Genome-Annotated Bacterial Collection of the Barley Rhizosphere Microbiota.

A culture collection of 41 bacteria isolated from the rhizosphere of cultivated barley (Hordeum vulgare subsp. vulgare) is available at the Division of Plant Sciences, University of Dundee (UK). The data include information on genes putatively implicated in nitrogen fixation, HCN channels, phosphate solubilization, and linked whole-genome sequences.

Biofilm hydrophobicity in environmental isolates of Bacillus subtilis.

Biofilms are communities of bacteria that are attached to a surface and surrounded by an extracellular matrix. The extracellular matrix protects the community from stressors in the environment, making biofilms robust. The Gram-positive soil bacterium Bacillus subtilis, particularly the isolate NCIB 3610, is widely used as a model for studying biofilm formation. B. subtilis NCIB 3610 forms colony biofilms that are architecturally complex and highly hydrophobic. The hydrophobicity is linked, in part, to the localisation of the protein BslA at the surface of the biofilm, which provides the community with increased resistance to biocides. As most of our knowledge about B. subtilis biofilm formation comes from one isolate, it is unclear if biofilm hydrophobicity is a widely distributed feature of the species. To address this knowledge gap, we collated a library of B. subtilis soil isolates and acquired their whole genome sequences. We used our novel isolates to examine biofilm hydrophobicity and found that, although BslA is encoded and produced by all isolates in our collection, hydrophobicity is not a universal feature of B. subtilis colony biofilms. To test whether the matrix exopolymer poly γ-glutamic acid could be masking hydrophobicity in our hydrophilic isolates, we constructed deletion mutants and found, contrary to our hypothesis, that the presence of poly γ-glutamic acid was not the reason for the observed hydrophilicity. This study highlights the natural variation in the properties of biofilms formed by different isolates and the importance of using a more diverse range of isolates as representatives of a species.

Automated Phylogenetic Analysis Using Best Reciprocal BLAST.

Reconstruction of the evolutionary history of specific protein-coding genes is an essential component of the biological sciences toolkit and relies on identification of orthologs (a gene in different organisms related by vertical descent from a common ancestor and usually presumed to have the same or similar function) and paralogs (a gene related to another in the same organism by descent from a single ancestral gene which may, or may not, retain the same/similar function) across a range of taxa. While obviously essential for the reconstruction of evolutionary histories, ortholog identification is of importance for protein expression, modeling for drug discovery programs, identification of critical residues and other studies. Here we describe an automated system for searching for orthologs and paralogs in eukaryotic organisms. Unlike manual methods the system is fast, requiring minimal user input while still being highly configurable.

EMSY expression affects multiple components of the skin barrier with relevance to atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a common, complex, and highly heritable inflammatory skin disease. Genome-wide association studies offer opportunities to identify molecular targets for drug development. A risk locus on chromosome 11q13.5 lies between 2 candidate genes, EMSY and LRRC32 (leucine-rich repeat-containing 32) but the functional mechanisms affecting risk of AD remain unclear. OBJECTIVES: We sought to apply a combination of genomic and molecular analytic techniques to investigate which genes are responsible for genetic risk at this locus and to define mechanisms contributing to atopic skin disease. METHODS: We used interrogation of available genomic and chromosome conformation data in keratinocytes, small interfering RNA (siRNA)-mediated knockdown in skin organotypic culture and functional assessment of barrier parameters, mass spectrometric global proteomic analysis and quantitative lipid analysis, electron microscopy of organotypic skin, and immunohistochemistry of human skin samples. RESULTS: Genomic data indicate active promoters in the genome-wide association study locus and upstream of EMSY; EMSY, LRRC32, and intergenic variants all appear to be within a single topologically associating domain. siRNA-knockdown of EMSY in organotypic culture leads to enhanced development of barrier function, reflecting increased expression of structural and functional proteins, including filaggrin and filaggrin-2, as well as long-chain ceramides. Conversely, overexpression of EMSY in keratinocytes leads to a reduction in markers of barrier formation. Skin biopsy samples from patients with AD show greater EMSY staining in the nucleus, which is consistent with an increased functional effect of this transcriptional control protein. CONCLUSION: Our findings demonstrate an important role for EMSY in transcriptional regulation and skin barrier formation, supporting EMSY inhibition as a therapeutic approach.

