Genetic and phylogenetic evidence for horizontal gene transfer among ecologically disparate groups of marine Vibrio

Vibrio represents a diverse bacterial genus found in different niches of the marine environment, including numerous genera of marine sponges (phylum Porifera), inhabiting different depths and regions of benthic seas, that are potentially important in driving adaptive change among Vibrio spp. Using 16S rRNA gene sequencing, a previous study showed that sponge‐derived (SD) vibrios clustered with their mainstream counterparts present in shallow, coastal ecosystems, suggesting a genetic relatedness between these populations. Sequences from the topA, ftsZ, mreB, rpoD, rctB and toxR genes were used to investigate the degree of relatedness existing between these two separate populations by examining their phylogenetic and genetic disparity. Phylogenies were constructed from the concatenated sequences of the six housekeeping genes using maximum‐parsimony, maximum‐likelihood and neighbour‐joining algorithms. Genetic recombination was evaluated using the incongruence length difference test, Split decomposition and measuring overall compatibility of sites. This combined technical approach provided evidence that SD Vibrio strains are largely genetically homologous to their shallow‐water counterparts. Moreover, the analyses conducted support the existence of extensive horizontal gene transfer between these two groups, supporting the idea of a single panmictic population structure among vibrios from two seemingly distinct, marine environments.     

