Complete genome sequence of Acidimicrobium ferrooxidans type strain (ICP)

Acidimicrobium ferrooxidans (Clark and Norris 1996) is the sole and type species of the genus, which until recently was the only genus within the actinobacterial family Acidimicrobiaceae and in the order Acidomicrobiales. Rapid oxidation of iron pyrite during autotrophic growth in the absence of an enhanced CO(2) concentration is characteristic for A. ferrooxidans. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the order Acidomicrobiales, and the 2,158,157 bp long single replicon genome with its 2038 protein coding and 54 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Tooth wear in captive wild ruminant species differs from that of free-ranging conspecifics

The mesowear method evaluates the wear patterns of herbivore cheek teeth by visually evaluating the facet development of the occlusal surfaces. It thus allows classification of most herbivorous ungulates into browsers, grazers or intermediate feeders, due to the fact that in grazers, tooth wear is characterized by a comparatively high degree of abrasion, most probably due to the presence of silicacious phytoliths in grasses, a higher amount of dust and grit adhering to their forage, or both. It has been suggested that excessive tooth wear could be a particularly limiting factor in the husbandry of captive large browsing species, and major tooth wear was demonstrated in captive as compared to free-ranging giraffe. If this increased tooth wear in captivity was an effect of feeding type and diets fed, then it would be expected that other browsing species are affected in a similar manner. In order to test this hypothesis, we investigated the dental mesowear pattern in captive individuals of 19 ruminant species and compared the results to data on free-ranging animals. Compared to free-ranging populations, captive browsers show a significantly more abrasion-dominated tooth wear signal. The reverse applies to captive grazers, which tend to show a less abrasion-dominated wear in captivity. Captive ruminants were generally more homogenous in their wear signature than free-ranging ruminants. If grit contamination in the natural habitat is a major cause of dental wear in grazers, then diets in captivity, although similar in botanical composition, most likely contain less abrasives due to feeding hygiene. If dental wear is one of the major factors limiting longevity, then captive grazers should achieve longer lifespans than both captive browsers and free-ranging grazers. In particular with respect to browsers, the results suggest that captive feeding regimes could be improved.     

Reversible Phosphorylation as a Molecular Switch to Regulate Plasma Membrane Targeting of Acylated SH4 Domain Proteins

Acylated SH4 domains represent N-terminal targeting signals that anchor peripheral membrane proteins such as Src kinases in the inner leaflet of plasma membranes. Here we provide evidence for a novel regulatory mechanism that may control the levels of SH4 proteins being associated with plasma membranes. Using a fusion protein of the SH4 domain of Leishmania HASPB and GFP as a model system, we demonstrate that threonine 6 is a substrate for phosphorylation. Substitution of threonine 6 by glutamate (to mimic a phosphothreonine residue) resulted in a dramatic redistribution from plasma membranes to intracellular sites with a particular accumulation in a perinuclear region. As shown by both pharmacological inhibition and RNAi-mediated down-regulation of the threonine/ serine-specific phosphatases PP1 and PP2A, recycling back to the plasma membrane required dephosphorylation of threonine 6. We provide evidence that a cycle of phosphorylation and dephosphorylation may also be involved in intracellular targeting of other SH4 proteins such as the Src kinase Yes.     

Rhizospheric streptomycetes as potential biocontrol agents of <Emphasis Type="Italic">Fusarium</Emphasis> and <Emphasis Type="Italic">Armillaria</Emphasis> pine rot and as PGPR for <Emphasis Type="Italic">Pinus taeda</Emphasis>

Pinus taeda is one of the main timber trees in Brazil, occupying 1.8 million ha with an annual productivity of 25–30 m³ ha⁻¹. Another important species is Araucaria angustifolia, belonging to the fragile Rainforest biome, which for decades has been a major source of timber in Brazil. Some diseases that affect the roots and/or the stem of these trees and cause “damping-off” of the seedlings, with economic and environmental losses for the forest sector, are caused by the plant pathogenic fungi Fusarium sp. or Armillaria sp. This research project intended to isolate actinobacteria from the Araucaria rhizosphere, which present an antagonistic effect against these fungi. After the selection of the best pathogen inhibitors, morphologic characteristics, enzyme production, and their effect on the growth of Pinus taeda were studied. The actinobacteria were tested for their antagonistic capacity against Fusarium sp. in Petri plates with PDA as substrate. The inhibition zone was measured after 3, 5, 7, and 10 days. Of all the isolates tested, only two of them maintained inhibition zones up to 4 mm for 10 days. The inhibition of Armillaria sp. was tested in liquid medium and also in Petri dishes through the evaluation of the number of the fungal rhizomorphs in dual culture with the actinobacteria. It was found that all five isolates were able to inhibit the rhizomorph production, with the best performance of the isolate A43, which was capable of inhibiting both fungi, Fusarium and Armillaria. In a greenhouse experiment, the effect of five isolates on the growth of Pinus taeda seedlings was tested. Plant height, stem diameter, root and shoot dry matter were determined. The Streptomyces isolate A43 doubled plant growth. These results may lead to the development of new technologies in the identification of still unknown bacterial metabolites and new management techniques to control forest plant diseases.     

