Phage Diversity in a Methanogenic Digester

It has been shown that phages are present in natural and engineered ecosystems and influence the structure and performance of prokaryotic communities. However, little has been known about phages occurring in anaerobic ecosystems, including those in methanogenic digesters for waste treatment. This study investigated phages produced in an upflow anaerobic sludge blanket methanogenic digester treating brewery wastes. Phage-like particles (PLPs) in the influent and effluent of the digester were concentrated and purified by sequential filtration and quantified and characterized by transmission electron microscopy (TEM), fluorescence assay, and field inversion gel electrophoresis (FIGE). Results indicate that numbers of PLPs in the effluent of the digester exceeded 1 × 10⁹ L⁻¹ and at least 10 times greater than those in the influent, suggesting that substantial amounts of PLPs were produced in the digester. A production rate of the PLPs was estimated at least 5.2 × 10⁷ PLPs day⁻¹ L⁻¹. TEM and FIGE showed that a variety of phages were produced in the digester, including those affiliated with Siphoviridae, Myoviridae, and Cystoviridae.     

海南植物区系的多样性

记载海南维管束植物的多样性,表明蕨类植物区系已有古生代和中生代的孑遗,又有白垩纪以来的现代蕨类,它们都存在着许多特有种;裸子植物则以泛热带成分及华夏成分为主;有花植物包括全球植物区系8个植物区的成分,而以热带成分及全球性分布成分最多。最后,文章分析了海南植物区系多样性的地史背景及自然条件的因素。     

海南植物区系的多样性

记载海南维管束植物的多样性,表明蕨类植物区系已有古生代和中生代的孑遗,又有白垩纪以来的现代蕨类,它们都存在着许多特有种;裸子植物则以泛热带成分及华夏成分为主;有花植物包括全球植物区系8个植物区的成分,而以热带成分全球性分布成分最多。最后,文章分析了海南植物区系多样性的地史背景及自然条件的因素。     

Diversity and Population Dynamics of Methanogenic Bacteria in a Granular Consortium

Upflow anaerobic sludge blanket bioreactor granules were used as an experimental model microbial consortium to study the dynamics and distribution of methanogens. Immunologic methods revealed a considerable diversity of methanogens that was greater in mesophilic granules than in the same granules 4 months after a temperature shift from 38 to 55°C. During this period, the sizes of the methanogenic subpopulations changed with distinctive profiles after the initial reduction caused by the shift. Methanogens antigenically related to Methanobrevibacter smithii PS and ALI, Methanobacterium hungatei JF1, and Methanosarcina thermophila TM1 increased rapidly, reached a short plateau, and then fell to lower concentrations that persisted for the duration of the experiment. A methanogen related to Methanogenium cariaci JR1 followed a similar profile at the beginning, but it soon diminished below detection levels. Methanothrix rods weakly related to the strain Opfikon increased rapidly, reaching a high-level, long-lasting plateau. Two methanogens related to Methanobrevibacter arboriphilus AZ and Methanobacterium thermoautotrophicum ΔH emerged from very low levels before the temperature shift and multiplied to attain their highest numbers 4 months after the shift. Histochemistry and immunohistochemistry revealed thick layers, globular clusters, and lawns of variable density which were distinctive of the methanogens related to M. thermoautotrophicum ΔH, M. thermophila TM1, and M. arboriphilus AZ and M. soehngenii Opfikon, respectively, in thin sections of granules grown at 55°C for 4 months. Mesophilic granules showed a different pattern of methanogenic subpopulations.     

Spike-Triggered Covariance Analysis Reveals Phenomenological Diversity of Contrast Adaptation in the Retina