Correlative Imaging of Fluorescent Proteins in Resin-Embedded Plant Material1

Fluorescent proteins (FPs) were developed for live-cell imaging and have revolutionized cell biology. However, not all plant tissues are accessible to live imaging using confocal microscopy, necessitating alternative approaches for protein localization. An example is the phloem, a tissue embedded deep within plant organs and sensitive to damage. To facilitate accurate localization of FPs within recalcitrant tissues, we developed a simple method for retaining FPs after resin embedding. This method is based on low-temperature fixation and dehydration, followed by embedding in London Resin White, and avoids the need for cryosections. We show that a palette of FPs can be localized in plant tissues while retaining good structural cell preservation, and that the polymerized block face can be counterstained with cell wall probes. Using this method we have been able to image green fluorescent protein-labeled plasmodesmata to a depth of more than 40 μm beneath the resin surface. Using correlative light and electron microscopy of the phloem, we were able to locate the same FP-labeled sieve elements in semithin and ultrathin sections. Sections were amenable to antibody labeling, and allowed a combination of confocal and superresolution imaging (three-dimensional-structured illumination microscopy) on the same cells. These correlative imaging methods should find several uses in plant cell biology.The localization of fluorescent proteins (FPs) in cells and tissues has become one of the major tools in cell biology (Tsien, 1998; Shaner et al., 2005). Advances in confocal microscopy have meant that many proteins can be tagged with appropriate fluorescent markers and tracked as they move within and between cells (Chapman et al., 2005). Additional approaches involving photobleaching and photoactivation of FPs have opened up new avenues for exploring protein dynamics and turnover within cells (Lippincott-Schwartz et al., 2003). However, not all cells are amenable to live-cell imaging, which in plants is usually restricted to surface cells such as the leaf epidermis. An example is the phloem. The delicate nature of sieve elements and companion cells, which are under substantial hydrostatic pressure, has made studies of the fine structure of these cells particularly difficult (Knoblauch and van Bel, 1998). Despite this, significant advances have been made in imaging the phloem through inventive use of imaging protocols that allow living sieve elements to be observed as they translocate assimilates (for review, see Knoblauch and Oparka, 2012). However, determining the precise localization of the plethora of proteins located within the sieve element (SE)-companion cell (CC) complex remains a technical challenge. The phloem is the conduit for long-distance movement of macromolecules in plants, including viral genomes. For several viruses, the entry into the SE-CC complex is a crucial step that determines the capacity for long-distance movement. Identifying the cell types within the phloem that restrict the movement of some viruses is technically challenging due to the small size of phloem cells and their location deep within plant organs (Nelson and van Bel, 1998).The problems associated with imaging proteins in phloem tissues prompted us to explore methods for retaining the fluorescence of tagged proteins within tissues not normally amenable to confocal imaging. Previously, we used superresolution imaging techniques on fixed phloem tissues sectioned on a Vibroslice, providing information on the association between a viral movement protein (MP) and plasmodesmata (PD) within the SE-CC complex (Fitzgibbon et al., 2010). However, we wished to explore the same cells using correlative light and electron microscopy (CLEM), necessitating the development of methods that would allow sequential imaging of cells using fluorescence microscopy and transmission electron microscopy (TEM). To this end, we developed a protocol that retains fluorescent proteins through aldehyde fixation and resin embedding.In the last 10 years there has been significant interest in imaging fluorescent proteins in semithin sections (for review, see Cortese et al., 2009). Luby-Phelps and colleagues (2003) first described a method for retaining GFP fluorescence after fixation and resin embedding, but their method has not seen widespread application. The advent of superresolution imaging techniques (for review, see Bell and Oparka, 2011) has stimulated considerable interest in this field as the retention of fluorescence in thin sections means that cells can be imaged using techniques such as photoactivation light microscopy and stochastic optical reconstruction microscopy, allowing a lateral resolution of less than 10 nm to be achieved (Subach et al., 2009; Xu et al., 2012). A number of studies have described CLEM on the same cells (Luby-Phelps et al., 2003; Betzig et al., 2006; Watanabe et al., 2011). Advances in this field were reviewed recently (Jahn et al., 2012; see contributions in Muller-Reichert and Verkade, 2012). For example, Pfeiffer et al. (2003) were able to image SEs and CCs using high-pressure freezing, followed by freeze substitution in acetone and resin embedding. They then used thick optical sections of the tissue to locate cells of interest, and these were subsequently imaged using TEM. However, there have been few attempts to retain FPs in resin-embedded plant tissues. Thompson and Wolniak (2008) described the retention of mCitrine fused to an SE-plasma membrane protein in glycol methacrylate sections. The fluorescent signal was stable using wide-field microscopy but bleached rapidly under the confocal microscope.To date, cryosections have been the preferred choice for CLEM in mammalian tissues (Watanabe et al., 2011). Recently, Lee et al. (2011) chemically fixed Arabidopsis (Arabidopsis thaliana) seedlings, cut 50-μm sections, and examined these with a confocal microscope. After confocal mapping the sections were embedded in resin and thin sectioned. These authors were able to locate the same PD pit fields using confocal and TEM, providing important information on the localization of a novel PD protein. As general rule, cryosectioning is a time-consuming process, and subcellular details may be obscured in cryosections because of poor tissue contrast (Watanabe et al., 2011). A major problem with imaging FPs in resin sections has been that GFP and its derivatives are quenched by the acidic, oxidizing conditions required for fixation, dehydration, and embedding of delicate specimens (Tsien, 1998; Keene et al., 2008). Recently, however, Watanabe et al. (2011) explored the retention of FPs in Caenorhabditis elegans cells after fixation by different aldehydes and embedding media. These authors tested a range of resins and found that Citrine and tandem dimer Eos (tdEos) could be retained in methacrylate plastic sections. This material was difficult to cut thinly (<70 nm) compared to epoxy-based resins, but the authors obtained valuable correlative images using stimulated emission depletion microscopy and photoactivation light microscopy followed by low-voltage scanning electron microscopy.Because the retention of fluorescent proteins may differ between plant and animal cells, we explored a number of approaches for retaining fluorescent proteins in resin. Using low-temperature conditions (<8°C) during fixation and dehydration, we could retain strong fluorescence prior to tissue embedding. We also explored different embedding media and found that tissue could be effectively polymerized in London Resin (LR) White while retaining sufficient fluorescence for confocal imaging. Using water-dipping lenses, we were able to detect fluorescent proteins in optical sections up to 40 μm below the surface of the block face. Ultrathin sections from the same blocks showed good structural preservation and allowed CLEM. Subsequently, we cut 1- to 2-μm sections and examined these using confocal microscopy and three-dimensional-structured illumination microscopy (3D-SIM). Sections could be counterstained with a number of conventional fluorophores and antibodies, allowing colocalization studies. These simple methods allow successive imaging of FPs with the light and electron microscope, combining the strengths of both imaging platforms. We believe this approach will have significant utility for tissues that are recalcitrant to conventional confocal imaging.     

