Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

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.

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

PLoS Pathogens ◽

10.1371/journal.ppat.1007652 ◽

2019 ◽

Vol 15 (8) ◽

pp. e1007652 ◽

Cited By ~ 11

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

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

PLoS Genetics ◽

10.1371/journal.pgen.1004782 ◽

2014 ◽

Vol 10 (11) ◽

pp. e1004782 ◽

Cited By ~ 85

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

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Transcriptomic Analysis of Shiga Toxin-Producing Escherichia coli during Initial Contact with Cattle Colonic Explants

Microorganisms ◽

10.3390/microorganisms8111662 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1662

Author(s):

Zachary R. Stromberg ◽

Rick E. Masonbrink ◽

Melha Mellata

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Bacterial Colonization ◽

Coli Strain ◽

Fold Change ◽

Initial Contact ◽

Lon Protease ◽

E Coli ◽

Log2 Fold Change ◽

Colonization Factors

Foodborne pathogens are a public health threat globally. Shiga toxin-producing Escherichia coli (STEC), particularly O26, O111, and O157 STEC, are often associated with foodborne illness in humans. To create effective preharvest interventions, it is critical to understand which factors STEC strains use to colonize the gastrointestinal tract of cattle, which serves as the reservoir for these pathogens. Several colonization factors are known, but little is understood about initial STEC colonization factors. Our objective was to identify these factors via contrasting gene expression between nonpathogenic E. coli and STEC. Colonic explants were inoculated with nonpathogenic E. coli strain MG1655 or STEC strains (O26, O111, or O157), bacterial colonization levels were determined, and RNA was isolated and sequenced. STEC strains adhered to colonic explants at numerically but not significantly higher levels compared to MG1655. After incubation with colonic explants, flagellin (fliC) was upregulated (log2 fold-change = 4.0, p < 0.0001) in O157 STEC, and collectively, Lon protease (lon) was upregulated (log2 fold-change = 3.6, p = 0.0009) in STEC strains compared to MG1655. These results demonstrate that H7 flagellum and Lon protease may play roles in early colonization and could be potential targets to reduce colonization in cattle.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa204 ◽

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.

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

Journal of Bacteriology ◽

10.1128/jb.00548-20 ◽

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814130116 ◽

2019 ◽

Vol 116 (4) ◽

pp. 1394-1403 ◽

Cited By ~ 26

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.

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Multiple in vivo passages enhance the ability of a clinical Helicobacter pylori isolate to colonize the stomach of Mongolian gerbils and to induce gastritis

Laboratory Animals ◽

10.1258/0023677053739800 ◽

2005 ◽

Vol 39 (2) ◽

pp. 221-229 ◽

Cited By ~ 10

Author(s):

A Bleich ◽

I Köhn ◽

S Glage ◽

W Beil ◽

S Wagner ◽

...

Keyword(s):

Helicobacter Pylori ◽

Bacterial Colonization ◽

Underlying Mechanism ◽

Mongolian Gerbils ◽

Histopathological Changes ◽

Genetic Changes ◽

Colonization Ability ◽

Colonization Factors ◽

H Pylori

The Mongolian gerbil is an excellent animal model for Helicobacter pylori-induced gastritis in humans. In this study, initially low colonization rates of the H. pylori strains ATCC 43504, SS1, or HP87 inoculated into gerbils caused difficulties in establishing this model. In order to increase the colonization ability and pathogenicity, the clinical HP87 isolate was selected for adaptation to the gerbil stomach by multiple in vivo passages through gerbils. Development of gastritis was examined histologically at 4–52 weeks after infection. The proportion of gerbils which tested positive for H. pylori by culture at four weeks after inoculation gradually increased from 11.1% of gerbils inoculated with HP87 without prior in vivo passage (P0) to 100% of gerbils inoculated with HP87 with seven in vivo passages (P7). In addition, adaptation of HP87 resulted in more severe histopathological changes. Gerbils infected with adapted HP87 (P7) exhibited severe infiltration by monomorphonuclear and polymorphonuclear leukocytes in the mucosa, submucosa, and subserosa of the gastric antrum, as well as epithelial changes consisting of hyperplasia, erosion, and ulceration. Histopathological changes increased in severity from four to 52 weeks after infection. Adaptation of HP87 during its passages through gerbils could be due to genetic changes in bacterial colonization factors. Identification of these changes might be useful to understand the underlying mechanism of gastric adaptation and pathogenesis of H. pylori.

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