Use of next-generation sequencing in the CHAT study (acute HCV in HIV): effect of baseline resistance-associated NS3 variants on treatment failure.

Background The epidemic of acute HCV infection among HIV-infected men who have sex with men (MSM) is ongoing. Transmission of drug-resistant variants (DRVs) after HCV treatment failure could pose a major threat to the effectiveness of future therapies. We determined the baseline prevalence of pre-existing DRVs in the HCV NS3 protease gene and their effects on the addition of telaprevir (TVR) to standard pegylated interferon and ribavirin (PEG-IFN/RBV) for acute HCV infection in individuals enrolled in a multicentre randomized controlled trial (2013 and 2014). Methods The HCV NS3 viral protease was analyzed using Sanger and next-generation sequencing (NGS) for DRVs at baseline (n = 31), and at viral breakthrough following TVR-based treatment (n = 3) or PEG-IFN/RBV alone (n = 2). Results Sequence analysis indicated that all individuals were infected with HCV genotype 1a. Complete (100%) concordance was seen between Sanger and NGS for high levels of mutant viral populations. The simeprevir-associated Q80K variant was present at high frequency in the German samples (7/11-64%) and infrequently in the UK samples (1/20-5%). In the three TVR-based treatment failures, V36M/l and R155K/T emerged, but not R155G which was detectable at low levels in two individuals at baseline. Failure rate at week 24 was 26.7% (with baseline DRVs) vs. 6.3% (without baseline DRVs), p = 0.17). Comparison of sequences pre- and post-therapy in 5 who failed therapy revealed the emergence of not previously described variants V193G, E176K, P189S (on TVR), and V181S in one instance each. Conclusion The presence of baseline DRVs for the NS3 protease gene of HCV genotype 1a did not appear to predict treatment failure in our patient cohort. Where detected, Q80K was present at high levels (>98%), but had no effect on outcomes and remained high after failure.

Structure and evolution of barley powdery mildew effector candidates.

BACKGROUND: Protein effectors of pathogenicity are instrumental in modulating host immunity and disease resistance. The powdery mildew pathogen of grasses Blumeria graminis causes one of the most important diseases of cereal crops. B. graminis is an obligate biotrophic pathogen and as such has an absolute requirement to suppress or avoid host immunity if it is to survive and cause disease. RESULTS: Here we characterise a superfamily predicted to be the full complement of Candidates for Secreted Effector Proteins (CSEPs) in the fungal barley powdery mildew parasite B. graminis f.sp. hordei. The 491 genes encoding these proteins constitute over 7% of this pathogen's annotated genes and most were grouped into 72 families of up to 59 members. They were predominantly expressed in the intracellular feeding structures called haustoria, and proteins specifically associated with the haustoria were identified by large-scale mass spectrometry-based proteomics. There are two major types of effector families: one comprises shorter proteins (100-150 amino acids), with a high relative expression level in the haustoria and evidence of extensive diversifying selection between paralogs; the second type consists of longer proteins (300-400 amino acids), with lower levels of differential expression and evidence of purifying selection between paralogs. An analysis of the predicted protein structures underscores their overall similarity to known fungal effectors, but also highlights unexpected structural affinities to ribonucleases throughout the entire effector super-family. Candidate effector genes belonging to the same family are loosely clustered in the genome and are associated with repetitive DNA derived from retro-transposons. CONCLUSIONS: We employed the full complement of genomic, transcriptomic and proteomic analyses as well as structural prediction methods to identify and characterize the members of the CSEPs superfamily in B. graminis f.sp. hordei. Based on relative intron position and the distribution of CSEPs with a ribonuclease-like domain in the phylogenetic tree we hypothesize that the associated genes originated from an ancestral gene, encoding a secreted ribonuclease, duplicated successively by repetitive DNA-driven processes and diversified during the evolution of the grass and cereal powdery mildew lineage.

