scholarly journals The RNA degradosome promotes tRNA quality control through clearance of hypomodified tRNA

2019 ◽  
Vol 116 (4) ◽  
pp. 1394-1403 ◽  
Author(s):  
Satoshi Kimura ◽  
Matthew K. Waldor
Keyword(s):  
Quality Control ◽  
Elongation Factor ◽  
Trna Modification ◽  
Rapid Degradation ◽  
Transposon Insertion ◽  
Specific Manner ◽  
Suppressor Mutants ◽  
Rna Degradosome ◽  
Biochemical Analyses ◽  
Transposon Insertion Sequencing

The factors and mechanisms that govern tRNA stability in bacteria are not well understood. Here, we investigated the influence of posttranscriptional modification of bacterial tRNAs (tRNA modification) on tRNA stability. We focused on ThiI-generated 4-thiouridine (s4U), a modification found in bacterial and archaeal tRNAs. Comprehensive quantification ofVibrio choleraetRNAs revealed that the abundance of some tRNAs is decreased in a ΔthiIstrain in a stationary phase-specific manner. Multiple mechanisms, including rapid degradation of a subset of hypomodified tRNAs, account for the reduced abundance of tRNAs in the absence ofthiI. Through transposon insertion sequencing, we identified additional tRNA modifications that promote tRNA stability and bacterial viability. Genetic analysis of suppressor mutants as well as biochemical analyses revealed that rapid degradation of hypomodified tRNA is mediated by the RNA degradosome. Elongation factor Tu seems to compete with the RNA degradosome, protecting aminoacyl tRNAs from decay. Together, our observations describe a previously unrecognized bacterial tRNA quality control system in which hypomodification sensitizes tRNAs to decay mediated by the RNA degradosome.

scholarly journals Reproducible and accessible analysis of transposon insertion sequencing in Galaxy for qualitative essentiality analyses

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Delphine Larivière ◽  
Laura Wickham ◽  
Kenneth Keiler ◽  
Anton Nekrutenko ◽  
Keyword(s):  
Data Analysis ◽  
Promoter Sequence ◽  
Entire Genome ◽  
Link Type ◽  
Transposon Insertion ◽  
Control Procedures ◽  
Reproducible Analysis ◽  
Using Data ◽  
Transposon Insertion Sequencing ◽  
The Impact

Abstract Background Significant progress has been made in advancing and standardizing tools for human genomic and biomedical research. Yet, the field of next-generation sequencing (NGS) analysis for microorganisms (including multiple pathogens) remains fragmented, lacks accessible and reusable tools, is hindered by local computational resource limitations, and does not offer widely accepted standards. One such “problem areas” is the analysis of Transposon Insertion Sequencing (TIS) data. TIS allows probing of almost the entire genome of a microorganism by introducing random insertions of transposon-derived constructs. The impact of the insertions on the survival and growth under specific conditions provides precise information about genes affecting specific phenotypic characteristics. A wide array of tools has been developed to analyze TIS data. Among the variety of options available, it is often difficult to identify which one can provide a reliable and reproducible analysis. Results Here we sought to understand the challenges and propose reliable practices for the analysis of TIS experiments. Using data from two recent TIS studies, we have developed a series of workflows that include multiple tools for data de-multiplexing, promoter sequence identification, transposon flank alignment, and read count repartition across the genome. Particular attention was paid to quality control procedures, such as determining the optimal tool parameters for the analysis and removal of contamination. Conclusions Our work provides an assessment of the currently available tools for TIS data analysis. It offers ready to use workflows that can be invoked by anyone in the world using our public Galaxy platform (https://usegalaxy.org). To lower the entry barriers, we have also developed interactive tutorials explaining details of TIS data analysis procedures at https://bit.ly/gxy-tis.

scholarly journals Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization

2019 ◽  
Vol 15 (8) ◽  
pp. e1007652 ◽  
Author(s):  
Alyson R. Warr ◽  
Troy P. Hubbard ◽  
Diana Munera ◽  
Carlos J. Blondel ◽  
Pia Abel zur Wiesch ◽  
...  
Keyword(s):  
Intestinal Colonization ◽  
Transposon Insertion ◽  
Transposon Insertion Sequencing

scholarly journals ARTIST: High-Resolution Genome-Wide Assessment of Fitness Using Transposon-Insertion Sequencing

