Short read sequence typing (SRST): multi-locus sequence types from short reads

ABSTRACT: BACKGROUND: Multi-locus sequence typing (MLST) has become the gold standard for population analyses of bacterial pathogens. This method focuses on the sequences of a small number of loci (usually seven) to divide the population and is simple, robust and facilitates comparison of results between laboratories and over time. Over the last decade, researchers and population health specialists have invested substantial effort in building up public MLST databases for nearly 100 different bacterial species, and these databases contain a wealth of important information linked to MLST sequence types such as time and place of isolation of isolation, host or niche, serotype and even clinical or drug resistance profiles. Recent advances in sequencing technology mean it is increasingly feasible to perform bacterial population analysis at the whole genome level. This offers massive gains in resolving power and genetic profiling compared to MLST, and will eventually replace MLST for bacterial typing and population analysis. However given the wealth of data currently available in MLST databases, it is crucial to maintain backwards compatibility with MLST schemes so that new genome analyses can be understood in their proper historical context. RESULTS: We present a software tool, SRST, for quick and accurate retrieval of sequence types from short read sets, using inputs easily downloaded from public databases. SRST assigns alleles using read mapping and an allele assignment score incorporating sequence coverage and variability, to determine the most likely allele. Analysis of over 3,500 loci in more than 500 publicly accessible Illumina read sets showed SRST to be highly accurate at allele assignment. SRST output is compatible with common analysis tools such as eBURST, Clonal Frame or PhyloViz, allowing easy comparison between novel genome data and MLST data. Alignment, fastq and pileup files can also be generated for novel alleles. CONCLUSIONS: SRST is a novel software tool for accurate assignment of sequence types using short read data. Several uses for the tool are demonstrated, including quality control for high-throughput sequencing projects, plasmid MLST and analysis of genomic data during outbreak investigation. SRST is open-source, requires Python, BWA and SamTools, and is available from http://srst.sourceforge.net.     

On the use of weather data in ecological studies along altitudinal and latitudinal gradients

Global warming has created a need for studies along climatic gradients to assess the effects of temperature on ecological processes. Altitudinal and latitudinal gradients are often used as such, usually in combination with air temperature data from the closest weather station recorded at 1.5–2 m above the ground. However, many ecological processes occur in, at, or right above the soil surface. To evaluate how representative the commonly used weather station data are for the microclimate relevant for soil surface biota, we compared weather station temperatures for an altitudinal (500–900 m a.s.l.) and a latitudinal gradient (49–68°N) with data obtained by temperature sensors placed right below the soil surface at five sites along these gradients. The mean annual temperatures obtained from weather stations and adjusted using a lapse rate of ?5.5°C km^?1 were between 3.8°C lower and 1.6°C higher than those recorded by the temperature sensors at the soil surface, depending on the position along the gradients. The monthly mean temperatures were up to 10°C warmer or 5°C colder at the soil surface. The within‐site variation in accumulated temperature was as high as would be expected from a 300 m change in altitude or from a 4° change in latitude or a climate change scenario corresponding to warming of 1.6–3.8°C. Thus, these differences introduced by the decoupling are significant from a climate change perspective, and the results demonstrate the need for incorporating microclimatic variation when conducting studies along altitudinal or latitudinal gradients. We emphasize the need for using relevant temperature data in climate impact studies and further call for more studies describing the soil surface microclimate, which is crucial for much of the biota.     

Plant species richness belowground: higher richness and new patterns revealed by next-generation sequencing

Variation in plant species richness has been described using only aboveground vegetation. The species richness of roots and rhizomes has never been compared with aboveground richness in natural plant communities. We made direct comparisons of grassland plant richness in identical volumes (0.1 × 0.1 × 0.1 m) above and below the soil surface, using conventional species identification to measure aboveground richness and 454 sequencing of the chloroplast trnL(UAA) intron to measure belowground richness. We described above- and belowground richness at multiple spatial scales (from a neighbourhood scale of centimetres to a community scale of hundreds of metres), and related variation in richness to soil fertility. Tests using reference material indicated that 454 sequencing captured patterns of species composition and abundance with acceptable accuracy. At neighbourhood scales, belowground richness was up to two times greater than aboveground richness. The relationship between above- and belowground richness was significantly different from linear: beyond a certain level of belowground richness, aboveground richness did not increase further. Belowground richness also exceeded that of aboveground at the community scale, indicating that some species are temporarily dormant and absent aboveground. Similar to other grassland studies, aboveground richness declined with increasing soil fertility; in contrast, the number of species found only belowground increased significantly with fertility. These results indicate that conventional aboveground studies of plant richness may overlook many coexisting species, and that belowground richness becomes relatively more important in conditions where aboveground richness decreases. Measuring plant belowground richness can considerably alter perceptions of biodiversity and its responses to natural and anthropogenic factors.     