Root colonization by Piriformospora indica enhances grain yield in barley under diverse nutrient regimes by accelerating plant development

The basidiomycete fungus Piriformospora indica colonizes roots of a broad range of mono- and dicotyledonous plants. It confers enhanced growth, improves resistance against biotic and tolerance to abiotic stress, and enhances grain yield in barley. To analyze mechanisms underlying P. indica-induced improved grain yield in a crop plant, the influence of different soil nutrient levels and enhanced biotic stress were tested under outdoor conditions. Higher grain yield was induced by the fungus independent of different phosphate and nitrogen fertilization levels. In plants challenged with the root rot-causing fungus Fusarium graminearum, P. indica was able to induce a similar magnitude of yield increase as in unchallenged plants. In contrast to the arbuscular mycorrhiza fungus Glomus mosseae, total phosphate contents of host plant roots and shoots were not significantly affected by P. indica. On the other hand, barley plants colonised with the endophyte developed faster, and were characterized by a higher photosynthetic activity at low light intensities. Together with the increased root formation early in development these factors contribute to faster development of ears as well as the production of more tillers per plant. The results indicate that the positive effect of P. indica on grain yield is due to accelerated growth of barley plants early in development, while improved phosphate supply—a central mechanism of host plant fortification by arbuscular mycorrhizal fungi—was not observed in the P. indica-barley symbiosis.     

Increased Activity of the Vacuolar Monosaccharide Transporter TMT1 Alters Cellular Sugar Partitioning,Sugar Signaling,and Seed Yield in Arabidopsis