PLoS Genetics ◽  
2014 ◽  
Vol 10 (11) ◽  
pp. e1004782 ◽  
Author(s):  
Justin R. Pritchard ◽  
Michael C. Chao ◽  
Sören Abel ◽  
Brigid M. Davis ◽  
Catherine Baranowski ◽  
...  
Keyword(s):  
High Resolution ◽  
Transposon Insertion ◽  
Genome Wide ◽  
Transposon Insertion Sequencing

scholarly journals Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

2020 ◽  
Vol 48 (8) ◽  
pp. 4585-4600
Author(s):  
Gabriel A Suárez ◽  
Kyle R Dugan ◽  
Brian A Renda ◽  
Sean P Leonard ◽  
Lakshmi Suryateja Gangavarapu ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Single Gene ◽  
Acinetobacter Baylyi ◽  
Gene Deletions ◽  
Multiple Gene ◽  
Transposon Insertion ◽  
A Genome ◽  
Acinetobacter Baylyi Adp1 ◽  
Transposon Insertion Sequencing ◽  
Improved Methods

Abstract One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

scholarly journals Transposon Insertion Sequencing in a Clinical Isolate of Legionella pneumophila Identifies Essential Genes and Determinants of Natural Transformation

2020 ◽  
Vol 203 (3) ◽  
Author(s):  
Léo Hardy ◽  
Pierre-Alexandre Juan ◽  
Bénédicte Coupat-Goutaland ◽  
Xavier Charpentier
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Legionella Pneumophila ◽  
Genetic Basis ◽  
Natural Transformation ◽  
Essential Genes ◽  
Content Type ◽  
Transposon Insertion ◽  
Life Traits ◽  
Transposon Insertion Sequencing

ABSTRACT Legionella pneumophila is a Gram-negative bacterium ubiquitous in freshwater environments which, if inhaled, can cause a severe pneumonia in humans. The emergence of L. pneumophila is linked to several traits selected in the environment, the acquisition of some of which involved intra- and interkingdom horizontal gene transfer events. Transposon insertion sequencing (TIS) is a powerful method to identify the genetic basis of selectable traits as well as to identify fitness determinants and essential genes, which are possible antibiotic targets. TIS has not yet been used to its full power in L. pneumophila, possibly because of the difficulty of obtaining a high-saturation transposon insertion library. Indeed, we found that isolates of sequence type 1 (ST1), which includes the commonly used laboratory strains, are poorly permissive to saturating mutagenesis by conjugation-mediated transposon delivery. In contrast, we obtained high-saturation libraries in non-ST1 clinical isolates, offering the prospect of using TIS on unaltered L. pneumophila strains. Focusing on one of them, we then used TIS to identify essential genes in L. pneumophila. We also revealed that TIS could be used to identify genes controlling vertical transmission of mobile genetic elements. We then applied TIS to identify all the genes required for L. pneumophila to develop competence and undergo natural transformation, defining the set of major and minor type IV pilins that are engaged in DNA uptake. This work paves the way for the functional exploration of the L. pneumophila genome by TIS and the identification of the genetic basis of other life traits of this species. IMPORTANCE Legionella pneumophila is the etiologic agent of a severe form of nosocomial and community-acquired pneumonia in humans. The environmental life traits of L. pneumophila are essential to its ability to accidentally infect humans. A comprehensive identification of their genetic basis could be obtained through the use of transposon insertion sequencing. However, this powerful approach had not been fully implemented in L. pneumophila. Here, we describe the successful implementation of the transposon-sequencing approach in a clinical isolate of L. pneumophila. We identify essential genes, potential drug targets, and genes required for horizontal gene transfer by natural transformation. This work represents an important step toward identifying the genetic basis of the many life traits of this environmental and pathogenic species.

