Emerging findings from studies of phospholipase D in model organisms (and a short update on phosphatidic acid effectors)

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to generate phosphatidic acid and choline. Historically, much PLD work has been conducted in mammalian settings although genes encoding enzymes of this family have been identified in all eukaryotic organisms. Recently, important insights on PLD function are emerging from work in yeast, but much less is known about PLD in other organisms. In this review we will summarize what is known about phospholipase D in several model organisms, including C. elegans, D. discoideum, D. rerio and D. melanogaster. In the cases where knockouts are available (C. elegans, Dictyostelium and Drosophila) the PLD gene(s) appear not to be essential for viability, but several studies are beginning to identify pathways where this activity has a role. Given that the proteins in model organisms are very similar to their mammalian counterparts, we expect that future studies in model organisms will complement and extend ongoing work in mammalian settings. At the end of this review we will also provide a short update on phosphatidic acid targets, a topic last reviewed in 2006.     

Comparative analysis of the relationship between translation efficiency and sequence features of endogenous proteins in multiple organisms

The translation efficiency of protein genes is known to be affected by sequence features. Previous studies have found that various sequence features based on codon usage and mRNA secondary structure contribute to translation efficiency. However, most studies have focused on a specific organism, usually a model organism such as Escherichia coli or Saccharomyces cerevisiae. Here, we investigate whether the relationship between translation efficiency and sequence features is conserved among multiple organisms using publicly available ribosome profiling data and RNA-Seq data. We analyze nine organisms from various taxa: Staphylococcus aureus, five species of Streptomyces, two strains of E. coli, and S. cerevisiae. We reveal that the relationship between translation efficiency and sequence features differs across organisms, partly reflecting their taxonomy. The codon adaptation index shows high correlation in all analyzed organisms. Our study provides an insight into the diversity and commonality of sequence determinants of protein expression in these organisms.     

Genome-Wide Screen in Saccharomyces cerevisiae Identifies Vacuolar Protein Sorting,Autophagy, Biosynthetic,and tRNA Methylation Genes Involved in Life Span Regulation

The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.     

Potential distributional shifts in North America of allelopathic invasive plant species under climate change models

Predictive studies play a crucial role in the study of biological invasions of terrestrial plants under possible climate change scenarios. Invasive species are recognized for their ability to modify soil microbial communities and influence ecosystem dynamics. Here, we focused on six species of allelopathic flowering plants—Ailanthus altissima, Casuarina equisetifolia, Centaurea stoebe ssp. micranthos, Dioscorea bulbifera, Lantana camara, and Schinus terebinthifolia—that are invasive in North America and examined their potential to spread further during projected climate change. We used Species Distribution Models (SDMs) to predict future suitable areas for these species in North America under several proposed future climate models. ENMEval and Maxent were used to develop SDMs, estimate current distributions, and predict future areas of suitable climate for each species. Areas with the greatest predicted suitable climate in the future include the northeastern and the coastal northwestern regions of North America. Range size estimations demonstrate the possibility of extreme range loss for these invasives in the southeastern United States, while new areas may become suitable in the northeastern United States and southeastern Canada. These findings show an overall northward shift of suitable climate during the next few decades, given projected changes in temperature and precipitation. Our results can be utilized to analyze potential shifts in the distribution of these invasive species and may aid in the development of conservation and management plans to target and control dissemination in areas at higher risk for potential future invasion by these allelopathic species.     

