15 research outputs found
Toxin-Antitoxin systems eliminate defective cells and preserve symmetry in Bacillus subtilis biofilms.
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A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes
The ability to simultaneously characterize the bacterial and host expression programs during infection would facilitate a comprehensive understanding of pathogen-host interactions. While RNA-Seq has greatly advanced our ability to study the transcriptomes of prokaryote and eukaryotes separately, limitations in existing protocols for generating and analyzing RNA-Seq data have hindered simultaneous profiling of host and bacterial pathogen transcripts from the same sample. Here we provide a detailed protocol for simultaneous analysis of host and bacterial transcripts by RNA-Seq. Importantly, this protocol details the steps required for efficient host and bacteria lysis, barcoding of samples, technical advances in sample preparation for low yield sample inputs and a computational pipeline to analyze both mammalian and microbial reads from mixed hostpathogen RNA-Seq data. Sample preparation takes 3 d from cultured cells to pooled libraries. Data analysis takes an additional day. Compared with previous methods, the protocol detailed here provides a sensitive, facile and generalizable approach, suitable for large-scale studies, which will enable the field to obtain in-depth analysis of hostpathogen interactions in infection models
A Comprehensive tRNA Deletion Library Unravels the Genetic Architecture of the tRNA Pool
<div><p>Deciphering the architecture of the tRNA pool is a prime challenge in translation research, as tRNAs govern the efficiency and accuracy of the process. Towards this challenge, we created a systematic tRNA deletion library in <i>Saccharomyces cerevisiae</i>, aimed at dissecting the specific contribution of each tRNA gene to the tRNA pool and to the cell's fitness. By harnessing this resource, we observed that the majority of tRNA deletions show no appreciable phenotype in rich medium, yet under more challenging conditions, additional phenotypes were observed. Robustness to tRNA gene deletion was often facilitated through extensive backup compensation within and between tRNA families. Interestingly, we found that within tRNA families, genes carrying identical anti-codons can contribute differently to the cellular fitness, suggesting the importance of the genomic surrounding to tRNA expression. Characterization of the transcriptome response to deletions of tRNA genes exposed two disparate patterns: in single-copy families, deletions elicited a stress response; in deletions of genes from multi-copy families, expression of the translation machinery increased. Our results uncover the complex architecture of the tRNA pool and pave the way towards complete understanding of their role in cell physiology.</p></div
Changes in the tRNA pool affect protein folding.
<p>(AβC) Relative growth rate (compare to wild-type) of the following five deletion strain: <i>tL(GAG)G</i> (blue), <i>tR(CCU)J</i> (red), <i>tiM(CAU)C</i> (green), <i>tH(GUG)G1</i> (magenta) and <i>tR(UCU)M2</i> (cyan). Strains were grown in media supplemented with increasing concentrations of the following proteotoxic agent: AZC (A) Tunicamycin (B) DTT (C). (D) Percentage of cells that contain puncta in the populations of the above strains. (E) Percentage of cells that contain puncta in the populations of the above strains following treatment with 2.5 mM AZC. Data are presented as mean of 3 biological repetitions +/β SEM, in each repetition 500 cells were counted. (*) P<0.001 by Students <i>t</i>-test. (FβG) Images of representative fields for the wild-type and <i>tR(CCU)J</i> deletion strain, without treatment (F) and following treatment with 2.5 mM AZC (G).</p
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Toxin-Antitoxin systems eliminate defective cells and preserve symmetry in Bacillus subtilis biofilms.
Toxin-antitoxin modules are gene pairs encoding a toxin and its antitoxin, and are found on the chromosomes of many bacteria, including pathogens. Here, we characterize the specific contribution of the TxpA and YqcG toxins in elimination of defective cells from developing Bacillus subtilis biofilms. On nutrient limitation, defective cells accumulated in the biofilm breaking its symmetry. Deletion of the toxins resulted in accumulation of morphologically abnormal cells, and interfered with the proper development of the multicellular community. Dual physiological responses are of significance for TxpA and YqcG activation: nitrogen deprivation enhances the transcription of both TxpA and YqcG toxins, and simultaneously sensitizes the biofilm cells to their activity. Furthermore, we demonstrate that while both toxins when overexpressed affect the morphology of the developing biofilm, the toxin TxpA can act to lyse and dissolve pre-established B. subtilis biofilms
Screening the tRNA deletion library across various growth conditions.
<p>(A) Percent of strains exhibiting a growth yield phenotype in various conditions. The color indicates the type of phenotype: impaired (blue) or improved (red). (B) Percent of strains exhibiting a growth rate phenotype in various conditions. (CβD) The Ο values measured for both the growth yield (C) and the growth rate (D) for all deletion strains across six conditions. The color bar indicates the Ο values, red denoting improvement and blue impairment. Each row denotes a tRNA deletion strain and each column denotes different growth condition. Strains are ordered on the y-axis according to amino acids (denoted by letter) and further separated into families (denoted by lines within the amino-acid box). Black rows denote lethal strains. Gray rows indicate strains for which the respective value was not measured.</p
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Toxin-Antitoxin systems eliminate defective cells and preserve symmetry in Bacillus subtilis biofilms.