When visual contrast changes, retinal ganglion cells adapt by adjusting their sensitivity as well as their temporal filtering characteristics. The latter has classically been described by contrast-induced gain changes that depend on temporal frequency. Here, we explored a new perspective on contrast-induced changes in temporal filtering by using spike-triggered covariance analysis to extract multiple parallel temporal filters for individual ganglion cells. Based on multielectrode-array recordings from ganglion cells in the isolated salamander retina, we found that contrast adaptation of temporal filtering can largely be captured by contrast-invariant sets of filters with contrast-dependent weights. Moreover, differences among the ganglion cells in the filter sets and their contrast-dependent contributions allowed us to phenomenologically distinguish three types of filter changes. The first type is characterized by newly emerging features at higher contrast, which can be reproduced by computational models that contain response-triggered gain-control mechanisms. The second type follows from stronger adaptation in the Off pathway as compared to the On pathway in On-Off-type ganglion cells. Finally, we found that, in a subset of neurons, contrast-induced filter changes are governed by particularly strong spike-timing dynamics, in particular by pronounced stimulus-dependent latency shifts that can be observed in these cells. Together, our results show that the contrast dependence of temporal filtering in retinal ganglion cells has a multifaceted phenomenology and that a multi-filter analysis can provide a useful basis for capturing the underlying signal-processing dynamics.     

Examination of Food Chain-Derived Listeria monocytogenes Strains of Different Serotypes Reveals Considerable Diversity in inlA Genotypes,Mutability, and Adaptation to Cold Temperatures

Listeria monocytogenes strains belonging to serotypes 1/2a and 4b are frequently linked to listeriosis. While inlA mutations leading to premature stop codons (PMSCs) and attenuated virulence are common in 1/2a, they are rare in serotype 4b. We observed PMSCs in 35% of L. monocytogenes isolates (n = 54) recovered from the British Columbia food supply, including serotypes 1/2a (30%), 1/2c (100%), and 3a (100%), and a 3-codon deletion (amino acid positions 738 to 740) seen in 57% of 4b isolates from fish-processing facilities. Caco-2 invasion assays showed that two isolates with the deletion were significantly more invasive than EGD-SmR (P < 0.0001) and were either as (FF19-1) or more (FE13-1) invasive than a clinical control strain (08-5578) (P = 0.006). To examine whether serotype 1/2a was more likely to acquire mutations than other serotypes, strains were plated on agar with rifampin, revealing 4b isolates to be significantly more mutable than 1/2a, 1/2c, and 3a serotypes (P = 0.0002). We also examined the ability of 33 strains to adapt to cold temperature following a downshift from 37°C to 4°C. Overall, three distinct cold-adapting groups (CAG) were observed: 46% were fast (<70 h), 39% were intermediate (70 to 200 h), and 15% were slow (>200 h) adaptors. Intermediate CAG strains (70%) more frequently possessed inlA PMSCs than did fast (20%) and slow (10%) CAGs; in contrast, 87% of fast adaptors lacked inlA PMSCs. In conclusion, we report food chain-derived 1/2a and 4b serotypes with a 3-codon deletion possessing invasive behavior and the novel association of inlA genotypes encoding a full-length InlA with fast cold-adaptation phenotypes.     

Evidence of Increased Diversity of Methanogenic Archaea with Plant Extract Supplementation

This study evaluated the effects of selected essential oils on archaeal communities using the ovine rumen model. Forty weaned Canadian Arcott ewes, fed with barley-based diet, were allotted to one of three essential oil supplementation treatments or a control (10 ewes per treatment) for 13 weeks. The treatments were cinnamaldehyde, garlic oil, juniper berry oil, and a control with no additive. Rumen content was sampled after slaughter and grouped by treatment by combining subsamples from each animal. DNA was extracted from the pooled samples and analyzed for methanogenic archaea using quantitative polymerase chain reaction, denaturing gradient gel electrophoresis, cloning, and sequencing. Our results suggest that the total copy number of archaeal 16S rRNA was not significantly affected by the treatments. The phylogenetic analysis indicated a trend toward an increased diversity of methanogenic archaea related to Methanosphaera stadtmanae, Methanobrevibacter smithii, and some uncultured groups with cinnamaldehyde, garlic, and juniper berry oil supplementation. The trends in the diversity of methanogenic archaea observed with the essential oil supplementation may have resulted from changes in associated protozoal species. Supplementation of ruminant diets with essential oils may alter the diversity of rumen methanogens without affecting the methanogenic capacity of the rumen.     

