Inorganic polyphosphates in extremophiles and their possible functions

Many extremophilic microorganisms are polyextremophiles, being confronted with more than one stress condition. For instance, some thermoacidophilic microorganisms are in addition capable to resist very high metal concentrations. Most likely, they have developed special adaptations to thrive in their living environments. Inorganic polyphosphate (polyP) is a molecule considered to be primitive in its origin and ubiquitous in nature. It has many roles besides being a reservoir for inorganic phosphate and energy. Of special interest are those functions related to survival under stressing conditions in all kinds of cells. PolyP may therefore have a fundamental part in extremophilic microorganism's endurance. Evidence for a role of polyP in the continued existence under acidic conditions, high concentrations of toxic heavy metals and elevated salt concentrations are reviewed in the present work. Actual evidence suggests that polyP may provide mechanistic alternatives in tuning microbial fitness for the adaptation under stressful environmental situations and may be of crucial relevance amongst extremophiles. The enzymes involved in polyP metabolism show structure conservation amongst bacteria and archaea. However, the lack of a canonical polyP synthase in Crenarchaea, which greatly accumulate polyP, strongly suggests that in this phylum a different enzyme may be in charge of its synthesis.     

A Highlights from MBoC Selection: Chromosome replication and segregation govern the biogenesis and inheritance of inorganic polyphosphate granules

Prokaryotes and eukaryotes synthesize long chains of orthophosphate, known as polyphosphate (polyP), which form dense granules within the cell. PolyP regulates myriad cellular functions and is often localized to specific subcellular addresses through mechanisms that remain undefined. In this study, we present a molecular-level analysis of polyP subcellular localization in the model bacterium Caulobacter crescentus. We demonstrate that biogenesis and localization of polyP is controlled as a function of the cell cycle, which ensures regular partitioning of granules between mother and daughter. The enzyme polyphosphate kinase 1 (Ppk1) is required for granule production, colocalizes with granules, and dynamically localizes to the sites of new granule synthesis in nascent daughter cells. Localization of Ppk1 within the cell requires an intact catalytic active site and a short, positively charged tail at the C-terminus of the protein. The processes of chromosome replication and segregation govern both the number and position of Ppk1/polyP complexes within the cell. We propose a multistep model in which the chromosome establishes sites of polyP coalescence, which recruit Ppk1 to promote the in situ synthesis of large granules. These findings underscore the importance of both chromosome dynamics and discrete protein localization as organizing factors in bacterial cell biology.     

Large-scale comparative genomic analyses of cytoplasmic membrane transport systems in prokaryotes

The recent advancements in genome sequencing make it possible for the comparative analyses of essential cellular processes like transport in organisms across the three domains of life. Membrane transporters play crucial roles in fundamental cellular processes and functions in prokaryotic systems. Between 3 and 16% of open reading frames in prokaryotic genomes were predicted to encode membrane transport proteins, emphasizing the importance of transporters in their lifestyles. Hierarchical clustering of phylogenetic profiles of transporter families, which are derived from the presence or absence of a certain transporter family, showed distinct clustering patterns for obligate intracellular organisms, plant/soil-associated microbes and autotrophs. Obligate intracellular organisms possess the fewest types and number of transporters presumably due to their relatively stable living environment, while plant/soil-associated organisms generally encode the largest variety and number of transporters. A group of autotrophs are clustered together largely due to their absence of transporters for carbohydrate and organic nutrients and the presence of transporters for inorganic nutrients. Inside of each group, organisms are further clustered by their phylogenetic properties. These findings strongly suggest the correlation of transporter profiles to both evolutionary history and the overall physiology and lifestyles of the organisms.     

Trypanosoma cruzi: a stage-specific calpain-like protein is induced after various kinds of stress

Calpains are calcium-dependent cysteine proteinases found in all living organisms and are involved in diverse cellular processes. Calpain-like proteins have been reported after in silico analysis of the Tritryps genome and are believed to play important roles in cell functions of trypanosomatids. We describe the characterization of a member of this family, which is differentially expressed during the life-cycle of Trypanosoma cruzi.     