Selection and validation of reference genes for real-time RT-PCR studies in the non-model species Delomys sublineatus, an endemic Brazilian rodent

Quantitative real-time RT-PCR (qRT-PCR) is a sensitive technique for gene expression analysis. A critical factor for creating reliable data in relative quantification is the normalization of the expression data of genes of interest. Therefore the needed normalization factor is calculated out of the expression data of co-amplified genes that are stable expressed in the certain sample material, the so-called reference genes. In this study, we demonstrate the important process of validating potential reference genes using a non-model species. As there are almost no sequences known of the Pallid Atlantic Forest Rat (Delomys sublineatus), a rodent used as indicator species in conservation studies of the endangered Brazilian rainforest, suitable primer sets are more problematic to find than in model species. Out of nine tested primer sets designed for the fully sequenced Mus musculus, five could be used for the establishment of a proper running SYBR-Green assay and validation of their constant expression. qRT-PCR results of 12 cDNAs of Delomys livers were analyzed with three different validation software programs: BestKeeper, NormFinder and geNorm. Our approach showed that out of the five (Sdha, Canx, Pgk1, Actb and Actg1) potential reference genes, the first four should be used for accurate normalization in further relative quantification analyses. Transferring data from close-by model organisms makes high sensitive real-time RT-PCR applicable even to free-ranging non-model organisms. Our approach might be suitable for other non-model organisms.     

Molecular biology of extremophiles: recent progress on the hyperthermophilic archaeon Sulfolobus

Extremophiles are microorganisms that flourish in habitats of extreme temperature, pH, salinity, or pressure. All extreme environments are dominated by microorganisms belonging to Archaea, the third domain of life, evolutionary distinct from Bacteria and Eucarya. Over the past few years the biology of extremophilic Archaea has stimulated a lot of interest, aimed at understanding at molecular level the adaptation to their life conditions, as well as their evolutionary relationships to other organisms. Here, we review recent insights in the molecular biology of thermoacidophilic Archaea of the genus Sulfolobus, which has been used as a model system for biochemical, structural, and genetic studies in Archaea and extremophiles in general. With the recent completion of the genome sequence of Sulfolobus solfataricus it is expected that these organisms will contribute new discoveries in the near future. This revised version was published online in August 2006 with corrections to the Cover Date.     

The changing epitome of species identification – DNA barcoding

The discipline taxonomy (the science of naming and classifying organisms, the original bioinformatics and a basis for all biology) is fundamentally important in ensuring the quality of life of future human generation on the earth; yet over the past few decades, the teaching and research funding in taxonomy have declined because of its classical way of practice which lead the discipline many a times to a subject of opinion, and this ultimately gave birth to several problems and challenges, and therefore the taxonomist became an endangered race in the era of genomics. Now taxonomy suddenly became fashionable again due to revolutionary approaches in taxonomy called DNA barcoding (a novel technology to provide rapid, accurate, and automated species identifications using short orthologous DNA sequences). In DNA barcoding, complete data set can be obtained from a single specimen irrespective to morphological or life stage characters. The core idea of DNA barcoding is based on the fact that the highly conserved stretches of DNA, either coding or non coding regions, vary at very minor degree during the evolution within the species. Sequences suggested to be useful in DNA barcoding include cytoplasmic mitochondrial DNA (e.g. cox1) and chloroplast DNA (e.g. rbcL, trnL-F, matK, ndhF, and atpB rbcL), and nuclear DNA (ITS, and house keeping genes e.g. gapdh). The plant DNA barcoding is now transitioning the epitome of species identification; and thus, ultimately helping in the molecularization of taxonomy, a need of the hour. The ‘DNA barcodes’ show promise in providing a practical, standardized, species-level identification tool that can be used for biodiversity assessment, life history and ecological studies, forensic analysis, and many more.     

The Rate and Molecular Spectrum of Spontaneous Mutations in the GC-Rich Multichromosome Genome of Burkholderia cenocepacia

Spontaneous mutations are ultimately essential for evolutionary change and are also the root cause of many diseases. However, until recently, both biological and technical barriers have prevented detailed analyses of mutation profiles, constraining our understanding of the mutation process to a few model organisms and leaving major gaps in our understanding of the role of genome content and structure on mutation. Here, we present a genome-wide view of the molecular mutation spectrum in Burkholderia cenocepacia, a clinically relevant pathogen with high %GC content and multiple chromosomes. We find that B. cenocepacia has low genome-wide mutation rates with insertion–deletion mutations biased toward deletions, consistent with the idea that deletion pressure reduces prokaryotic genome sizes. Unlike prior studies of other organisms, mutations in B. cenocepacia are not AT biased, which suggests that at least some genomes with high %GC content experience unusual base-substitution mutation pressure. Importantly, we also observe variation in both the rates and spectra of mutations among chromosomes and elevated G:C > T:A transversions in late-replicating regions. Thus, although some patterns of mutation appear to be highly conserved across cellular life, others vary between species and even between chromosomes of the same species, potentially influencing the evolution of nucleotide composition and genome architecture.     

