, and 2.01) in NuoE subunit. These clusters were assigned to clusters N3 and N1a, respectively. Based on the site-directed mutagenesis experiments, we identified that cluster N3 is ligated to the 351Cx2Cx2Cx40C398 motif.     

Uroplakin III, a novel Src substrate in Xenopus egg rafts, is a target for sperm protease essential for fertilization

In a previous study, we identified Xenopus egg uroplakin III (xUPIII), a single-transmembrane protein that localized to lipid/membrane rafts and was tyrosine-phosphorylated upon fertilization. An antibody against the xUPIII extracellular domain abolishes fertilization, suggesting that xUPIII acts not only as tyrosine kinase substrate but also as a receptor for sperm. Previously, it has been shown that the protease cathepsin B can promote a transient Ca2+ release and egg activation as seen in fertilized eggs (Mizote, A., Okamoto, S., Iwao, Y., 1999. Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization. Dev. Biol. 208, 79-92). Here, we show that activation of Xenopus eggs by cathepsin B is accompanied by tyrosine phosphorylation of egg-raft-associated Src, phospholipase Cgamma, and xUPIII. Cathepsin B also promotes a partial digestion of xUPIII both in vitro and in vivo. A synthetic xUPIII-GRR peptide, which contains a potential proteolytic site, inhibits the cathepsin-B-mediated proteolysis and tyrosine phosphorylation of xUPIII and egg activation. Importantly, this peptide also inhibits sperm-induced tyrosine phosphorylation of xUPIII and egg activation. Protease activity that digests xUPIII in an xUPIII-GRR peptide-sensitive manner is present in Xenopus sperm. Several protease inhibitors, which have been identified to be inhibitory toward Xenopus fertilization, are shown to inhibit sperm-induced tyrosine phosphorylation of xUPIII. Uroplakin Ib, a tetraspanin UP member, is found to be associated with xUPIII in egg rafts. Our results highlight novel mechanisms of fertilization signaling by which xUPIII serves as a potential target for sperm protease essential for fertilization.     

Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm

A cytosolic sperm protein(s), referred to as sperm factor (SF), is delivered into eggs by the sperm during mammalian fertilization to induce repetitive increases in the intracellular concentration of free Ca2+ ([Ca2+]i) that are referred to as [Ca2+]i oscillations. [Ca2+]i oscillations are essential for egg activation and early embryonic development. Recent evidence shows that the novel sperm-specific phospholipase C (PLC), PLCzeta, may be the long sought after [Ca2+]i oscillation-inducing SF. Here, we demonstrate the complete extraction of SF from porcine sperm and show that regardless of the method of extraction a single molecule/complex appears to be responsible for the [Ca2+]i oscillation-inducing activity of these extracts. Consistent with this notion, all sperm fractions that induced [Ca2+]i oscillations, including FPLC-purified fractions, exhibited high in vitro PLC activity at basal Ca2+ levels (0.1-5 microM), a hallmark of PLCzeta. Notably, we detected immunoreactive 72-kDa PLCzeta in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLCzeta. Nonetheless, in the latter fractions, proteolytic fragments, presumably corresponding to cleaved forms of PLCzeta, were detected by immunoblotting. Therefore, our findings corroborate the hypothesis that a sperm-specific PLC is the main component of the [Ca2+]i oscillation-inducing activity of sperm but provide evidence that the presence of 72-kDa PLCzeta does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions.     

X-ray spectroscopy of the Mn4Ca cluster in the water-oxidation complex of Photosystem II

The water-oxidation complex of Photosystem II (PS II) contains a heteronuclear cluster of 4 Mn atoms and a Ca atom. Ligands to the metal cluster involve bridging O atoms, and O and N atoms from amino acid side-chains of the D1 polypeptide of PS II, with likely additional contributions from water and CP43. Although moderate resolution X-ray diffraction-based structures of PS II have been reported recently, and the location of the Mn₄Ca cluster has been identified, the structures are not resolved at the atomic level. X-ray absorption (XAS), emission (XES), resonant inelastic X-ray scattering (RIXS) and extended X-ray absorption fine structure (EXAFS) provide independent and potentially highly accurate sources of structural and oxidation-state information. When combined with polarized X-ray studies of oriented membranes or single-crystals of PS II, a more detailed picture of the cluster and its disposition in PS II is obtained.