The extent to which vacuolar sugar transport activity affects molecular, cellular, and developmental processes in Arabidopsis (Arabidopsis thaliana) is unknown. Electrophysiological analysis revealed that overexpression of the tonoplast monosaccharide transporter TMT1 in a tmt1-2::tDNA mutant led to increased proton-coupled monosaccharide import into isolated mesophyll vacuoles in comparison with wild-type vacuoles. TMT1 overexpressor mutants grew faster than wild-type plants on soil and in high-glucose (Glc)-containing liquid medium. These effects were correlated with increased vacuolar monosaccharide compartmentation, as revealed by nonaqueous fractionation and by chlorophyll_ab-binding protein1 and nitrate reductase1 gene expression studies. Soil-grown TMT1 overexpressor plants respired less Glc than wild-type plants and only about half the amount of Glc respired by tmt1-2::tDNA mutants. In sum, these data show that TMT activity in wild-type plants limits vacuolar monosaccharide loading. Remarkably, TMT1 overexpressor mutants produced larger seeds and greater total seed yield, which was associated with increased lipid and protein content. These changes in seed properties were correlated with slightly decreased nocturnal CO₂ release and increased sugar export rates from detached source leaves. The SUC2 gene, which codes for a sucrose transporter that may be critical for phloem loading in leaves, has been identified as Glc repressed. Thus, the observation that SUC2 mRNA increased slightly in TMT1 overexpressor leaves, characterized by lowered cytosolic Glc levels than wild-type leaves, provided further evidence of a stimulated source capacity. In summary, increased TMT activity in Arabidopsis induced modified subcellular sugar compartmentation, altered cellular sugar sensing, affected assimilate allocation, increased the biomass of Arabidopsis seeds, and accelerated early plant development.Sugars fulfill an extraordinarily wide range of functions in plants as well as in other organisms. They serve as valuable energy resources that are easy to store and remobilize. Sugars are required for the synthesis of cell walls and carbohydrate polymers. They are also necessary for starch accumulation and serve as precursors for a range of primary and secondary plant intermediates. From a chemical point of view, sugars represent a large class of metabolites. Among the prominent members in higher plants are the monosaccharides Glc and Fru and the disaccharide Suc (ap Rees, 1994).In contrast to heterotrophic organisms, plants are able to synthesize sugars de novo and to degrade them via oxidative or fermentative metabolism (Heldt, 2005). Net sugar accumulation in plants takes place during the day, whereas net degradation of stored carbohydrate reserves takes place the following night. In higher plants, autotrophic and heterotrophic organs appear to be interconnected by phloem for long-distance transport of sugars (Ruiz-Medrano et al., 2001). Accordingly, sugars must be transported within cells, between cells, and between plant organs. Given these factors, along with the outstanding importance of sugars, it is not surprising that plants sense intracellular sugar availability and use this information to coordinate the expression of many genes (Koch, 1996; Moore et al., 2003).In Arabidopsis (Arabidopsis thaliana), about 60 genes code for putative monosaccharide transport proteins and about 10 genes encode predicted disaccharide carriers (Lalonde et al., 2004). Transport of neutral sugars has been monitored across the plasma membrane, the chloroplast envelope, and the vacuolar membrane (Weber et al., 2000; Niittylä et al., 2004; Martinoia et al., 2007). So far, all sugar carriers residing in the plant plasma membrane have been characterized to catalyze proton-coupled sugar movement (Sauer, 1992; Büttner and Sauer, 2000; Carpaneto et al., 2005). In contrast, both facilitated diffusion and proton-driven antiport mechanisms have been described for monosaccharide and Suc transport across the vacuolar membrane (Thom and Komor, 1984; Daie and Wilusz, 1987; Martinoia et al., 1987; Shiratake et al., 1997; Neuhaus, 2007).In plants, vacuoles fulfill critical functions in the long-term and temporary storage of sugars, sugar alcohols, and other primary metabolites such as carboxylates and amino acids (Dietz et al., 1990; Rentsch and Martinoia, 1991; Martinoia and Rentsch, 1992; Emmerlich et al., 2003). Recently, the first solute carriers responsible for vacuolar Suc and inositol transport have been identified (Endler et al., 2006; Schneider et al., 2008). In addition, TMT (for tonoplast monosaccharide transporter) and VGT (for vacuolar Glc transporter) were the first vacuolar carrier proteins proven to have transport capacity for both Glc and Fru (Wormit et al., 2006; Aluri and Büttner, 2007).TMT exists in three isoforms in Arabidopsis (TMT1–TMT3), and orthologs have been found in other plant species like grapevine (Vitis vinifera), barley (Hordeum vulgare), and rice (Oryza sativa; Wormit et al., 2006). In Arabidopsis, the genes TMT1 and TMT2 are expressed in various tissues, whereas TMT3 is hardly expressed throughout the entire plant life cycle (Wormit et al., 2006). Interestingly, TMT1 and TMT2 are induced by Glc, salt, drought, and cold stress (Wormit et al., 2006), and vacuoles isolated from a TMT1 loss-of-function (T-DNA) Arabidopsis mutant showed reduced Glc import capacity in comparison with corresponding wild-type organelles (Wormit et al., 2006). Moreover, after transfer into the cold, these mutant leaves showed impaired ability to accumulate Glc and Fru, underscoring the in vivo function of TMT under selected conditions (Wormit et al., 2006).However, it is unknown to what extent overexpression of a vacuolar sugar carrier affects subcellular sugar allocation in Arabidopsis. In addition, whether increased vacuolar sugar transport influences sugar signaling, plant development, or organ properties has not been determined. Thus, it is unknown how important controlled activity of vacuolar monosaccharide transport is to plant development or physiological properties. To reveal whether TMT activity affects these processes, we created TMT1-overexpressing Arabidopsis lines and analyzed their physiological and molecular feedbacks.     

Mitochondrial Malate Dehydrogenase Lowers Leaf Respiration and Alters Photorespiration and Plant Growth in Arabidopsis