A New Method for Genome-wide Marker Development and Genotyping Holds Great Promise for Molecular Primatology

Over the last two decades primatologists have benefited from the use of numerous molecular markers to study various aspects of primate behavior and evolutionary history. However, most of the studies to date have been based on a single locus, usually mitochondrial DNA, or a few nuclear markers, e.g., microsatellites. Unfortunately, the use of such markers not only is unable to address successfully important questions in primate population genetics and phylogenetics (mainly because of the discordance between gene tree and species tree), but also their development is often a time-consuming and expensive task. The advent of next-generation sequencing allows researchers to generate large amounts of genomic data for nonmodel organisms. However, whole genome sequencing is still cost prohibitive for most primate species. We here introduce a second-generation sequencing technique for genotyping thousands of genome-wide markers for nonmodel organisms. Restriction site–associated DNA sequencing (RAD-seq) reduces the complexity of the genome and allows inexpensive and fast discovery of thousands of markers in many individuals. Here, we describe the principles of this technique and we demonstrate its application in five primates, Microcebus sp., Cebus sp., Theropithecus gelada, Pan troglodytes, and Homo sapiens, representing some of the major lineages within the order. Despite technical and bioinformatic challenges, RAD-seq is a promising method for multilocus phylogenetic and population genetic studies in primates, particularly in young clades in which a high number of orthologous regions are likely to be found across populations or species.     

Simple Microfluidic Devices for in vivo Imaging of C. elegans,Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques ^1-3. Microfluidic devices have been used for sub-cellular imaging ^4,5, in vivo laser microsurgery ^2,6 and cellular imaging ^4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction ^5,8, tapered channels ^6,7,9, deformable membranes ^2-4,10, suction with additional cooling ⁵, anesthetic gas ¹¹, temperature sensitive gels ¹², cyanoacrylate glue ¹³ and anesthetics such as levamisole ^14,15. Commonly used anesthetics influence synaptic transmission ^16,17 and are known to have detrimental effects on sub-cellular neuronal transport ⁴. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 μm to ~800 μm for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F⁴).Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.     

Application and commercialization of nematodes

While nematodes are most commonly known for their negative impact on plants, animals, and humans, there are a number of species which are commercially explored. This review highlights some of the most important success stories for the application of nematodes. They are used as bioindicators in ecological and toxicity studies, as model organisms for elucidating fundamental biological questions and for high throughput screening of drugs. Besides these indirect uses, direct applications include the use of Beddingia siricidicola against a major forest pest and the commercialization of Steinernema, Heterorhabditis, and Phasmarhabditis as biological pest control products. New directions for the commercialization of nematodes are the use as living food, specifically loaded with essential nutrients for various fish and shrimp larvae. Even human parasites or closely related species have been successfully used for curing autoimmune disorders and are currently in the process of being developed as drugs. With the striving development of life sciences, we are likely to see more applications for nematodes in the future. A prerequisite is that we continue to explore the vast number of yet undiscovered nematode species.     

