Expression, purification and secondary structure analysis of Saccharomyces cerevisiae vacuolar membrane H+-ATPase subunit F (Vma7p).

The vacuolar H+-ATPase is an acid pump found in virtually all eukaryotic cells. It shares a common macromolecular organization with the F1F0-ATPase, and some V-ATPase subunits are structural and functional homologues of F-ATPase components. However, the vacuolar complex contains several subunits which do not resemble F-ATPase subunits at the sequence level, and which currently have no specific function assigned. One example is subunit F, the Vma7p polypeptide of Saccharomyces cerevisiae. A recombinant form of Vma7p was expressed in Escherichia coli and purified to homogeneity. Mass spectroscopy confirmed a mass of 13460 Da for Vma7p, and dynamic light scattering showed that the polypeptide was globular and monodisperse even at high concentrations. Analysis of secondary structure by circular dichroism and FTIR showed that Vma7p comprises 30% alpha-helix and 32-42% beta-sheet. The protein fold recognition programme 'Threader 2' produced highly significant matches between Vma7p and five alpha-beta sandwich folds. Relative proportions of secondary structure elements within these folds were broadly consistent with the spectroscopic data. Although Vma7p does not share sequence similarity with the F-ATPase epsilon subunit, the analysis suggests that the polypeptides not only have similar masses and assemble into homologous core complexes, but also share similar secondary structures. It is possible that the two polypeptides are homologous and perform similar functions within their respective ATPases. The production of high yields of homogeneous, folded, monodisperse protein will facilitate high resolution crystallography and NMR spectroscopy studies.     

Late winter-to-summer change in ocean acidification state in Kongsfjorden,with implications for calcifying organisms

Late winter-to-summer changes (April to July) in ocean acidification state, calcium carbonate (CaCO₃) saturation for aragonite (Ω _a) and calcite (Ω _c) and biogeochemical properties were investigated in 2013 and 2014 in Kongsfjorden, Svalbard. We investigated physical (salinity, temperature) and chemical (carbonate system, nutrient) properties in the water column from the glacier front in the fjord to the west Spitsbergen shelf. The average range of Ω _a in the upper 50 m in the fjord in winter was 1.59–1.74 and in summer 1.65–2.66. The lowest Ω _a (1.5) was close to the reported critical threshold for aragonite-forming organisms such as the pteropod Limacina helicina. In summer 2013, Ω _a, pH_T and salinity were generally lower than in 2014 as a result of a larger influence of high-CO₂ water from the coastal current and less Atlantic water. The inner fjord was influenced by glacial water in summer which decreased Ω _a by 0.7. Biological CO₂ consumption based on a winter-to summer decrease in nitrate was larger in 2014 than in 2013, suggesting more primary production in 2014. The influence of freshwater decreased Ω _a by the same amount as the biological effect increased Ω _a. The seasonal increase in temperature only played a minor role on the increase of Ω _a. The biological effect showed more inter-annual variability than the effect of freshwater. Based on this study, we suggest that changes in the inflow of different water masses and freshwater directly influence ocean acidification state, but also indirectly affect the biological drivers of carbonate chemistry in the fjord.     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-V-ATPase

Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an alpha-helical conformation for peptide MTM7 and in DMSO three alpha-helical regions are identified by 2D 1H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an alpha-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Effects on phytoplankton of nutrients added in conjunction with acidification

1. The effects of nitric acidification on phytoplankton were studied in a small, eperimentally manipulated, oligotrophic lake (L302N) in the Eperimental Lakes Area of Canada. The focus was altered after 9 years of acidification to investigate the possibility of using nutrient additions to stimulate recovery, followed by a controlled incremental recovery, in which the pH was increased to a predetermined target level. 2. Five years of additions of HNO₃ to L302N reduced its pH from 6.5 to 6.1. Nitrate concentration increased because the algal community was severely P deficient. The phytoplankton community structure and productivity were not significantly affected by these additions. 3. The phytoplankton community was significantly affected when pH was subsequently decreased over three successive years from 6.1 to 5.1 by the addition of HCl. Dominance shifted from chrysophytes to a co-dominance of chlorophytes and dinoflagellates, which altered the size structure of the community. Species diversity significantly decreased, although phytoplankton productivity remained unchanged. 4. At pH 5.1 nitrate and sulphate additions were made, creating conditions like those in lakes in eastern North America, which receive high loadings of nitrogen from the atmosphere. The phytoplankton assemblage shifted to dominance by small coccoidal chlorophytes. However, biomass and productivity were unaffected. 5. Finally, phosphate, as phosphoric acid, was added, along with nitrate and sulphate, to the epilimnion, which stimulated internal alkalinity generation and productivity. It is concluded that CO₂ concentrations and the form of N (nitrate vs. ammonia) affect algal composition but that P determines algal biomass and productivity. Chlorophytes were found to be good competitors for P when N and CO₂ were high; it is epected that cyanobacteria would be more competitive for P in low CO₂ systems. Conversely, dinoflagellates are most competitive in systems with low pH and high P, such as that which occurred in L302N. Although the P additions reduced N concentrations and created alkalinity, this is not a recommended remedial procedure in acidified lakes because it enhanced dinoflagellate abundance, which has been associated with fish kills. 6. When all additions ceased, the pH of L302N recovered from 5.1 to 5.8, chrysophytes and chlorophytes became more abundant and dinoflagellates decreased in abundance. Phytoplankton biomass decreased and species diversity increased. Phytoplankton productivity remained unchanged     