Generation of RNA pseudoknot structures with topological genus filtration

In this paper we present a sampling framework for RNA structures of fixed topological genus. We introduce a novel, linear time, uniform sampling algorithm for RNA structures of fixed topological genus g  , for arbitrary g>0$g>0$. Furthermore we develop a linear time sampling algorithm for RNA structures of fixed topological genus g   that are weighted by a simplified, loop-based energy functional. For this process the partition function of the energy functional has to be computed once, which has O(n²)$O(n^2)$ time complexity.     

Linking the evolution of habitat choice to ecosystem functioning: direct and indirect effects of pond-reproducing fire salamanders on aquatic-terrestrial subsidies

Shifts in life history traits and in the behaviour of species can potentially alter ecosystem functioning. The reproduction of the central European fire salamander (Salamandra salamandra), which usually deposits its larvae in first-order streams, in small pool and pond-like habitats, is an example of a recent local adaptation in this species. Here we aimed to quantify the direct and indirect effects of the predatory larvae on the aquatic food webs in the ponds and on the flux of matter between the ponds and adjacent terrestrial habitats. Our estimates are based on biomass data of the present pond fauna as well as on the analysis of stomach content data, growth rates and population dynamics of the salamander larvae in pond habitats. By their deposition of larvae in early spring, female fire salamanders import between 0.07 and 2.86 g dry mass m^?2 larval biomass into the ponds. Due to high mortality rates in the larval phase and the relatively small size at metamorphosis of the pond-adapted salamanders compared to stream-adapted ones, the biomass export of the metamorphosed salamanders clearly falls below the initial biomass import. Catastrophic events such as high water temperatures and low oxygen levels may even occasionally result in mass mortalities of salamander larvae and thus in a net 100 % import of the salamander biomass into the pond food webs. Indirect effects further accelerate this net import of matter into the aquatic habitat, e.g. the feeding of salamanders on aquatic insect larvae with the emergence of terrestrial adults—thus preventing export—and on terrestrial organisms that fall on the water surface (supporting import). This study demonstrates that the adaptation of salamanders to pond reproduction can alter food web linkages across ecosystem boundaries by enhancing the flux of materials and energy from terrestrial (i.e. forest) to the aquatic (i.e. pond) habitat.     

A comparison of the strength of biodiversity effects across multiple functions

In order to predict which ecosystem functions are most at risk from biodiversity loss, meta-analyses have generalised results from biodiversity experiments over different sites and ecosystem types. In contrast, comparing the strength of biodiversity effects across a large number of ecosystem processes measured in a single experiment permits more direct comparisons. Here, we present an analysis of 418 separate measures of 38 ecosystem processes. Overall, 45 % of processes were significantly affected by plant species richness, suggesting that, while diversity affects a large number of processes not all respond to biodiversity. We therefore compared the strength of plant diversity effects between different categories of ecosystem processes, grouping processes according to the year of measurement, their biogeochemical cycle, trophic level and compartment (above- or belowground) and according to whether they were measures of biodiversity or other ecosystem processes, biotic or abiotic and static or dynamic. Overall, and for several individual processes, we found that biodiversity effects became stronger over time. Measures of the carbon cycle were also affected more strongly by plant species richness than were the measures associated with the nitrogen cycle. Further, we found greater plant species richness effects on measures of biodiversity than on other processes. The differential effects of plant diversity on the various types of ecosystem processes indicate that future research and political effort should shift from a general debate about whether biodiversity loss impairs ecosystem functions to focussing on the specific functions of interest and ways to preserve them individually or in combination.     

