Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase

Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins. (C) 2010 Elsevier B.V. All rights reserved.

Biochemical and electron microscopic characterization of the F1F0 ATP Synthase from the hyperthermophilic eubacterium Aquifex aeolicus

The F1F0 ATP synthase has been purified from the hyperthermophilic eubacterium Aquifex aeolicus and characterized. Its subunits have been identified by MALDI-mass spectrometry through peptide mass fingerprinting and MS/MS. It contains the canonical subunits alpha, beta, gamma, delta and epsilon of F-1 and subunits a and c of F-0. Two versions of the b subunit were found, which show a low sequence homology to each other. Most likely they form a heterodimer. An electron microscopic single particle analysis revealed clear structural details, including two stalks connecting F-1 and F-0. In several orientations the central stalk appears to be tilted and/or kinked. It is unclear whether there is a direct connection between the peripheral stalk and the 6 subunit. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Biochemical compositions of two dominant bloom- forming species isolated from the Yangtze River Estuary in response to different nutrient conditions

Variations of cellular total lipid, total carbohydrate and total protein content of two dominant bloom-forming species (Skeletonema costatum and Prorocentrum donghaiense) isolated from the Yangtze River Estuary were examined under six different nutrient conditions in batch cultures. Daily samples were collected to estimate the cell growth, nutrient concentration and three biochemical compositions content during 7 days for S. costatum and the same sampling procedure was done every other day during 10 days for P. donghaiense. Results showed that for S. costatum, cellular total lipid content increased under phosphorus (P) limitation, but not for nitrogen (N) limitation; cellular carbohydrate were accumulated under both N and P limitation: cellular total protein content of low nutrient concentration treatments were significantly lower than that of high nutrient concentration treatments. For P. donghaiense, both cellular total lipid content and total carbohydrate content were greatly elevated as a result of N and P exhaustion, but cellular total protein content had no significant changes under nutrient limitation. In addition, the capability of accumulation of three biochemical constituents of P. donghaiense was much stronger than that of S. costatum. Pearson correlation showed that for both species, the biochemical composition of three constituents (lipid, carbohydrate and protein) had no significant relationship with extracellular N concentration, but had positive correlation with extracellular and intracellular P concentration. The capability of two species to accumulate cellular total lipid and carbohydrate under nutrient limitation may help them accommodate the fluctuating nutrient condition of the Yangtze River Estuary. The different responses of two species of cellular biochemical compositions content under different nutrient conditions may provide some evidence to explain the temporal characteristic of blooms Caused by two species in the Yangtze River Estuary. (C) 2008 Elsevier B.V. All rights reserved.

Variability in radiocarbon ages of biochemical compound classes of high molecular weight dissolved organic matter in estuaries

High molecular weight dissolved organic matter (HMW-DOM, > 1000 Da) represents a major fraction (> 30%) of dissolved organic carbon (DOC) in the ocean and thus plays an important role in the global biogeochemical cycling of carbon and many other elements. Its organic sources and formation mechanisms, however, are still not well understood especially in estuarine and coastal regions where multiple natural and anthropogenic sources contribute to total HMW-DOM. In this paper we report our measurements of natural radiocarbon (C-14) abundances and stable carbon isotope (C-13) compositions of the major biochemical compound classes: amino acids, carbohydrates and lipids separated from eight HMW-DOM samples collected from five US estuaries as part of our on-going study of sources, distribution and transport of chromophoric dissolved organic matter (CDOM) in estuarine and coastal waters. Distinct differences in both C-14 and C-13 values were found among the bulk HMW-DOM samples as well as the individual compound classes. Radiocarbon ages of the major compound classes varied by as much as 27,000 years in a single sample. The calculated average radiocarbon ages of the compound fractions of HMW-DOM indicate that the total lipid fraction is very "old", while the acid-insoluble fraction is slightly younger. Total amino acid and carbohydrate fractions, however, have relatively modern apparent C-14 ages. The significant variability in C-14 ages among the compound classes indicates not only multiple organic carbon sources but also different formation and turnover pathways controlling the cycling of different biochemical components of HMW-DOM in estuarine and coastal waters. (c) 2006 Elsevier Ltd. All rights reserved.