Malate dehydrogenase (MDH) catalyzes a reversible NAD⁺-dependent-dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments. To explore the role of mitochondrial MDH (mMDH) in Arabidopsis (Arabidopsis thaliana), knockout single and double mutants for the highly expressed mMDH1 and lower expressed mMDH2 isoforms were constructed and analyzed. A mmdh1mmdh2 mutant has no detectable mMDH activity but is viable, albeit small and slow growing. Quantitative proteome analysis of mitochondria shows changes in other mitochondrial NAD-linked dehydrogenases, indicating a reorganization of such enzymes in the mitochondrial matrix. The slow-growing mmdh1mmdh2 mutant has elevated leaf respiration rate in the dark and light, without loss of photosynthetic capacity, suggesting that mMDH normally uses NADH to reduce oxaloacetate to malate, which is then exported to the cytosol, rather than to drive mitochondrial respiration. Increased respiratory rate in leaves can account in part for the low net CO₂ assimilation and slow growth rate of mmdh1mmdh2. Loss of mMDH also affects photorespiration, as evidenced by a lower postillumination burst, alterations in CO₂ assimilation/intercellular CO₂ curves at low CO₂, and the light-dependent elevated concentration of photorespiratory metabolites. Complementation of mmdh1mmdh2 with an mMDH cDNA recovered mMDH activity, suppressed respiratory rate, ameliorated changes to photorespiration, and increased plant growth. A previously established inverse correlation between mMDH and ascorbate content in tomato (Solanum lycopersicum) has been consolidated in Arabidopsis and may potentially be linked to decreased galactonolactone dehydrogenase content in mitochondria in the mutant. Overall, a central yet complex role for mMDH emerges in the partitioning of carbon and energy in leaves, providing new directions for bioengineering of plant growth rate and a new insight into the molecular mechanisms linking respiration and photosynthesis in plants.Plant tissues contain multiple isoforms of malate dehydrogenase (l-malate-NAD-oxidoreductase [MDH]; EC 1.1.1.37) that catalyze the interconversion of malate and oxaloacetate (OAA) coupled to reduction or oxidation of the NAD pool. These isoforms are encoded by separate genes in plants and have been shown to possess distinct kinetic properties as well as subcellular targeting and physiological functions (Gietl, 1992). While the MDH reaction is reversible, it strongly favors the reduction of OAA. The direction of the reaction in vivo depends on substrate/product ratios and the NAD redox state, and it can vary even in the same tissue due to prevailing physiological conditions. Isoforms operate in mitochondria, chloroplasts, peroxisomes, and the cytosol, but due to the ready transport and utilization of malate and OAA and the availability of NAD, this reaction can cooperate across compartments and is the basis for malate/OAA shuttling of reducing equivalents in many different metabolic schemes of plant cellular function (Krömer, 1995). It is clear, however, that the exchange through the membranes is strictly controlled, since large redox differences in NAD(H) pools exist between compartments (Igamberdiev and Gardeström, 2003).The mitochondrial MDH (mMDH) is thought to operate in at least three different pathways in plants. First, it is a classical tricarboxylic acid (TCA) cycle enzyme that oxidizes the malate product from the fumarase reaction to OAA for the citrate synthase-dependent condensation with acetyl-CoA to form citrate. Second, it is considered to operate in the reverse direction during the conversion of Gly to Ser by reducing OAA to malate and providing a supply of NAD⁺ for Gly decarboxylase (Journet et al., 1981). Third, in a more specialized pathway in C4 plants, it provides a supply of CO₂ for fixation in bundle sheath chloroplasts by reducing OAA (generated from Asp transported from mesophyll cells) into malate that is then decarboxylated by NAD-malic enzyme (NAD-ME) to CO₂ and pyruvate (Hatch and Osmond, 1976). Plant mitochondria can support TCA cycle activity with malate as the sole substrate due to MDH and NAD-ME, both ubiquitous in plants (Palmer, 1984). OAA is readily transported both into and out of isolated plant mitochondria (Douce and Bonner, 1972), in contrast to mammalian mitochondria, which are essentially impermeable to this organic acid.While these three mMDH schemes and metabolic schemes for other MDH isoforms are plausible, widely accepted, and consistent with a range of biochemical studies, the depletion, removal, and overexpression of specific MDH isoforms in plants have led to surprising insights into MDH roles in vivo. For example, the peroxisomal MDH (PMDH) was until recently generally considered to be involved in the synthesis of NADH for hydroxypyruvate reduction in the photorespiratory cycle and for the oxidation of NADH generated during β-oxidation of fatty acids, but its potential role in the oxidation of malate in the glyoxylate cycle was unclear. However, studies of the double knockout of PMDH in Arabidopsis (Arabidopsis thaliana) showed that while PMDH is essential for β-oxidation, its removal does not impair glyoxylate cycle activity (Pracharoenwattana et al., 2007) and has only a limited impact on hydroxypyruvate reduction (Cousins et al., 2008).Changes in mMDH have been reported both through the study of spontaneous mutants and the expression of antisense constructs. Spontaneous null mutants of mMDH1 in soybean (Glycine max) are linked to a yellow foliage phenotype and are associated with the removal of two of the three mMDH isoforms (Imsande et al., 2001). Expression of an antisense fragment of mMDH in tomato (Solanum lycopersicum), driven by the 35S promoter, lowered mMDH protein in mitochondria, decreased total cellular MDH by approximately 60%, but had a positive impact on photosynthetic activity, CO₂ assimilation rate, and total plant dry matter in long-day-grown plants (Nunes-Nesi et al., 2005). A range of carbohydrates also accumulated in the tomato antisense plants, as did redox-related compounds such as ascorbate. The increase in ascorbate content may be linked to the enhancement of photosynthesis, as ascorbate feeding to leaves can also increase photosynthetic performance (Nunes-Nesi et al., 2005). This link is not absolute, however, given that short-day-grown antisense tomato plants had stunted growth, which was potentially due to impaired photosynthesis, but still had elevated levels of ascorbate due to a higher ratio of reduction of the ascorbate pool compared with the wild type (Nunes-Nesi et al., 2008). Analysis of roots from these antisense tomato plants revealed a negative impact of mMDH loss, leading to a lower root dry weight and lower root respiratory rate (van der Merwe et al., 2009). This implies a distinct impact of mMDH loss on roots and shoots. Overexpression of cytosolic MDH led to a 4-fold elevation of root organic acids in alfalfa (Medicago sativa) plants and high rates of organic acid exudation that increased aluminum tolerance through metal chelation in the soil (Tesfaye et al., 2001). These studies imply that there is a complex form of functional redundancy between MDH isoforms in different compartments, allowing MDH in separate locations to maintain specific pathways via malate/OAA shuttling, or that a range of redox requirements that have been linked to MDH in accepted metabolic schemes are incorrect and other reactions couple NAD/NADH pool homeostasis. In addition, these studies clearly show that changes in the amount of MDH isoforms can alter metabolic flux into a range of organic acids and have far-reaching effects on plant growth and development.To better understand the importance of the mMDH and to determine if plants are viable without any mMDH isoforms due either to the role of NAD-ME and/or malate/OAA shuttling to other compartments, we have constructed and analyzed mMDH mutants in Arabidopsis. A major and a minor MDH isoform exist in Arabidopsis mitochondria, evidenced by differing levels of gene expression and differing protein abundance (Lee et al., 2008). We hypothesized that if mMDH works in concert with other MDH isoforms and is responsible for the reduction of OAA to malate for export from the mitochondrion, then if we remove mMDH, not only would the loss of extramitochondrial malate and the slowing of Gly decarboxylation limit photorespiratory carbon flux, but oxidation of NADH remaining in the mitochondrion could lead to elevated leaf respiration and alteration in plant growth. We found that not only did mutants have low photorespiratory flux, but they also increased respiration and had slow growth due to lowered net CO₂ assimilation. The previously established correlation between mMDH abundance, photosynthetic performance, and foliar ascorbate levels was also investigated. Elevated levels of the metabolite were found in Arabidopsis, consolidating the work done in tomato (Nunes-Nesi et al., 2005). Proteomic analyses, followed by immunodetection studies, unearthed altered abundance of the terminal enzyme of the ascorbate biosynthetic pathway, galactono-1,4-lactone dehydrogenase (GLDH), as a mechanistic element in the phenomenon linked directly to mitochondrial function.     

Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines

The epithelial–mesenchymal transition (EMT) is regarded as an important step in cancer metastasis. Snail, a master regulator of EMT, has been recently proposed to act additionally as a cell survival factor and inducer of motility. We have investigated the function of Snail (SNAI1) in prostate cancer cells by downregulating its expression via short (21-mer) interfering RNA (siRNA) and measuring the consequences on EMT markers, cell viability, death, cell cycle, senescence, attachment, and invasivity. Of eight carcinoma cell lines, the prostate carcinoma cell lines LNCaP and PC-3 showed the highest and moderate expression of SNAI1 mRNA, respectively, as measured by quantitative RT-PCR. Long-term knockdown of Snail induced a severe decline in cell numbers in LNCaP and PC-3 and caspase activity was accordingly enhanced in both cell lines. In addition, suppression of Snail expression induced senescence in LNCaP cells. SNAI1-siRNA-treated cells did not tolerate detachment from the extracellular matrix, probably due to downregulation of integrin α6. Expression of E-cadherin, vimentin, and fibronectin was also affected. Invasiveness of PC-3 cells was not significantly diminished by Snail knockdown. Our data suggest that Snail acts primarily as a survival factor and inhibitor of cellular senescence in prostate cancer cell lines. We therefore propose that Snail can act as early driver of prostate cancer progression.