Polyphosphate is involved in cell cycle progression and genomic stability in Saccharomyces cerevisiae

Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4Δ) or hydrolyse polyP (ppn1Δ, ppx1Δ) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.     

Cytoskeleton and plant organogenesis

The functions of microtubules and actin filaments during various processes that are essential for the growth, reproduction and survival of single plant cells have been well characterized. A large number of plant structural cytoskeletal or cytoskeleton-associated proteins, as well as genes encoding such proteins, have been identified. Although many of these genes and proteins have been partially characterized with respect to their functions, a coherent picture of how they interact to execute cytoskeletal functions in plant cells has yet to emerge. Cytoskeleton-controlled cellular processes are expected to play crucial roles during plant cell differentiation and organogenesis, but what exactly these roles are has only been investigated in a limited number of studies in the whole plant context. The intent of this review is to discuss the results of these studies in the light of what is known about the cellular functions of the plant cytoskeleton, and about the proteins and genes that are required for them. Directions are outlined for future work to advance our understanding of how the cytoskeleton contributes to plant organogenesis and development.     

Extracellular ATP: a potential regulator of plant cell death

Adenosine 5′‐triphosphate (ATP) has been regarded as an intracellular energy currency molecule for many years. In recent decades, it has been determined that ATP is released into the extracellular milieu by animal, plant and microbial cells. In animal cells, this extracellular ATP (eATP) functions as a signalling compound to mediate many cellular processes through its interaction with membrane‐associated receptor proteins. It has also been reported that eATP is a signalling molecule required for the regulation of plant growth, development and responses to environmental stimuli. Recently, the first plant receptor for eATP was identified in Arabidopsis thaliana. Interestingly, some studies have shown that eATP is of particular importance in the control of plant cell death. In this review article, we summarize and discuss the theoretical and experimental advances that have been made with regard to the roles and mechanisms of eATP in plant cell death. We also make an attempt to address some speculative aspects to help develop and expand future research in this area.     

多聚磷酸相关蛋白结构及生物学功能

多聚磷酸（polyphosphate,polyP）是由几个到数百个磷酸基通过高能磷酸酐键连接而成的链状多聚体,存在于所有细胞生物中.多聚磷酸相关蛋白包括多聚磷酸相关酶和多聚磷酸结合蛋白.多聚磷酸相关酶如多聚磷酸激酶（polyphosphate kinase,PPK）催化polyPn生成polyPn+1的可逆反应;外切聚磷酸酶（exopolyphosphatase,PPX）、内切聚磷酸酶（endopolyphosphatase,PPN）能将polyP水解成磷酸残基;多聚磷酸依赖的激酶将polyP的磷转移到生物小分子上,如葡萄糖和烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide,NAD）,使其分别磷酸化为6 磷酸葡萄糖和烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate,NADP）.多聚磷酸结合蛋白可与多聚磷酸结合,发挥各种生物学功能.本文将简要介绍多聚磷酸相关蛋白的结构与主要生物学功能,以阐述多聚磷酸参与的细胞内生化过程.     

Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes

Inorganic polyphosphate (polyP) has been identified and measured in human platelets. Millimolar levels (in terms of Pi residues) of short chain polyP were found. The presence of polyP of approximately 70-75 phosphate units was identified by 31P NMR and by urea-polyacrylamide gel electrophoresis of platelet extracts. An analysis of human platelet dense granules, purified using metrizamide gradient centrifugation, indicated that polyP was preferentially located in these organelles. This was confirmed by visualization of polyP in the dense granules using 4',6-diamidino-2-phenylindole and by its release together with pyrophosphate and serotonin upon thrombin stimulation of intact platelets. Dense granules were also shown to contain large amounts of calcium and potassium and both bafilomycin A1-sensitive ATPase and pyrophosphatase activities. In agreement with these results, when human platelets were loaded with the fluorescent calcium indicator Fura-2 acetoxymethyl ester to measure their intracellular Ca2+ concentration ([Ca2+]i), they were shown to possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by the following: 1) the increase in [Ca2+]i induced by nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2 and 2) the effect of ionomycin, which could not take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by the previous addition of nigericin, monensin, or NH4Cl. All of these characteristics of the platelet dense granules, together with their known acidity and high density (both by weight and by electron microscopy), are similar to those of acidocalcisomes (volutin granules, polyP bodies) of bacteria and unicellular eukaryotes. The results suggest that acidocalcisomes have been conserved during evolution from bacteria to humans.     