Identification of quantitative trait loci for growth-related traits in the Pacific abalone Haliotis discus hannai Ino

The locations and effects of quantitative trait loci (QTL) were estimated for nine characters for growth-related traits in the Pacific abalone (Haliotis discus hannai Ino) using a randomly amplified polymorphic DNA (RAPD), amplification fragment length polymorphism (AFLP) and SSR genetic linkage map. Twenty-eight putatively significant QTLs (LOD > 2.4) were detected for nine traits (shell length, shell width, total weight, shell weight, weight of soft part, muscle weight, gonad and digestive gland weight, mantle weight and gill weight). The percentage of phenotypic variation explained by a single QTL ranged from 8.0% to 35.9%. The significant correlations (P < 0.001) were found among all the growth-related traits, and Pearson's correlation coefficients were more than 0.81. For the female map, the QTL for growth were concentrated on groups 1 and 4 linkage maps. On the male map, the QTL that influenced growth-related traits gathered on the groups 1 and 9 linkage maps. Genetic linkage map construction and QTL analysis for growth-related traits are the basis for the marker-assisted selection and will eventually improve production and quality of the Pacific abalone.

Genetic parameter estimates for growth traits at early stage of Pacific abalone, Haliotis discus hannai Ino

Heritability and genetic and phenotypic correlations were estimated for juvenile growth traits of Pacific abalone Haliotis discus hannai Ino. The estimates were calculated from shell length and shell width measurements on progeny resulting from 12 half-sib families and 36 full-sib families obtained using artificial fertilization of mating three females to each male. The measurements were taken at 10, 20 and 30 d after fertilization. It was found that heritability estimates based on sire component ranged from 0.23 to 0.36 for shell length and 0.21 to 0.32 for shell width. Heritability estimates from dam component were larger than those from sire component at three ages, indicating presence of maternal effects, non-additive genetic effects and common environmental effects. Phenotypic correlations were significant at three ages (P < 0.05), with values of 0.92, 0.93 and 0.92, respectively. Genetic correlations from the paternal half-sib correlation analysis were highly positive at three ages, with values of 0.50, 0.78 and 0.81, respectively. The results suggest that selective breeding is an effective approach to improving growth traits of Pacific abalone stocks.

Inbreeding depression for various traits in two cultured populations of the American bay scallop, Argopecten irradians irradians Lamarck (1819) introduced into China

This paper examines the effect of inbreeding level of population on the magnitude of inbreeding depression expressed by comparing them between two cultured populations (A and B) in the hermaphroditic animal of the bay scallop Argopecten irradians irradians. Population A is expected to have less genetic variations and higher inbreeding level due to longer cultured history (20 generations) and less "ancestral" individuals (26 individuals) than population B due to shorter cultured history (4 generations) and more "ancestral" individuals (406 individuals). Two groups within each population were produced, one using self-fertilization and one using mass-mating within the same population. Selfed offspring (AS and BS) from two populations both had lower fitness components than their mass-mated counterparts (AM and BM) and exhibited inbreeding depression for all examined traits, e.g. lower hatching, less viability and slower growth, indicating that inbreeding depression is a common feature in this animal. Fitness components in all traits of offspring from population A significantly differed those from population B and the magnitude of inbreeding depression for all traits in population A with higher inbreeding level was significantly smaller than that in population B with lower inbreeding level, indicating that both fitness components and magnitude of inbreeding depression were significantly affected by inbreeding level of populations and genetic load harbored in population A may be partially purged through inbreeding. Moreover, the magnitude of inbreeding depression in the two populations both varied among traits and life history stages. The present results support the partial-dominance hypothesis of inbreeding depression. (C) 2008 Elsevier B.V. All rights reserved.

