van Opijnen T, Bodi KL, Camilli A.. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6: 767-772

Howard Hughes Medical Institute, and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA.
Nature Methods (Impact Factor: 32.07). 09/2009; 6(10):767-72. DOI: 10.1038/nmeth.1377
Source: PubMed


Biological pathways are structured in complex networks of interacting genes. Solving the architecture of such networks may provide valuable information, such as how microorganisms cause disease. Here we present a method (Tn-seq) for accurately determining quantitative genetic interactions on a genome-wide scale in microorganisms. Tn-seq is based on the assembly of a saturated Mariner transposon insertion library. After library selection, changes in frequency of each insertion mutant are determined by sequencing the flanking regions en masse. These changes are used to calculate each mutant's fitness. Using this approach, we determined fitness for each gene of Streptococcus pneumoniae, a causative agent of pneumonia and meningitis. A genome-wide screen for genetic interactions of five query genes identified both alleviating and aggravating interactions that could be divided into seven distinct categories. Owing to the wide activity of the Mariner transposon, Tn-seq has the potential to contribute to the exploration of complex pathways across many different species.

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    • "59 Epistasis has also been studied in vivo. For example, in yeast, the effects of specific mutations on 60 fitness can be rapidly analyzed in the background of thousands of individual gene knockouts 61 using the epistatic mini-array profile (E-MAP) approach (Schuldiner et al. 2005), or synthetic 62 genetic analysis (SGA, Tong et al. 2001; van Opijnen et al. 2009). While epistasis mapping by 63 these approaches has been extremely useful for detecting physiological connections between 64 gene products, it is not well suited to investigate intragenic epistasis, or to comprehensively 65 screen point mutants. "
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    ABSTRACT: Mutations are the source of evolutionary variation. The interactions of multiple mutations can have important effects on fitness and evolutionary trajectories. We have recently described the distribution of fitness effects of all single mutations for a nine amino acid region of yeast Hsp90 (Hsp82) implicated in substrate binding. Here, we report and discuss the distribution of intragenic epistatic effects within this region in seven Hsp90 point mutant backgrounds of neutral to slightly deleterious effect, resulting in an analysis of more than 1000 double-mutants. We find negative epistasis between substitutions to be common, and positive epistasis to be rare - resulting in a pattern that indicates a drastic change in the distribution of fitness effects one step away from the wild type. This can be well explained by a concave relationship between phenotype and genotype (i.e., a concave shape of the local fitness landscape), suggesting mutational robustness intrinsic to the local sequence space. Structural analyses indicate that, in this region, epistatic effects are most pronounced when a solvent-inaccessible position is involved in the interaction. In contrast, all 18 observations of positive epistasis involved at least one mutation at a solvent-exposed position. By combining the analysis of evolutionary and biophysical properties of an epistatic landscape, these results contribute to a more detailed understanding of the complexity of protein evolution.
    Molecular Biology and Evolution 11/2014; 32(1). DOI:10.1093/molbev/msu301 · 9.11 Impact Factor
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    • "One of the potential perils of using next generation sequencing, with its current reliance on short read sequences is that transposition events may be missed. Therefore, employing methods that specifically identify novel insertion sites of retrotransposon (Gabriel et al. 2006; Mularoni et al. 2012; van Opijnen et al. 2009) remains an important aspect of analyzing genomes of evolved lineages. "
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    ABSTRACT: Two central problems in biology are determining the molecular basis of adaptive evolution and understanding how cells regulate their growth. The chemostat is a device for culturing cells that provides great utility in tackling both of these problems: it enables precise control of the selective pressure under which organisms evolve and it facilitates experimental control of cell growth rate. The aim of this review is to synthesize results from studies of the functional basis of adaptive evolution in long-term chemostat selections using Escherichia coli and Saccharomyces cerevisiae. We describe the principle of the chemostat, provide a summary of studies of experimental evolution in chemostats and use these studies to assess our current understanding of selection in the chemostat. Functional studies of adaptive evolution in chemostats provide a unique means of interrogating the genetic networks that control cell growth, which complements functional genomic approaches and quantitative trait loci (QTL) mapping in natural populations. An integrated approach to the study of adaptive evolution that accounts for both molecular function and evolutionary processes is critical to advancing our understanding of evolution. By renewing efforts to integrate these two research programs experimental evolution in chemostats is ideally suited to extending this functional synthesis to the study of genetic networks.This article is protected by copyright. All rights reserved.
    FEMS microbiology reviews 08/2014; 39(1). DOI:10.1111/1574-6976.12082 · 13.24 Impact Factor
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    • "RW1 transcriptomics in contaminated sand SK Moreno-Forero and JR van der Meer ratio of the abundance of transposon insertions in exponentially growing RW1 populations in sand (vs the control of the transposon abundance distribution in the starter transposon library), with gene lists appearing in Supplementary Tables S14 and S15. The transposon scanning approach is based on the premise that any transposon insertion in a gene affecting growth under a specific set of growth conditions will lead to a lower abundance (or even disappearance) of that transposon mutant population in the metapopulation of all mutants (Gawronski et al., 2009; van Opijnen et al., 2009; Roggo et al., 2013). Under exclusion of essential genes and only focussing on those genes with higher expression in sand, one can conclude from GO classification that a wide range of metabolic processes is specific for growth in soil (Table 3), and a further range of gene functions is implicated in specific survival in soil upon cessation of growth (cellular homeostasis, nutrient scavenging, stress response, Table 3). "
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    ABSTRACT: The efficacy of inoculation of single pure bacterial cultures into complex microbiomes, for example, in order to achieve increased pollutant degradation rates in contaminated material (that is, bioaugmentation), has been frustrated by insufficient knowledge on the behaviour of the inoculated bacteria under the specific abiotic and biotic boundary conditions. Here we present a comprehensive analysis of genome-wide gene expression of the bacterium Sphingomonas wittichii RW1 in contaminated non-sterile sand, compared with regular suspended batch growth in liquid culture. RW1 is a well-known bacterium capable of mineralizing dibenzodioxins and dibenzofurans. We tested the reactions of the cells both during the immediate transition phase from liquid culture to sand with or without dibenzofuran, as well as during growth and stationary phase in sand. Cells during transition show stationary phase characteristics, evidence for stress and for nutrient scavenging, and adjust their primary metabolism if they were not precultured on the same contaminant as found in the soil. Cells growing and surviving in sand degrade dibenzofuran but display a very different transcriptome signature as in liquid or in liquid culture exposed to chemicals inducing drought stress, and we obtain evidence for numerous 'soil-specific' expressed genes. Studies focusing on inoculation efficacy should test behaviour under conditions as closely as possible mimicking the intended microbiome conditions.
    The ISME Journal 06/2014; 9(1). DOI:10.1038/ismej.2014.101 · 9.30 Impact Factor
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