N-myristoyltransferase in the leukocytic development processes

The lipidic modification of proteins has recently been shown to be of immense importance, although many of the roles of these modifications remain as yet unidentified. One of such key modifications occurring on several proteins is the covalent addition of a 14-carbon long saturated fatty acid, a process termed myristoylation. Myristoylation can occur during both co-translational protein synthesis and posttranslationally, confers lipophilicity to protein molecules, and controls protein functions. The protein myristoylation process is catalyzed by the enzyme N-myristoyltransferase (NMT), which exists as two isoforms: NMT1 and NMT2. NMT1 is essential for growth and development, during which rapid cellular proliferation is required, in a variety of organisms. NMT1 is also reported to be elevated in many cancerous states, which also involve rapid cellular growth, albeit in an unwanted and uncontrolled manner. The delineation of myristoylation-dependent cellular functions is still in a state of infancy, and many of the roles of the myristoylated proteins remain to be established. The development of cells of the leukocytic lineage represents a phase of rapid growth and development, and we have observed that NMT1 plays a role in this process. The current review outlines the roles of NMT1 in the growth and differentiation of the cells of leukocytic origin. The described studies clearly demonstrate the roles of NMT1 in the regulation of the developmental processes of the leukocytes cells and provide a basis for further research with the aim of unraveling the roles of protein myristoylation in both cellular and physiological context.     

Pyrroloquinoline-quinone and its versatile roles in biological processes

Pyrroloquinoline-quinine (PQQ) was initially characterized as a redox cofactor for membrane-bound dehydrogenases in the bacterial system. Subsequently, PQQ was shown to be an antioxidant protecting the living cells from oxidative damage in vivo and the biomolecules from artificially produced reaction oxygen species in vitro. The presence of PQQ has been documented from different biological samples. It functions as a nutrient and vitamin for supporting the growth and protection of living cells under stress. Recently, the role of PQQ has also been shown as a bio-control agent for plant fungal pathogens, an inducer for proteins kinases involved in cellular differentiation of mammalian cells and as a redox sensor leading to development of biosensor. Recent reviews published on PQQ and enzymes requiring this cofactor have brought forth the case specific roles of PQQ. This review covers the comprehensive information on various aspects of PQQ known till date. These include the roles of PQQ in the regulation of cellular growth and differentiation in mammalian system, as a nutrient and vitamin in stress tolerance, in crop productivity through increasing the availability of insoluble phosphate and as a bio-control agent, and as a redox agent leading to the biosensor development. Most recent findings correlating the exceptionally high redox recycling ability of PQQ to its potential as anti-neurodegenerative, anticancer and pharmacological agents, and as a signalling molecule have been distinctly brought out. This review discusses different findings suggesting the versatility in PQQ functions and provides the most plausible intellectual basis to the ubiquitous roles of this compound in a large number of biological processes, as a nutrient and a perspective vitamin.     

