Recombination-based tools for introducing targeted genomic mutations in Mycobacterium tuberculosis are not efficient due to higher rate of illegitimate recombination compared with homologous DNA exchange. Moreover, involvement of multiple steps and specialized reagents make these tools cost ineffective. Here we introduce a novel clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) approach that efficiently represses expression of target genes in mycobacteria. CRISPRi system involves co-expression of the catalytically dead form of RNA-guided DNA endonuclease from the type II CRISPR system known as dCas9 and the small guide RNA specific to a target sequence, resulting in the DNA recognition complex that interferes with the transcription of corresponding DNA sequence. We show that co-expression of the codon-optimized dCas9 of S. pyogenes with sequence-specific guide RNA results in complete repression of individual or multiple targets in mycobacteria. CRISPRi thus offers a simple, rapid and cost-effective tool for selective control of gene expression in mycobacteria.

Key messageExploitation of data from a ryegrass breeding program has enabled rapid development and implementation of genomic selection for sward-based biomass yield with a twofold-to-threefold increase in genetic gain.AbstractGenomic selection, which uses genome-wide sequence polymorphism data and quantitative genetics techniques to predict plant performance, has large potential for the improvement in pasture plants. Major factors influencing the accuracy of genomic selection include the size of reference populations, trait heritability values and the genetic diversity of breeding populations. Global diversity of the important forage species perennial ryegrass is high and so would require a large reference population in order to achieve moderate accuracies of genomic selection. However, diversity of germplasm within a breeding program is likely to be lower. In addition, de novo construction and characterisation of reference populations are a logistically complex process. Consequently, historical phenotypic records for seasonal biomass yield and heading date over a 18-year period within a commercial perennial ryegrass breeding program have been accessed, and target populations have been characterised with a high-density transcriptome-based genotyping-by-sequencing assay. Ability to predict observed phenotypic performance in each successive year was assessed by using all synthetic populations from previous years as a reference population. Moderate and high accuracies were achieved for the two traits, respectively, consistent with broad-sense heritability values. The present study represents the first demonstration and validation of genomic selection for seasonal biomass yield within a diverse commercial breeding program across multiple years. These results, supported by previous simulation studies, demonstrate the ability to predict sward-based phenotypic performance early in the process of individual plant selection, so shortening the breeding cycle, increasing the rate of genetic gain and allowing rapid adoption in ryegrass improvement programs.Electronic supplementary materialThe online version of this article (10.1007/s00122-018-3121-7) contains supplementary material, which is available to authorized users.

The YidC–Oxa1–Alb3 preprotein translocases play a vital role in membrane insertion of proteins in eukaryotes and bacteria. In a recent study we observed that Rv3921c, which encodes putative YidC translocase in Mycobacterium tuberculosis (Mtb), is essential for in vitro growth of bacteria. However, the exact function of this particular protein remains to identify in mycobacterial pathogens. By performing a systematic study here we show that YidC of Mtb is an envelope protein, which is required for production of ATP and maintenance of cellular redox balance. Drastic effects of depletion of Rv3921c on the expression of hypoxic genes, ATP synthases, and many proteins of central metabolic and respiratory pathways shed a significant light on the function of YidC towards controlling respiratory metabolism in Mtb. Association of YidC with proteins such as succinate dehydrogenases and ubiquinol-cytochrome C reductase further confirms its role in respiration. Finally we demonstrate that YidC is required for the intracellular survival of Mtb in human macrophages.

