Genome sequence of Phaeobacter caeruleus type strain (DSM 24564T), a surface-associated member of the marine Roseobacter clade

In 2009 Phaeobacter caeruleus was described as a novel species affiliated with the marine Roseobacter clade, which, in turn, belongs to the class Alphaproteobacteria. The genus Phaeobacter is well known for members that produce various secondary metabolites. Here we report of putative quorum sensing systems, based on the finding of six N-acyl-homoserine lactone synthetases, and show that the blue color of P. caeruleus is probably due to the production of the secondary metabolite indigoidine. Therefore, P. caeruleus might have inhibitory effects on other bacteria. In this study the genome of the type strain DSM 24564^T was sequenced, annotated and characterized. The 5,344,419 bp long genome with its seven plasmids contains 5,227 protein-coding genes (3,904 with a predicted function) and 108 RNA genes.     

Nep1-like proteins as a target for plant pathogen control

The lack of efficient methods to control the major diseases of crops most important to agriculture leads to huge economic losses and seriously threatens global food security. Many of the most important microbial plant pathogens, including bacteria, fungi, and oomycetes, secrete necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), which critically contribute to the virulence and spread of the disease. NLPs are cytotoxic to eudicot plants, as they disturb the plant plasma membrane by binding to specific plant membrane sphingolipid receptors. Their pivotal role in plant infection and broad taxonomic distribution makes NLPs a promising target for the development of novel phytopharmaceutical compounds. To identify compounds that bind to NLPs from the oomycetes Pythium aphanidermatum and Phytophthora parasitica, a library of 587 small molecules, most of which are commercially unavailable, was screened by surface plasmon resonance. Importantly, compounds that exhibited the highest affinity to NLPs were also found to inhibit NLP-mediated necrosis in tobacco leaves and Phytophthora infestans growth on potato leaves. Saturation transfer difference-nuclear magnetic resonance and molecular modelling of the most promising compound, anthranilic acid derivative, confirmed stable binding to the NLP protein, which resulted in decreased necrotic activity and reduced ion leakage from tobacco leaves. We, therefore, confirmed that NLPs are an appealing target for the development of novel phytopharmaceutical agents and strategies, which aim to directly interfere with the function of these major microbial virulence factors. The compounds identified in this study represent lead structures for further optimization and antimicrobial product development.     

Snapshots of native pre-50S ribosomes reveal a biogenesis factor network and evolutionary specialization

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Acyl carriers used as substrates by the desaturases and elongases involved in very long-chain polyunsaturated fatty acids biosynthesis reconstituted in yeast

The health benefits attributed to very long-chain polyunsaturated fatty acids and the long term goal to produce them in transgenic oilseed crops have led to the cloning of all the genes coding for the desaturases and elongases involved in their biosynthesis. The encoded activities have been confirmed in vivo by heterologous expression, but very little is known about the actual acyl substrates involved in these pathways. Using a Delta 6-elongase and front-end desaturases from different organisms, we have reconstituted in Saccharomyces cerevisiae the biosynthesis of arachidonic acid from exogenously supplied linoleic acid in order to identify these acyl carriers. Acyl-CoA measurements strongly suggest that the elongation step involved in polyunsaturated fatty acids biosynthesis is taking place within the acyl-CoA pool. In contrast, detailed analyses of lipids revealed that the two desaturation steps (Delta 5 and Delta 6) occur predominantly at the sn-2 position of phosphatidylcholine when using Delta 5- and Delta 6-desaturases from lower plants, fungi, worms, and algae. The specificity of these Delta 6-desaturases for the fatty acid acylated at this particular position as well as a limiting re-equilibration with the acyl-CoA pool result in the accumulation of gamma-linolenic acid at the sn-2 position of phosphatidylcholine and prevent efficient arachidonic acid biosynthesis in yeast. We confirm by using a similar experimental approach that, in contrast, the human Delta 6-desaturase uses linoleoyl-CoA as substrate, which results in high efficiency of the subsequent elongation step. In addition, we report that Delta 12-desaturases have no specificity toward the lipid polar headgroup or the sn-position.     

Comparing High‐throughput Platforms for Sequencing the V4 Region of SSU‐rDNA in Environmental Microbial Eukaryotic Diversity Surveys

High‐throughput sequencing platforms are continuing to increase resulting read lengths, which is allowing for a deeper and more accurate depiction of environmental microbial diversity. With the nascent Reagent Kit v3, Illumina MiSeq now has the ability to sequence the eukaryotic hyper‐variable V4 region of the SSU‐rDNA locus with paired‐end reads. Using DNA collected from soils with analyses of strictly‐ and nearly identical amplicons, here we ask how the new Illumina MiSeq data compares with what we can obtain with Roche/454 GS FLX with regard to quantity and quality, presence and absence, and abundance perspectives. We show that there is an easy qualitative transition from the Roche/454 to the Illumina MiSeq platforms. The ease of this transition is more nuanced quantitatively for low‐abundant amplicons, although estimates of abundances are known to also vary within platforms.     

Simultaneous ex vivo quantification of antigen-specific CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cell responses using in vitro transcribed RNA

Assessment of antigen-specific T-cell responses has been greatly facilitated by development of ELISPOT and intracellular cytokine flow cytometry (CFC) assays. The use of autologous antigen presenting cells transfected with in vitro transcribed RNA as stimulators allows in principle quantification of antigen-specific T-cells independent of the knowledge of the epitopes. We describe here a cytokine secretion assay that enables simultaneous assessment of both antigen-specific CD4+ as well as CD8+ T-cells directly from clinical samples without the need for generation of dendritic cells. To this aim, bulk PBMCs were electroporated with RNA encoding the antigen fused to trafficking signal sequences derived from a MHC class I molecule and used as stimulators. With human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) as antigen we show that for measuring ex vivo T-cell responses in ELISPOT and CFC such stimulators are superior or at least equivalent to a pool of overlapping peptides representing the entire pp65 sequence as well as to untagged pp65 encoding RNA. This approach avoids the time consuming generation of dendritic cells as immune stimulators and, in particular when used in the context of the CFC, is robust, broadly applicable and fast.     

StreAM-$$T_g$$: algorithms for analyzing coarse grained RNA dynamics based on Markov models of connectivity-graphs

Background

In this work, we present a new coarse grained representation of RNA dynamics. It is based on adjacency matrices and their interactions patterns obtained from molecular dynamics simulations. RNA molecules are well-suited for this representation due to their composition which is mainly modular and assessable by the secondary structure alone. These interactions can be represented as adjacency matrices of k nucleotides. Based on those, we define transitions between states as changes in the adjacency matrices which form Markovian dynamics. The intense computational demand for deriving the transition probability matrices prompted us to develop StreAM-\(T_g\), a stream-based algorithm for generating such Markov models of k-vertex adjacency matrices representing the RNA.

Results

We benchmark StreAM-\(T_g\) (a) for random and RNA unit sphere dynamic graphs (b) for the robustness of our method against different parameters. Moreover, we address a riboswitch design problem by applying StreAM-\(T_g\) on six long term molecular dynamics simulation of a synthetic tetracycline dependent riboswitch (500 ns) in combination with five different antibiotics.

Conclusions

The proposed algorithm performs well on large simulated as well as real world dynamic graphs. Additionally, StreAM-\(T_g\) provides insights into nucleotide based RNA dynamics in comparison to conventional metrics like the root-mean square fluctuation. In the light of experimental data our results show important design opportunities for the riboswitch.