Influence of Three Contrasting Detrital Carbon Sources on Planktonic Bacterial Metabolism in a Mesotrophic Lake

Abstract Lakes receive organic carbon from a diversity of sources which vary in their contribution to planktonic microbial food webs. We conducted a mesocosm study to test the effects of three different detrital carbon sources (algae, aquatic macrophytes, terrestrial leaves) on several measures of microbial metabolism in a small meso-eutrophic lake (DOC ≈ 5 mg/L). Small DOC additions (ΔC < 1 mg/L) affected bacterial numbers, growth, and pathways of carbon acquisition. Macrophyte and leaf detritus significantly increased TDP and color, but bacterial densities initially (+12 h) were unaffected. After 168 h, densities in systems amended with terrestrial detritus were 60% less than in controls, while production rates in mesocosms with macrophyte detritus were 4-fold greater. Detritus treatments resulted in greater per-cell production rates either through stable cell numbers and greater growth rates (macrophyte-C) or lower densities with stable production rates (terrestrial-C). After only 12 h, rates of leucine aminopeptidase (LAPase) activity were 2.5× greater in macrophyte-C systems than in controls, but LAPase and β-N-acetylglucosamindase activities in systems amended with terrestrial-C were only 50% of rates in controls. After 168 h, β-xylosidase rates were significantly greater in communities with terrestrial and phytoplankton detritus. Microbial utilization of >20% of 102 carbon sources tested were affected by at least one detritus addition. Macrophyte-C had positive (6% of substrates) and negative (14%) effects on substrate use; terrestrial detritus had mainly positive effects. An ordination based on carbon-use profiles (+12 h) revealed a cluster of macrophyte-amended communities with greater use of psicose, lactulose, and succinamic acid; controls and algal-detritus systems were more effective in metabolizing two common sugars and cellobiose. After 168 h, communities receiving terrestrial detritus were most tightly clustered, exhibiting greater use of raffinose, pyroglutamic acid, and sebacic acid. Results suggest that pelagic bacterial communities respond to changes in organic carbon source rapidly and by different routes, including shifts in per-cell production rates and variations in degradation of a variety of compounds comprising the DOC pool. Received: 5 June 1998; Accepted: 24 August 1998     

The DIF1 gene of Arabidopsis is required for meiotic chromosome segregation and belongs to the REC8/RAD21 cohesin gene family

Cohesins are a group of conserved proteins responsible for cohesion between replicated sister chromatids during mitosis and meiosis and which are implicated in double-strand break repair and meiotic recombination. We describe here the identification and characterisation of an Arabidopsis gene - DETERMINATE, INFERTILE1 (DIF1), which is a homolog of the Schizosaccharomyces pombe REC8/RAD21 cohesin genes, and is essential for meiotic chromosome segregation. Five independent alleles of the DIF1 gene were isolated by transposon mutagenesis, and the mutants show complete male and female sterility. Pollen mother cells (PMCs) of dif1 mutants show multiple meiotic defects which are represented by univalent chromosomes and chromosome fragmentation at metaphase I, and acentric fragments and chromatin bridges in meiosis I and II. Consequently, chromosome segregation is strongly affected, resulting in meiotic products of uneven size, shape and of variable ploidy. The similarities in phenotype, and the sequence homology between DIF1 and the REC8/RAD21 cohesins suggests that cohesin function is largely conserved between eukaryotes and highlights the essential role cohesins play in plant meiosis.     

Function and redundancy of the chaplin cell surface proteins in aerial hypha formation, rodlet assembly, and viability in Streptomyces coelicolor

The chaplins are a family of eight secreted proteins that are critical for raising aerial hyphae in Streptomyces coelicolor. These eight chaplins can be separated into two main groups: the long chaplins (ChpA to -C) and the short chaplins (ChpD to -H). The short chaplins can be further subdivided on the basis of their abilities to form intramolecular disulfide bonds: ChpD, -F, -G, and -H contain two Cys residues, while ChpE has none. A "minimal chaplin strain" containing only chpC, chpE, and chpH was constructed and was found to raise a substantial aerial mycelium. This strain was used to examine the roles of specific chaplins. Within this strain, the Cys-containing ChpH was identified as the major polymerization unit contributing to aerial hypha formation and assembly of an intricate rodlet ultrastructure on the aerial surfaces, and the two Cys residues were determined to be critical for its function. ChpC augmented aerial hypha formation and rodlet assembly, likely by anchoring the short chaplins to the cell surface, while ChpE was essential for the viability of wild-type S. coelicolor. Interestingly, the lethal effects of a chpE null mutation could be suppressed by the loss of the other chaplins, the inactivation of the twin arginine translocation (Tat) secretion pathway, or the loss of the rodlins.