环境DNA技术在地下生态学中的应用

地下生态过程是生态系统结构、功能和过程研究中最不确定的因素。由于技术和方法的限制,作为"黑箱"的地下生态系统已经成为限制生态学发展的瓶颈,也是未来生态学发展的主要方向。环境DNA技术,是指从土壤等环境样品中直接提取DNA片段,然后通过DNA测序技术来定性或定量化目标生物,以确定目标生物在生态系统中的分布及功能特征。环境DNA技术已成功用于地下生态过程的研究。目前,环境DNA技术在土壤微生物多样性及其功能方面的研究相对成熟,克服了土壤微生物研究中不能培养的问题,可以有效地分析土壤微生物的群落组成、多样性及空间分布,尤其是宏基因组学技术的发展,使得微生物生态功能方面的研究成为可能;而且,环境DNA技术已经在土壤动物生态学的研究中得到了初步应用,可快速分析土壤动物的多样性及其分布特征,更有效地鉴定出未知的或稀少的物种,鉴定土壤动物类群的幅度较宽;部分研究者通过提取分析土壤中DNA片段信息对生态系统植物多样性及植物分类进行了研究,其结果比传统的植物分类及物种多样性测定更精确,改变了以往对植物群落物种多样性模式的理解。同时,环境DNA技术克服传统根系研究方法中需要洗根、分根、只能测定单物种根系的局限,降低根系研究中细根区分的误差,并探索性地用于细根生物量的研究。主要综述了基于环境DNA技术的分子生物学方法在土壤微生物多样性及功能、土壤动物多样性、地下植物多样性及根系生态等地下生态过程研究中的应用进展。环境DNA技术对于以土壤微生物、土壤动物及地下植物根系为主体的地下生态学过程的研究具有革命性意义,并展现出良好的应用前景。可以预期,分子生物学技术与传统的生态学研究相结合将成为未来地下生态学研究的一个发展趋势。     

抗增殖蛋白研究进展

抗增殖蛋白作为一种高度保守的蛋白广泛分布于细菌、原虫、酵母等多种生物细胞中,主要定位于细胞膜、线粒体内膜、细胞核中,参与细胞增殖、分化、衰老、凋亡等多种生物学进程。由于抗增殖蛋白结构进化上的保守性和功能的多样性,正逐渐引起广泛的关注。该文正是基于抗增殖蛋白重要的生物学意义,通过对其基因结构和功能的分析,系统地阐述了它在生物体中发挥的重要作用,并对其应用前景作以展望。     

New insights into relationships between active and dormant organisms,phylogenetic diversity and ecosystem productivity

Marine microbes make up a key part of ocean food webs and drive ocean chemistry through a range of metabolic processes. A fundamental question in ecology is whether the diversity of organisms in a community shapes the ecological functions of that community. While there is substantial evidence to support a positive link between diversity and ecological productivity for macro‐organisms in terrestrial environments, this relationship has not previously been verified for marine microbial communities. One factor complicating the understanding of this relationship is that many marine microbes are dormant and are easily dispersed by ocean currents, making it difficult to ensure that the organisms found in a given environmental sample accurately reflect processes occurring in that environment. Another complication is that, due to microbes great range of genotypic and phenotypic variability, communities with distantly related species may have greater range of metabolic functions than communities have the same richness and evenness, but in which the species present are more closely related to each other. In this issue of Molecular Ecology, Galand et al. (2015) provide compelling evidence that the most metabolically active communities are those in which the nondormant portion of the microbial community has the highest phylogenetic diversity. They also illustrate that focusing on the active portion of the community allows for detection of temporal patterns in community structure that would not be otherwise evident. The authors’ point out that the presence of many dormant organisms that do not contribute to ecosystem functioning is a feature that makes microbial ecosystems fundamentally different from macro‐ecosystems and that this difference needs to be accounted for in microbial ecology theory.     

Growth of polychlorinated-biphenyl-degrading bacteria in the presence of biphenyl and chlorobiphenyls generates oxidative stress and massive accumulation of inorganic polyphosphate

Inorganic polyphosphate (polyP) plays a significant role in increasing bacterial cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. Using transmission electron microscopy of the polychlorinated biphenyl (PCB)-degrading bacterium Pseudomonas sp. strain B4 grown in defined medium with biphenyl as the sole carbon source, we observed large and abundant electron-dense granules at all stages of growth and following a shift from glucose to biphenyl or chlorobiphenyls. Using energy dispersive X-ray analysis and electron energy loss spectroscopy with an integrated energy-filtered transmission electron microscope, we demonstrated that these granules were mainly composed of phosphate. Using sensitive enzymatic methods to quantify cellular polyP, we confirmed that this polymer accumulates in PCB-degrading bacteria when they grow in the presence of biphenyl and chlorobiphenyls. Concomitant increases in the levels of the general stress protein GroEl and reactive oxygen species were also observed in chlorobiphenyl-grown cells, indicating that these bacteria adjust their physiology with a stress response when they are confronted with compounds that serve as carbon and energy sources and at the same time are chemical stressors.     