Next Generation Sequencing Reveals the Hidden Diversity of Zooplankton Assemblages

Background

Zooplankton play an important role in our oceans, in biogeochemical cycling and providing a food source for commercially important fish larvae. However, difficulties in correctly identifying zooplankton hinder our understanding of their roles in marine ecosystem functioning, and can prevent detection of long term changes in their community structure. The advent of massively parallel next generation sequencing technology allows DNA sequence data to be recovered directly from whole community samples. Here we assess the ability of such sequencing to quantify richness and diversity of a mixed zooplankton assemblage from a productive time series site in the Western English Channel.

Methodology/Principle Findings

Plankton net hauls (200 µm) were taken at the Western Channel Observatory station L4 in September 2010 and January 2011. These samples were analysed by microscopy and metagenetic analysis of the 18S nuclear small subunit ribosomal RNA gene using the 454 pyrosequencing platform. Following quality control a total of 419,041 sequences were obtained for all samples. The sequences clustered into 205 operational taxonomic units using a 97% similarity cut-off. Allocation of taxonomy by comparison with the National Centre for Biotechnology Information database identified 135 OTUs to species level, 11 to genus level and 1 to order, <2.5% of sequences were classified as unknowns. By comparison a skilled microscopic analyst was able to routinely enumerate only 58 taxonomic groups.

Conclusions

Metagenetics reveals a previously hidden taxonomic richness, especially for Copepoda and hard-to-identify meroplankton such as Bivalvia, Gastropoda and Polychaeta. It also reveals rare species and parasites. We conclude that Next Generation Sequencing of 18S amplicons is a powerful tool for elucidating the true diversity and species richness of zooplankton communities. While this approach allows for broad diversity assessments of plankton it may become increasingly attractive in future if sequence reference libraries of accurately identified individuals are better populated.     

内蒙古白粉菌区系多样性

为阐明内蒙古白粉菌区系组成特点、探讨其起源和演化,对其区系多样性进行了分析.区系中含有10种以上的属为自粉菌属(Erysiphe)、单囊白粉菌属(Podosphaera)和高氏白粉菌属(Golovinomyces),3属共104种,占总种数的84.55%.根据地理分布资料,内蒙古白粉菌可划分为世界广布种(13.82%)...     

大兴安岭南部山地苔藓植物区系多样性研究

于2009～2012年对大兴安岭南部山地6个自然保护区的苔藓植物进行了野外实地调查,对所采集的1 800余号标本进行室内分析鉴定,并计算分析其种类组成、区系地理成分、地区之间相似性系数和相异系数的欧氏距离比较,及其8种生境中苔藓植物多样性指数、丰富度指数、均匀度指数和优势度指数等,为保护区的管理和建设提供基础资料。结果表明:(1)大兴安岭南部山地6个自然保护区共有苔藓植物48科,134属,330种,其中苔类16科,21属,38种,1变种;藓类32科,113属,274种,14变种,2变型,1亚种。(2)首次发现中国新记录4种:长柄紫萼藓(Grimmia longirostris Hook.),毛尖连轴藓[Schistidium lancifolium(Kindb.)Blom.],平叶真藓(Bryumlaevifilum Syed.),凹叶毛灰藓[Homomallium adnatum(Hedw.)Broth.],首次发现本地区新记录12种。(3)以丛藓科为代表的旱生藓类在本区占有优势地位;区系地理成分以温带成分(60.79%)和东亚成分(16.19%)为主。(4)大兴安岭南部山地与兴安北部和燕山北部山地亲缘关系较近;生境条件的变化对苔藓植物多样性产生了一定的影响,湿润土生和石生生境为本区苔藓植物最丰富的生境,水生生境中苔藓植物分布最少。研究发现,大兴安岭南部山地苔藓植物区系是连接华北区、东北区、蒙新区和阴山地区的过渡性地带;与燕山北部和兴安北部相比,大兴安岭南部山地大面积苔藓植物地被层分布较少的主要原因是森林密度和湿度较低。因此,建立保护区对保护大兴安岭南部山地脆弱的森林、草原过渡生态系统,保护中国野生动植物类群及其他珍稀动植物的繁衍均具有十分重要的意义。     

A Genome-Wide Analysis of Populations from European Russia Reveals a New Pole of Genetic Diversity in Northern Europe