Mating system variation in Veronica (Plantaginaceae): inferences from pollen/ovule ratios and other reproductive traits

The pollen–ovule ratio (P/O) is commonly used to estimate the mode of sexual reproduction in flowering plants. In previous studies, a clear correspondence has been detected between this character and the degree of autogamy. We here investigate variation in this character and its expected correlates in the genus Veronica (Plantaginaceae). Pollen–ovule ratios of 45 species representing eleven percent of all the species in the genus were investigated and compared with results from crossing experiments from previous studies. In addition, multiple populations of 17 of the 45 studied species were sampled and a controlled‐environment experiment was conducted to evaluate the extent of intraspecific variation. Moreover, relationships between P/O and other primary and secondary reproductive characters of the Veronica flower were investigated in relation to a phylogenetic hypothesis in order to determine the phylogenetic constraints on reproductive characters. The differences in P/O among species correspond well to the diversity of mating systems in Veronica and correlate well with other floral characters such as corolla size. These characters together seem to allow a powerful and fast tool to infer mating systems. However, causes for intraspecific variation of P/O, such as different cytotypes, ecotypes or different growth conditions, need to be considered.     

Novel phage display-derived mycolic acid-specific antibodies with potential for tuberculosis diagnosis

Tuberculosis is a major cause of mortality and morbidity due to infectious disease. However, current clinical diagnostic methodologies such as PCR, sputum culture, or smear microscopy are not ideal. Antibody-based assays are a suitable alternative but require specific antibodies against a suitable biomarker. Mycolic acid, which has been found in patient sputum samples and comprises a large portion of the mycobacterial cell wall, is an ideal target. However, generating anti-lipid antibodies using traditional hybridoma methodologies is challenging and has limited the exploitation of this lipid as a diagnostic marker. We describe here the isolation and characterization of four anti-mycolic acid antibodies from a nonimmune antibody phage display library that can detect mycolic acids down to a limit of 4.5ng. All antibodies were specific for the methoxy subclass of mycolic acid with weak binding for α mycolic acid and did not show any binding to closely related lipids or other Mycobacterium tuberculosis (Mtb) derived lipids. We also determined the clinical utility of these antibodies based on their limit of detection for mycobacteria colony forming units (CFU). In combination with an optimized alkaline hydrolysis method for rapid lipid extraction, these antibodies can detect 10⁵ CFU of Mycobacterium bovis BCG, a close relative of Mtb and therefore represent a novel approach for the development of diagnostic assays for lipid biomarkers.     

Mechanism of Bacterial Oligosaccharyltransferase: IN VITRO QUANTIFICATION OF SEQUON BINDING AND CATALYSIS*

N-Linked glycosylation is an essential post-translational protein modification in the eukaryotic cell. The initial transfer of an oligosaccharide from a lipid carrier onto asparagine residues within a consensus sequon is catalyzed by oligosaccharyltransferase (OST). The first X-ray structure of a complete bacterial OST enzyme, Campylobacter lari PglB, was recently determined. To understand the mechanism of PglB, we have quantified sequon binding and glycosylation turnover in vitro using purified enzyme and fluorescently labeled, synthetic peptide substrates. Using fluorescence anisotropy, we determined a dissociation constant of 1.0 μm and a strict requirement for divalent metal ions for consensus (DQNAT) sequon binding. Using in-gel fluorescence detection, we quantified exceedingly low glycosylation rates that remained undetected using in vivo assays. We found that an alanine in the −2 sequon position, converting the bacterial sequon to a eukaryotic one, resulted in strongly lowered sequon binding, with in vitro turnover reduced 50,000-fold. A threonine is preferred over serine in the +2 sequon position, reflected by a 4-fold higher affinity and a 1.2-fold higher glycosylation rate. The interaction of the +2 sequon position with PglB is modulated by isoleucine 572. Our study demonstrates an intricate interplay of peptide and metal binding as the first step of protein N-glycosylation.