Strategies towards sequencing complex crop genomes.

A report on the Strategies for de novo assemblies of complex crop genomes workshop held at The Genome Analysis Centre, Norwich, UK, 8-10 October 2012.

c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress.

The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13-22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.

Comparative sequence analysis of wheat and barley powdery mildew fungi reveals gene colinearity, dates divergence and indicates host-pathogen co-evolution.

The two fungal pathogens Blumeria graminis f. sp. tritici (B.g. tritici) and hordei (B.g. hordei) cause powdery mildew specifically in wheat or barley. They have the same life cycle, but their growth is restricted to the respective host. Here, we compared the sequences of two loci in both cereal mildews to determine their divergence time and their relationship with the evolution of their hosts. We sequenced a total of 273.3kb derived from B.g. tritici BAC sequences and compared them with the orthologous regions in the B.g. hordei genome. Protein-coding genes were colinear and well conserved. In contrast, the intergenic regions showed very low conservation mostly due to different integration patterns of transposable elements. To estimate the divergence time of B.g. tritici and B.g. hordei, we used conserved intergenic sequences including orthologous transposable elements. This revealed that B.g. tritici and B.g. hordei have diverged about 10 million years ago (MYA), two million years after wheat and barley (12 MYA). These data suggest that B.g. tritici and B.g. hordei have co-evolved with their hosts during most of their evolutionary history after host divergence, possibly after a short phase of host expansion when the same pathogen could still grow on the two diverged hosts.

Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism.

Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.

WebACT: an online genome comparison suite.

Comparison of related genomes is an enormously powerful technique for explaining phenotypic differences and revealing recent evolutionary events. Genomes evolve through a host of mechanisms including long- and short-range intragenomic rearrangements, insertion of laterally acquired DNA, gene loss, and single-nucleotide polymorphisms. The Artemis Comparison Tool (ACT) was developed to enable the intuitive visualization of the consequences of such events in the context of two or more aligned genomes. WebACT is an online resource designed to allow the alignment of up to five genomic sequences within the ACT environment without the need for local software installation. Comparisons can be carried out between uploaded sequences, or those selected from the EMBL or RefSeq databases, using BLASTZ, MUMmer, or Basic Local Alignment Search Tool (BLAST). Precomputed comparisons can be selected from a database covering all the completed bacterial chromosome and plasmid sequences in the Genome Reviews database (1). This allows the rapid visualization of regions of interest, without the need to handle the full genome sequences. Here, we describe the process of using WebACT to prepare comparisons for visualization, and the selection of precomputed comparisons from the database. The use of ACT to view the selected comparison is then explored using examples from bacterial genomes.

Simultaneous suppression of multiple genes by single transgenes. Down-regulation of three unrelated lignin biosynthetic genes in tobacco.

Many reports now describe the manipulation of plant metabolism by suppressing the expression of single genes. The potential of such work could be greatly expanded if multiple genes could be coordinately suppressed. In the work presented here, we test a novel method for achieving this by using single chimeric constructs incorporating partial sense sequences for multiple genes to target suppression of two or three lignin biosynthetic enzymes. We compare this method with a more conventional approach to achieving the same end by crossing plants harboring different antisense transgenes. Our results indicate that crossing antisense plants is less straightforward and predictable in outcome than anticipated. Most progeny had higher levels of target enzyme activity than predicted and had lost the expected modifications to lignin structure. In comparison, plants transformed with the chimeric partial sense constructs had more consistent high level suppression of target enzymes and had significant changes to lignin content, structure, and composition. It was possible to suppress three target genes coordinately using a single chimeric construct. Our results indicate that chimeric silencing constructs offer great potential for the rapid and coordinate suppression of multiple genes on diverse biochemical pathways and that the technique therefore deserves to be adopted by other researchers.