Several studies examined the fine-scale structure of human genetic variation in Europe. However, the European sets analyzed represent mainly northern, western, central, and southern Europe. Here, we report an analysis of approximately 166,000 single nucleotide polymorphisms in populations from eastern (northeastern) Europe: four Russian populations from European Russia, and three populations from the northernmost Finno-Ugric ethnicities (Veps and two contrast groups of Komi people). These were compared with several reference European samples, including Finns, Estonians, Latvians, Poles, Czechs, Germans, and Italians. The results obtained demonstrated genetic heterogeneity of populations living in the region studied. Russians from the central part of European Russia (Tver, Murom, and Kursk) exhibited similarities with populations from central–eastern Europe, and were distant from Russian sample from the northern Russia (Mezen district, Archangelsk region). Komi samples, especially Izhemski Komi, were significantly different from all other populations studied. These can be considered as a second pole of genetic diversity in northern Europe (in addition to the pole, occupied by Finns), as they had a distinct ancestry component. Russians from Mezen and the Finnic-speaking Veps were positioned between the two poles, but differed from each other in the proportions of Komi and Finnic ancestries. In general, our data provides a more complete genetic map of Europe accounting for the diversity in its most eastern (northeastern) populations.     

Multilocus Sequence Analysis of Nectar Pseudomonads Reveals High Genetic Diversity and Contrasting Recombination Patterns

The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas ‘sensu stricto’ isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria.     

Pathways for Methanogenesis and Diversity of Methanogenic Archaea in Three Boreal Peatland Ecosystems

The main objectives of this study were to uncover the pathways used for methanogenesis in three different boreal peatland ecosystems and to describe the methanogenic populations involved. The mesotrophic fen had the lowest proportion of CH₄ produced from H₂-CO₂. The oligotrophic fen was the most hydrogenotrophic, followed by the ombrotrophic bog. Each site was characterized by a specific group of methanogenic sequences belonging to Methanosaeta spp. (mesotrophic fen), rice cluster-I (oligotrophic fen), and fen cluster (ombrotrophic bog).     

Quantitative Phosphoproteomics Analysis Reveals Broad Regulatory Role of Heparan Sulfate on Endothelial Signaling