Insights into archaeal chaperone machinery: a network-based approach

Molecular chaperones are a diverse group of proteins that ensure proteome integrity by helping the proteins fold correctly and maintain their native state, thus preventing their misfolding and subsequent aggregation. The chaperone machinery of archaeal organisms has been thought to closely resemble that found in humans, at least in terms of constituent players. Very few studies have been ventured into system-level analysis of chaperones and their functioning in archaeal cells. In this study, we attempted such an analysis of chaperone-assisted protein folding in archaeal organisms through network approach using Picrophilus torridus as model system. The study revealed that DnaK protein of Hsp70 system acts as hub in protein-protein interaction network. However, DnaK protein was present only in a subset of archaeal organisms and absent from many archaea, especially members of Crenarchaeota phylum. Therefore, a similar network was created for another archaeal organism, Sulfolobus solfataricus, a member of Crenarchaeota. The chaperone network of S. solfataricus suggested that thermosomes played an integral part of hub proteins in archaeal organisms, where DnaK was absent. We further compared the chaperone network of archaea with that found in eukaryotic systems, by creating a similar network for Homo sapiens. In the human chaperone network, the UBC protein, a part of ubiquitination system, was the most important module, and interestingly, this system is known to be absent in archaeal organisms. Comprehensive comparison of these networks leads to several interesting conclusions regarding similarities and differences within archaeal chaperone machinery in comparison to humans.     

Consolidated Bioprocessing of Lignocellulosic Feedstocks for Ethanol Fuel Production

Ethanol fuel can be produced renewably from numerous plant and waste materials, but harnessing the energy of lignocellulosic feedstocks has been particularly challenging in the development of this alternative fuel as a substitute for petroleum-based fuels. Consolidated bioprocessing has the potential to make the conversion of biomass to fuel an economical process by combining enzyme production, polysaccharide hydrolysis, and sugar fermentation into a single unit operation. This consolidation of steps takes advantage of the synergistic nature of enzyme systems but requires the use of one or a few organisms capable of producing highly efficient cellulolytic enzymes and fermenting most of the resulting sugars to ethanol with minimal byproduct formation while tolerating high levels of ethanol. In this review, conventional ethanol production, consolidated bioprocessing, and simultaneous saccharification and fermentation are described and compared. Several wild-type and genetically engineered microorganisms, including strains of Clostridium thermocellum, Saccharomyces cerevisiae, Klebsiella oxytoca, Escherichia coli, Flammulina velutipes, and Zymomonas mobilis, among others, are highlighted for their potential in consolidated bioprocessing. This review examines the favorable and undesirable qualities of these microorganisms and their enzyme systems, process engineering considerations for particular organisms, characteristics of cellulosomes, enzyme engineering strategies, progress in commercial development, and the impact of these topics on current and future research.     

Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP

The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales, Cycloclasticus and Colwellia, were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly ¹³C-labelled hydrocarbons, we identified several aliphatic (Alcanivorax, Marinobacter)- and polycyclic aromatic hydrocarbon (Alteromonas, Cycloclasticus, Colwellia)-degrading bacteria. We also isolated several strains (Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter and Pseudoalteromonas) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbon-degrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil.     

A Staging Scheme for the Development of the Moth Midge Clogmia albipunctata

Model organisms, such as Drosophila melanogaster, allow us to address a wide range of biological questions with experimental rigour. However, studies in model species need to be complemented by comparative studies if we are to fully understand the functional properties and evolutionary history of developmental processes. The establishment of new model organisms is crucial for this purpose. One of the first essential steps to establish a species as an experimental model is to carefully describe its life cycle and development. The resulting staging scheme serves as a framework for molecular studies, and allows us to homologise developmental processes between species. In this paper, we have characterised the life cycle and development of an emerging non-drosophilid dipteran model system: the moth midge Clogmia albipunctata. In particular, we focus on early embryogenesis (cleavage and blastoderm cycles before gastrulation), on formation and retraction of extraembryonic tissues, and on formation of the germ line. Considering the large evolutionary distance between the two species (approximately 250 million years), we find that the development of C. albipunctata is remarkably conserved compared to D. melanogaster. On the other hand, we detect significant differences in morphology and timing affecting the development of extraembryonic tissues and the germ line. Moreover, C. albipunctata shows several heterochronic shifts, and lacks head involution and associated processes during late stages of development.