Direct labeling of polyphosphate at the ultrastructural level in Saccharomyces cerevisiae by using the affinity of the polyphosphate binding domain of Escherichia coli exopolyphosphatase

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate and has many biological functions in prokaryotic and eukaryotic organisms. To investigate polyP localization, we developed a novel technique using the affinity of the recombinant polyphosphate binding domain (PPBD) of Escherichia coli exopolyphosphatase to polyP. An epitope-tagged PPBD was expressed and purified from E. coli. Equilibrium binding assay of PPBD revealed its high affinity for long-chain polyP and its weak affinity for short-chain polyP and nucleic acids. To directly demonstrate polyP localization in Saccharomyces cerevisiae on resin sections prepared by rapid freezing and freeze-substitution, specimens were labeled with PPBD containing an epitope tag and then the epitope tag was detected by an indirect immunocytochemical method. A goat anti-mouse immunoglobulin G antibody conjugated with Alexa 488 for laser confocal microscopy or with colloidal gold for transmission electron microscopy was used. When the S. cerevisiae was cultured in yeast extract-peptone-dextrose medium (10 mM phosphate) for 10 h, polyP was distributed in a dispersed fashion in vacuoles in successfully cryofixed cells. A few polyP signals of the labeling were sometimes observed in cytosol around vacuoles with electron microscopy. Under our experimental conditions, polyP granules were not observed. Therefore, it remains unclear whether the method can detect the granule form. The method directly demonstrated the localization of polyP at the electron microscopic level for the first time and enabled the visualization of polyP localization with much higher specificity and resolution than with other conventional methods.     

Calreticulin: conserved protein and diverse functions in plants

Calreticulin (CRT) is a key Ca²⁺-binding protein mainly resident in the endoplasmic reticulum (ER), which is highly conserved and extensively expressed in all eukaryotic organisms investigated. The protein plays important roles in a variety of cellular processes including Ca²⁺ signaling and protein folding. Although calreticulin has been well characterized in mammalian systems, increased investigations have demonstrated that plant CRTs have a number of specific properties different from their animal counterparts. Recent developments on plant CRTs have highlighted the significance of CRTs in plants growth and development as well as biotic and abiotic stress responses. There are at least two distinct groups of calreticulin isoforms in higher plants. Glycosylation of CRT was uniquely observed in plants. In this article, we will describe our current understanding of plant calreticulin gene family, protein structure, cellular localization, and diverse functions in plants. We also discuss the prospects of using this information for genetic improvements of crop plants.     

植物多胺代谢途径研究进展

多胺是一类小分子生物活性物质,广泛存在于生物体内,与植物的生长发育、衰老及抗逆性都有着密切的联系。目前,在植物中的多胺合成途径已经基本揭示,其生理作用在分子水平上逐步得到阐明。对多胺合成突变体和各种转基因植物的研究也使得人们更深入地了解了多胺以及其合成代谢相关酶在植物生长发育等生理过程中的重要作用。以下概述了植物多胺代谢途径,重点综述了代谢途径中各基因的功能及遗传操作的最新进展,并对将来的研究方向尤其是相关基因在植物抗逆境 (包括生物和非生物逆境) 基因工程方面的应用作了讨论。     

Cytoskeleton in plant development

The plant cytoskeleton has crucial functions in a number of cellular processes that are essential for cell morphogenesis, organogenesis and development. These functions have been intensively investigated using single cell model systems. With the recent characterization of plant mutants that show aberrant organogenesis resulting from primary defects in cytoskeletal organization, an integrated understanding of the importance of the cytoskeleton for plant development has begun to emerge. Newly established techniques that allow the non-destructive visualization of microtubules or actin filaments in living plant cells and organs will further advance this understanding.