Heparan sulfate (HS) is a linear, abundant, highly sulfated polysaccharide that expresses in the vasculature. Recent genetic studies documented that HS critically modulates various endothelial cell functions. However, elucidation of the underlying molecular mechanism has been challenging because of the presence of a large number of HS-binding ligands found in the examined experimental conditions. In this report, we used quantitative phosphoproteomics to examine the global HS-dependent signaling by comparing wild type and HS-deficient endothelial cells that were cultured in a serum-containing medium. A total of 7222 phosphopeptides, corresponding to 1179 proteins, were identified. Functional correlation analysis identified 25 HS-dependent functional networks, and the top five are related to cell morphology, cellular assembly and organization, cellular function and maintenance, cell-to-cell communication, inflammatory response and disorder, cell growth and proliferation, cell movement, and cellular survival and death. This is consistent with cell function studies showing that HS deficiency altered endothelial cell growth and mobility. Mining for the underlying molecular mechanisms further revealed that HS modulates signaling pathways critically related to cell adhesion, migration, and coagulation, including ILK, integrin, actin cytoskeleton organization, tight junction and thrombin signaling. Intriguingly, this analysis unexpectedly determined that the top HS-dependent signaling is the IGF-1 signaling pathway, which has not been known to be modulated by HS. In-depth analysis of growth factor signaling identified 22 HS-dependent growth factor/cytokine/growth hormone signaling pathways, including those both previously known, such as HGF and VEGF, and those unknown, such as IGF-1, erythropoietin, angiopoietin/Tie, IL-17A and growth hormones. Twelve of the identified 22 growth factor/cytokine/growth hormone signaling pathways, including IGF-1 and angiopoietin/Tie signaling, were alternatively confirmed in phospho-receptor tyrosine kinase array analysis. In summary, our SILAC-based quantitative phosphoproteomic analysis confirmed previous findings and also uncovered novel HS-dependent functional networks and signaling, revealing a much broader regulatory role of HS on endothelial signaling.Heparan sulfate (HS) is a linear, highly sulfated polysaccharide composed of glucosamine and hexauronic acid disaccharide repeating units (1). HS covalently attaches to core proteins to form HS proteoglycans (HSPG). Dictated by the location of the core proteins, HS chains present on cell surfaces, such as linking to syndecans and glypicans, and in the basement membrane by attaching to perlecan and agrin (1–3). HS biosynthesis is initiated by heterodimers formed by copolymerases Exostosin-1 (Ext1) and Exostosin-2 (Ext2) that elongate HS chains by alternatively adding glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) residues from their respective UDP-sugar nucleotide precursors. N-deacetylase/N-sulfotransferase (Ndst) initiates modification reactions that occur on newly assembled HS chains, including N-, 3-O, and 6-O sulfation of GlcNAc units (NS, 3S, 6S, respectively), epimerization of GlcA to iduronic acid (IdoA), and 2-O-sulfation of IdoA (2S). These modification reactions are incomplete, resulting in enormous structural diversity in mature HS and form a variety of ligand-binding sites to interact with a large number of protein ligands (1–3). The protein ligand-binding sites in HS often consist of relatively small tracts of variably sulfated glucosamine and uronic acid residues. For example, the antithrombin-binding site is composed of a specific pentasaccharide sequence: GlcNAc/NS(6S)-GlcA-GlcNS(6S)-GlcNS(3S6S)-IdoA(2S)-GlcNS(6S) (4). The FGF2 binding site is a short sulfated sequence with N- and 2-O-sulfation (5). Intriguingly, the generation of the ligand-binding sites is cell/tissue- and developmentally stage-specific, implying that the regulatory functions of HS occur in a temporal and spatial manner (6, 7).Endothelial cells are one of the major cellular components of blood vessels that form the inner monolayer endothelium of blood vessels. Under normal physiological conditions, endothelial cells maintain vascular homeostasis and respond to environmental changes to regulate inflammatory and immune response, vascular tone, coagulation, and fibrinolysis (8). Endothelial cells are also key players in angiogenesis that is finely tuned by the balance between pro- and anti-angiogenic factors (9). Angiogenesis plays an essential role in physiological conditions such as embryonic development, menstruation and wound healing, as well as in pathological conditions such as tumor growth, inflammatory disorders, eye diseases, stroke, etc (10). Therefore, understanding the mechanisms that control endothelial cell functions will greatly advance the development of effective treatment for vascular related human diseases.HS is expressed abundantly in the vasculature. Genetic studies have established that HS is essential for endothelial cell function under physiological and pathological conditions (11). We previously reported that endothelial-specific knockout of Ndst1 attenuates leukocyte adhesion and extravasation in inflammation (12), revealing that endothelial HS is required to facilitate leukocyte trafficking. This observation was confirmed by examining the same mutant mice by others (13, 14) and by endothelial-specific ablation of Ext1 (15) or HS 2-O-sulfotranserase (16). We also observed that deficiency of endothelial Ndst1 retards tumorigenesis (11), showing that endothelial HS critically promotes tumor angiogenesis. Recent studies by others also revealed that endothelial HS functions to promote vascular permeability (17) and tumor metastasis to lymph nodes (18). These studies illustrate HS to be a multifunctional molecule in vivo. To understand the underlying molecular mechanisms of HS on aforementioned endothelial cell functions, we examined the interactions of endothelial HS with one or several HS-binding ligands known to be involved in the studied biological functions. For example, we examined the interactions of endothelial HS with l-selectin and three chemokines involved in leukocyte trafficking (12), and the VEGF signaling in tumor angiogenesis (11). Similarly, only VEGF and neuropilin signaling were examined for their regulatory role of endothelial HS in vascular permeability (17), and only two chemokines were checked in the study of tumor metastasis to lymph nodes (18). This specific molecule targeting approach allowed us to understand some key underlying molecular mechanisms. However, in reality, a large number of HS-binding growth factors, morphogens, cytokines, and adhesion molecules are expected to co-express at the examined conditions and HS may selectively interact with a fraction of these HS-binding protein ligands to modulate endothelial functions (3, 19). To fully understand these molecular mechanisms, an unbiased analysis is essentially needed to determine the cell-specific and spatiotemporal regulatory roles of HS on endothelial cell function and related signaling.Regulatory post-translational modification represents a major cellular signaling mechanism that allows cells to sense environmental change. Among them, reversible phosphorylation controlled by kinases and phosphatases plays a central role in signaling in cell-cell or cell-environment communication (20). Global phosphoproteomics examines phosphorylation changes, and determines signaling regulation at a systemic level (21, 22). In this study, we applied the stable isotope labeling by amino acids (SILAC)¹-based global quantitative phosphoproteomics analysis to examine HS-dependent endothelial signaling by comparing wild type and HS-deficient endothelial cells that were cultured in serum-containing medium. A total of 7222 phosphopeptides corresponding to 1179 proteins were identified. Bioinformatic analysis of the identified proteins determined that HS deficiency altered cellular functions that are highly related to cell morphology, cellular assembly and organization, cellular function and maintenance, inflammatory response and disorder, cell growth and proliferation, cellular survival and death, consistent with cell function studies, which found that HS deficiency alters endothelial cell growth and mobility. Mining for the underlying molecular mechanisms revealed that HS modulates signaling pathways critically related to cell adhesion, migration, and coagulation, and found that HS critically modulates IGF-1 signaling. In-depth analysis of growth factor signaling identified 22 HS-dependent growth factor/cytokine/growth hormone signaling factors, including both previously known and unknown ones such as IGF-1, erythropoietin, angiopoietin/Tie, IL-17A, and growth hormones. Twelve of the identified 22 HS-dependent signaling factors were alternatively confirmed by phospho-receptor tyrosine kinase (p-RTK) array analysis. Therefore, our results revealed a broad and very complex regulatory role of HS on endothelial signaling.     