The role of leaf lobation in elongation responses to shade in the rosette-forming forb Serratula tinctoria (Asteraceae)

BACKGROUND AND AIMS: Lobed leaves are considered selectively advantageous in conditions of high irradiance. However, most studies have involved woody species, with only a few considering the role of leaf lobation in herbaceous rosette species. In this study, it is hypothesized that, in addition to its adaptive value in high light, leaf lobation may add to the function of petioles as vertical spacers in herbaceous species in conditions of strong competition for light. METHODS: To test this hypothesis, leaf development was examined under seasonally changing natural light conditions and a field experiment was conducted in which light climate was manipulated in a wooded meadow population of Serratula tinctoria. KEY RESULTS: No changes in leaf lobation were observed in response to experimental shading or different natural light conditions. However, in tall herbaceous vegetation, plants with highly lobed leaves achieved significantly greater vertical elongation than plants with less-lobed leaves. In contrast to herbaceous shade, tree shade had no effect on leaf elongation, suggesting differential responsiveness to competition from neighbouring herbs versus overhead shade. In shading treatments, imposed shade could only be responded to by the elongation of leaves that were produced late in development. CONCLUSIONS: The results show that extensive leaf lobation can enable greater leaf elongation in response to shade from surrounding herbaceous vegetation. The different morphological responses displayed by Serratula tinctoria to different types of shade demonstrate the importance of critically assessing experimental designs when investigating phenotypic plasticity in response to shade.     

Agricultural Policies Exacerbate Honeybee Pollination Service Supply-Demand Mismatches Across Europe

Declines in insect pollinators across Europe have raised concerns about the supply of pollination services to agriculture. Simultaneously, EU agricultural and biofuel policies have encouraged substantial growth in the cultivated area of insect pollinated crops across the continent. Using data from 41 European countries, this study demonstrates that the recommended number of honeybees required to provide crop pollination across Europe has risen 4.9 times as fast as honeybee stocks between 2005 and 2010. Consequently, honeybee stocks were insufficient to supply >90% of demands in 22 countries studied. These findings raise concerns about the capacity of many countries to cope with major losses of wild pollinators and highlight numerous critical gaps in current understanding of pollination service supplies and demands, pointing to a pressing need for further research into this issue.     

X Chromosome-linked Inhibitor of Apoptosis Regulates Cell Death Induction by Proapoptotic Receptor Agonists

Proapoptotic receptor agonists cause cellular demise through the activation of the extrinsic and intrinsic apoptotic pathways. Inhibitor of apoptosis (IAP) proteins block apoptosis induced by diverse stimuli. Here, we demonstrate that IAP antagonists in combination with Fas ligand (FasL) or the death receptor 5 (DR5) agonist antibody synergistically stimulate death in cancer cells and inhibit tumor growth. Single-agent activity of IAP antagonists relies on tumor necrosis factor-α signaling. By contrast, blockade of tumor necrosis factor-α does not affect the synergistic activity of IAP antagonists with FasL or DR5 agonist antibody. In most cancer cells, proapoptotic receptor agonist-induced cell death depends on amplifying the apoptotic signal via caspase-8-mediated activation of Bid and subsequent activation of the caspase-9-dependent mitochondrial apoptotic pathway. In the investigated cancer cell lines, induction of apoptosis by FasL or DR5 agonist antibody can be inhibited by knockdown of Bid. However, knockdown of X chromosome-linked IAP (XIAP) or antagonism of XIAP allows FasL or DR5 agonist antibody to induce activation of effector caspases efficiently without the need for mitochondrial amplification of the apoptotic signal and thus rescues the effect of Bid knockdown in these cells.     

Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP2(1)

A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.     

Cell Surface Mechanics Gate Embryonic Stem Cell Differentiation

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