Toxin-antitoxin modules are gene pairs encoding a toxin and its antitoxin, and are found on the chromosomes of many bacteria, including pathogens. Here, we characterize the specific contribution of the TxpA and YqcG toxins in elimination of defective cells from developing Bacillus subtilis biofilms. On nutrient limitation, defective cells accumulated in the biofilm breaking its symmetry. Deletion of the toxins resulted in accumulation of morphologically abnormal cells, and interfered with the proper development of the multicellular community. Dual physiological responses are of significance for TxpA and YqcG activation: nitrogen deprivation enhances the transcription of both TxpA and YqcG toxins, and simultaneously sensitizes the biofilm cells to their activity. Furthermore, we demonstrate that while both toxins when overexpressed affect the morphology of the developing biofilm, the toxin TxpA can act to lyse and dissolve pre-established B. subtilis biofilms
KEGG pathways differentiating between tRNA deletion sets.
<p>KEGG pathways <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004084#pgen.1004084-Kanehisa1" target="_blank">[49]</a> for which changes in genes expression are significantly different between the two groups of tRNA deletion strains: MC (multi-copy) group (<i>ΞtH(GUG)G1</i> and <i>ΞtR(UCU)M2</i>) vs. SC (single-copy) group (<i>ΞtL(GAG)G</i>, <i>ΞtR(CCU)J</i>, <i>ΞtiM(CAU)C</i>) calculated with GSEA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004084#pgen.1004084-Subramanian1" target="_blank">[51]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004084#pgen.1004084-Mootha1" target="_blank">[52]</a>. In the first column are pathways, which are higher in SC vs. MC and vice versa in the second column. The values are corrected for multiple hypothesis and the FDR q-values are indicated next to each pathway.</p
Differential contribution of identical tRNA gene copies.
<p>(AβB) Relative growth yield values of the tRNA deletion library strains in rich medium (A) and low glucose (B), sorted by anti-codon and amino-acid identity along the x-axis. Each dot along the vertical lines denotes the value (data are represented as mean of 3 biological repetitions +/β SEM) of a deletion strain of different tRNA gene of the respective family. The horizontal lines mark two standard deviations around the mean of the wild-type. Dots above or below these lines are considered non-normal phenotypes (see also Supplemental <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004084#pgen.1004084.s007" target="_blank">figure S7</a>). (C) Relative growth yield values (data are presented as mean of 3 biological repetitions +/β SEM) of various double deletion combinations consisting of: five <i>tR(UCU)</i> family members, <i>tR(CCU)</i> and <i>trm9</i> deletion strains as indicated on the x-axis, along with the five members of the <i>tR(UCU)</i> family each denoted by a different shape and color in the legend. (D) Relative growth yield of the five <i>tR(UCU)</i> members across different growth conditions, indicated on the x-axis. (E) Enrichment of conserved elements in tRNA genes divided according to phenotype observed in rich media for each growth parameter. Each column in the matrix denotes a conserved element as defined by <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004084#pgen.1004084-Giuliodori1" target="_blank">[42]</a>. Color bar indicates the βlog<sub>10</sub> of the hypergeometric p-value. (F) log10 E-value found by the MEME software for the most significant motif in a 9 bp window starting from the position indicated by the x-axis. The LOGOs of the two significant motifs are displayed below, next to a number indicating its position. Position 0 is the first position of the mature tRNA.</p
Extensive redundancy underlies robustness to tRNA gene deletion.
<p>(A) Schematic representation of the genetic interactions within and between tRNA families. Families are denoted by dark grey circles and grouped (black dashed line) according to their tRNA copy number. Each family is denoted by its anti-codon and amino-acid. A protein-coding gene i.e. <i>TRM9</i> is denoted by a grey box. Each filled circle indicates a tRNA deletion strain. The lines connecting the deletion strains denote a co-deletion of these genes (a multi-tRNAs deletion strain). The color of the filled circles and lines denote the severity of the growth phenotype for the respective strain: blue for normal growth, purple for impaired growth (worse than wild-type) and red for lethality. (B) Epistasis values for multi-tRNAs deletion strains which contain the deletion of <i>tL(GAG)</i> and either: one <i>tL(UAG)</i> gene, two <i>tL(UAG)</i> genes, <i>tL(CAA)</i> (which is a tRNA of different Leucine family), and <i>tW(CCA)</i> (which is a non-Leucine tRNA) as controls. (C) Epistasis values for multi-tRNAs deletion strains which contain the deletion of <i>trm9</i> with: the singleton <i>tR(CCU)</i>, and <i>tR(ACG)</i> which is a tRNA of different Arginine family and <i>tW(CCA)</i> which is a non- Arginine tRNA as controls. In both (B) and (C) epistasis values of the relative growth yield and growth rate are indicated in grey and green respectively. Data is presented as mean of 3 biological repetitions +/β SEM.</p