Analysis of Dengue Virus Genetic Diversity during Human and Mosquito Infection Reveals Genetic Constraints

Dengue viruses (DENV) cause debilitating and potentially life-threatening acute disease throughout the tropical world. While drug development efforts are underway, there are concerns that resistant strains will emerge rapidly. Indeed, antiviral drugs that target even conserved regions in other RNA viruses lose efficacy over time as the virus mutates. Here, we sought to determine if there are regions in the DENV genome that are not only evolutionarily conserved but genetically constrained in their ability to mutate and could hence serve as better antiviral targets. High-throughput sequencing of DENV-1 genome directly from twelve, paired dengue patients’ sera and then passaging these sera into the two primary mosquito vectors showed consistent and distinct sequence changes during infection. In particular, two residues in the NS5 protein coding sequence appear to be specifically acquired during infection in Ae. aegypti but not Ae. albopictus. Importantly, we identified a region within the NS3 protein coding sequence that is refractory to mutation during human and mosquito infection. Collectively, these findings provide fresh insights into antiviral targets and could serve as an approach to defining evolutionarily constrained regions for therapeutic targeting in other RNA viruses.     

Computational Modeling of Protein Stability: Quantitative Analysis Reveals Solutions to Pervasive Problems

1. Download : Download high-res image (151KB)
2. Download : Download full-size image

     

Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance

To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na⁺ sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H⁺-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H⁺-pump activity.     

Peptide Mapping Reveals Considerable Sequence Homology among the Three Polypeptide Subunits of G1 Storage Protein from French Bean Seed

The major storage protein, G1 globulin, of bean (cv. Tendergreen) seeds was subjected to limited proteolysis with trypsin, chymotrypsin, papain, proteinase K, and protease V8 and to cleavage with cyanogen bromide and 2-(2-nitrophenylsulfanyl)-3-methyl-3′bromoindolenine. Mapping of peptides separated from each of the three G1 subunits by polyacrylamide gel electrophoresis revealed that many proteolytic cleavage sites were present at similar positions on the subunits. Evidence was adduced that the G1 subunits are homologous in amino acid sequence for about 61% of their length. The remaining region (possibly COOH-terminal) of the subunits appears to be heterologous, with the α subunit bearing an additional methionine residue.