Decline in medicinal and forage species with warming is mediated by plant traits on the Tibetan Plateau

Experimental studies of how global changes and human activities affect plant diversity often focus on broad measures of diversity and discuss the implications of these changes for ecosystem function. We examined how experimental warming and grazing affected species within plant groups of direct importance to Tibetan pastoralists: medicinal plants used by humans and palatable plants consumed by livestock. Warming resulted in species losses from both the medicinal and palatable plant groups; however, differential relative vulnerability to warming occurred. With respect to the percent of warming-induced species losses, the overall plant community lost 27%, medicinal plants lost 21%, and non-medicinal plants lost 40% of species. Losses of palatable and non-palatable species were similar to losses in the overall plant community. The deep-rootedness of medicinal plants resulted in lowered sensitivity to warming, whereas the shallow-rootedness of non-medicinal plants resulted in greater sensitivity to warming; the variable rooting depth of palatable and non-palatable plants resulted in an intermediate response to warming. Predicting the vulnerability of plant groups to human activities can be enhanced by knowledge of plant traits, their response to specific drivers, and their distribution within plant groups. Knowledge of the mechanisms through which a driver operates, and the evolutionary interaction of plants with that driver, will aid predictions. Future steps to protect ecosystem services furnished by medicinal and palatable plants will be required under the novel stress of a warmer climate. Grazing may be an important tool in maintaining some of these services under future warming.

Molecular cloning, expression and biochemical property analysis of AtKP1, a kinesin gene from Arabidopsis thaliana

Kinesins are common in a variety of eukaryotic cells with diverse functions. A cDNA encoding a member of the Kinesin-14B subfamily is obtained using X-RACE technology and named AtKP1 (for Arabidopsis kinesin protein 1). This cDNA has a maximum open reading frame of 3.3 kb encoding a polypeptide of 1087 aa. Protein domain analysis shows that AtKP1 contains the motor domain and the calponin homology domain in the central and amino-terminal regions, respectively. The carboxyl-terminal region with 202 aa residues is diverse from other known kinesins. Northern blot analysis shows that AtKP1 is widely expressed at a higher level in seedlings than in mature plants. 2808 bp of the AtKP1 promoter region is cloned and fused to GUS. GUS expression driven by the AtKP1 promoter region shows that AtKP1 is mainly expressed in vasculature of young organs and young leaf trichomes, indicating that AtKP1 may participate in the differentiation or development of Arabidopsis thaliana vascular bundles and trichomes. A truncated AtKP1 protein containing the putative motor domain is expressed in E. coli and affinity-purified. In vitro characterizations indicate that the polypeptide has nucleotide-dependent microtubule-binding ability and microtubule-stimulated ATPase activity.

Capacity of soil to protect organic carbon and biochemical characteristics of density fractions in Ziwulin Haplic Greyxems soil

Physical protection is one of the important ways to stabilize organic carbon in soils. In order to understand the role of soils as a carbon sink or source in global climatic change and carbon cycles and properly manage soils as a carbon sink, we ought to know how many organic carbon (OC) in a given soil could be protected. By a density fractionation approach and ultrasonic technique, each soil sample was divided into three fractions: free light fraction (free-LF), occluded fraction (occluded-LF) and heavy fraction (HF). The obtained fractions were analyzed for total OC content, carbohydrate content and recalcitrant OC content. The results showed: (i) In the whole soil profile, dominance of OC consistently decreased in the following order: HF, free-LF, occluded-LF. This suggested that OC in soils were mostly protected. From 0-10 to 60-80 cm horizons, the OC in free-LF decreased from 25.27% to 3.72%, while OC in HF they were increased from 72.57% to 95.39%. The OC in occluded-LF was between 2.16% and 0.89%. (ii) Organic carbon recalcitrance in free-LF was similar to that in HF, and was even higher than that in HF below the surface horizon. This suggested that free-LF was not always the most fresh and non-decomposed fraction. OM quality of HF was higher than that of free-LF in the surface 10 cm below, namely the protected OM had higher quality than free OM in these horizons.