scholarly journals Unsupervised Learning Approach for Comparing Multiple Transposon Insertion Sequencing Studies

mSphere ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Troy P. Hubbard ◽  
Jonathan D. D’Gama ◽  
Gabriel Billings ◽  
Brigid M. Davis ◽  
Matthew K. Waldor
Keyword(s):  
Unsupervised Learning ◽  
Principal Component ◽  
Data Sets ◽  
Intestinal Colonization ◽  
Genetic Screens ◽  
Transposon Insertion ◽  
Screen Level ◽  
Forward Genetic Screens ◽  
Transposon Insertion Sequencing ◽  
Genome Scale

ABSTRACT Transposon insertion sequencing (TIS) is a widely used technique for conducting genome-scale forward genetic screens in bacteria. However, few methods enable comparison of TIS data across multiple replicates of a screen or across independent screens, including screens performed in different organisms. Here, we introduce a post hoc analytic framework, comparative TIS (CompTIS), which utilizes unsupervised learning to enable meta-analysis of multiple TIS data sets. CompTIS first implements screen-level principal-component analysis (PCA) and clustering to identify variation between the TIS screens. This initial screen-level analysis facilitates the selection of related screens for additional analyses, reveals the relatedness of complex environments based on growth phenotypes measured by TIS, and provides a useful quality control step. Subsequently, PCA is performed on genes to identify loci whose corresponding mutants lead to concordant/discordant phenotypes across all or in a subset of screens. We used CompTIS to analyze published intestinal colonization TIS data sets from two vibrio species. Gene-level analyses identified both pan-vibrio genes required for intestinal colonization and conserved genes that displayed species-specific requirements. CompTIS is applicable to virtually any combination of TIS screens and can be implemented without regard to either the number of screens or the methods used for upstream data analysis. IMPORTANCE Forward genetic screens are powerful tools for functional genomics. The comparison of similar forward genetic screens performed in different organisms enables the identification of genes with similar or different phenotypes across organisms. Transposon insertion sequencing is a widely used method for conducting genome-scale forward genetic screens in bacteria, yet few bioinformatic approaches have been developed to compare the results of screen replicates and different screens conducted across species or strains. Here, we used principal-component analysis (PCA) and hierarchical clustering, two unsupervised learning approaches, to analyze the relatedness of multiple in vivo screens of pathogenic vibrios. This analytic framework reveals both shared pan-vibrio requirements for intestinal colonization and strain-specific dependencies. Our findings suggest that PCA-based analytics will be a straightforward widely applicable approach for comparing diverse transposon insertion sequencing screens.

scholarly journals Defining the core essential genome ofPseudomonas aeruginosa

2019 ◽  
Vol 116 (20) ◽  
pp. 10072-10080 ◽  
Author(s):  
Bradley E. Poulsen ◽  
Rui Yang ◽  
Anne E. Clatworthy ◽  
Tiantian White ◽  
Sarah J. Osmulski ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Growth Conditions ◽  
Essential Genes ◽  
The Core ◽  
Transposon Insertion ◽  
Wide Range ◽  
Transposon Insertion Sequencing ◽  
Antibiotic Discovery

Genomics offered the promise of transforming antibiotic discovery by revealing many new essential genes as good targets, but the results fell short of the promise. While numerous factors contributed to the disappointing yield, one factor was that essential genes for a bacterial species were often defined based on a single or limited number of strains grown under a single or limited number of in vitro laboratory conditions. In fact, the essentiality of a gene can depend on both the genetic background and growth condition. We thus developed a strategy for more rigorously defining the core essential genome of a bacterial species by studying many pathogen strains and growth conditions. We assessed how many strains must be examined to converge on a set of core essential genes for a species. We used transposon insertion sequencing (Tn-Seq) to define essential genes in nine strains ofPseudomonas aeruginosaon five different media and developed a statistical model,FiTnEss, to classify genes as essential versus nonessential across all strain–medium combinations. We defined a set of 321 core essential genes, representing 6.6% of the genome. We determined that analysis of four strains was typically sufficient inP. aeruginosato converge on a set of core essential genes likely to be essential across the species across a wide range of conditions relevant to in vivo infection, and thus to represent attractive targets for novel drug discovery.

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