Whole genome sequencing offers genome wide, unbiased markers, and inexpensive library preparation. With the cost of sequencing decreasing rapidly, many plant genomes of modest size are amenable to skim whole genome resequencing (skim WGR). The use of skim WGR in diverse sample sets without the use of imputation was evaluated in silico in 149 canola samples representative of global diversity. Fastq files with an average of 10x coverage of the reference genome were used to generate skim samples representing 0.25x, 0.5x, 1x, 2x, 3x, 4x, and 5x sequencing coverage. Applying a pre-defined list of SNPs versus de novo SNP discovery was evaluated. As skim WGR is expected to result in some degree of insufficient allele sampling, all skim coverage levels were filtered at a range of minimum read depths from a relaxed minimum read depth of 2 to a stringent read depth of 5, resulting in 28 list-based SNP sets. As a broad recommendation, genotyping pre-defined SNPs between 1x and 2x coverage with relatively stringent depth filtering is appropriate for a diverse sample set of canola due to a balance between marker number, sufficient accuracy, and sequencing cost, but depends on the intended application. This was experimentally examined in two sample sets with different genetic backgrounds: 1x coverage of 1,590 individuals from 84 Australian spring type four-parent crosses aimed at maximizing diversity as well as one commercial F1 hybrid, and 2x coverage of 379 doubled haploids (DHs) derived from a subset of the four-parent crosses. To determine optimal coverage in a simpler genetic background, the DH sample sequence coverage was further down sampled in silico. The flexible and cost-effective nature of the protocol makes it highly applicable across a range of species and purposes.

The immune pathogenesis of dengue involves antibody production, B cell and T cell response and various pro-inflammatory and anti-inflammatory cytokines. VEGF, a potent permeability enhancing cytokine, is thought to play a pivotal role in mediating plasma leakage in DHF. It is a member of growing family of related proteins that includes VEGF B, VEGF C, VEGF D and placental growth factor. It promotes angiogenesis and vascular integrity. In addition to its role in promoting endothelial permeability & proliferation, it may contribute to inflammation and coagulation. This study was undertaken to investigate the role of VEGF in the patients with dengue infection. Sera were collected from 106 patients with various grades of dengue illness and 40 healthy controls and tested for VEGF levels using commercial ELISA kits. Viral serotypes were detected using specific primers. The results showed very low levels of VEGF (3.493 ± 1.982 pg/ml) in healthy controls. Levels of VEGF were higher in patients with severe dengue (428.170 ± 224.61 pg/ml) as compared to patients with non severe dengue with and without warning signs (290.407 ± 167.17 pg/ml). Significant correlation (p < 0.001) was found between raised VEGF levels and thrombocytopenia and raised haematocrit levels. The VEGF profile patterns discovered between the different phases of illness indicate an essential role in dengue pathogenesis and with further studies may serve as predictive markers for progression of dengue fever to severe dengue infection.

Bacterial P-loop GTPases belong to a family of proteins that selectively hydrolyze a small molecule guanosine tri-phosphate (GTP) to guanosine di-phosphate (GDP) and inorganic phosphate, and regulate several essential cellular activities such as cell division, chromosomal segregation and ribosomal assembly. A comparative genome sequence analysis of different mycobacterial species indicates the presence of multiple P-loop GTPases that exhibit highly conserved motifs. However, an exact function of most of these GTPases in mycobacteria remains elusive. In the present study we characterized the function of a P-loop GTPase in mycobacteria by employing an EngA homologue from Mycobacterium smegmatis, encoded by an open reading frame, designated as MSMEG_3738. Amino acid sequence alignment and phylogenetic analysis suggest that MSMEG_3738 (termed as EngAMS) is highly conserved in mycobacteria. Homology modeling of EngAMS reveals a cloverleaf structure comprising of α/β fold typical to EngA family of GTPases. Recombinant EngAMS purified from E. coli exhibits a GTP hydrolysis activity which is inhibited by the presence of GDP. Interestingly, the EngAMS protein is co-eluted with 16S and 23S ribosomal RNA during purification and exhibits association with 30S, 50S and 70S ribosomal subunits. Further studies demonstrate that GTP is essential for interaction of EngAMS with 50S subunit of ribosome and specifically C-terminal domains of EngAMS are required to facilitate this interaction. Moreover, EngAMS devoid of N-terminal region interacts well with 50S even in the absence of GTP, indicating a regulatory role of the N-terminal domain in EngAMS-50S interaction.

Studies have reported significant gender-related differences in serological tests for detection of NS1 antigen and IgM antibody used for diagnosing dengue fever. However, no such study has been undertaken in India though dengue fever is endemic in this country. Therefore, this study was planned to study the association of serological findings with gender in 700 patients suspected to be suffering from dengue fever in the Indian setting. Haematological parameters of seropositive patients were also studied. Seropositivity and haemorrhagic findings were significantly associated with the female gender. Positive NS1 antigen and IgM antibody results were significantly associated with females and males, respectively.

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