scholarly journals Elucidating essential genes in plant-associated Pseudomonas protegens Pf-5 using transposon insertion sequencing

2020 ◽  
Author(s):  
Belinda K Fabian ◽  
Christie Foster ◽  
Amy J Asher ◽  
Liam D. H. Elbourne ◽  
Amy K Cain ◽  
...  
Keyword(s):  
Essential Gene ◽  
Bacterial Pathogens ◽  
Essential Genes ◽  
Associated Bacteria ◽  
Gene Essentiality ◽  
Gene Set ◽  
Transposon Insertion ◽  
Gene Sets ◽  
Genome Wide ◽  
Transposon Insertion Sequencing

Gene essentiality studies have been performed on numerous bacterial pathogens, but essential gene sets have been determined for only a few plant-associated bacteria. Pseudomonas protegens Pf-5 is a plant-commensal, biocontrol bacteria that can control disease-causing pathogens on a wide range of crops. Work on Pf-5 has mostly focused on secondary metabolism and biocontrol genes, but genome-wide approaches such as high-throughput transposon mutagenesis have not yet been used in this species. Here we generated a dense P. protegens Pf-5 transposon mutant library and used transposon-directed insertion site sequencing (TraDIS) to identify 446 genes essential for growth on rich media. Genes required for fundamental cellular machinery were enriched in the essential gene set, while genes related to nutrient biosynthesis, stress responses and transport were under-represented. The majority of Pf-5 essential genes were part of the P. protegens core genome. Comparison of the essential gene set of Pf-5 with those of two plant associated pseudomonads, P. simiae and P. syringae, and the well-studied opportunistic human pathogen P. aeruginosa PA14 showed the four species share a large number of essential genes, but each species also had uniquely essential genes. Comparison of the Pf-5 in silico predicted and in vitro determined essential gene sets highlighted the essential cellular functions that are over- and underestimated by each method. Expanding essentiality studies into bacteria with a range of lifestyles may improve our understanding of the biological processes important for bacterial survival and growth. Importance Essential genes are those crucial for survival or normal growth rates in an organism. Essential gene sets have been identified in numerous bacterial pathogens, but only a few plant-associated bacteria. Employing genome-wide approaches, such as transposon insertion sequencing, allows for the concurrent analysis of all genes of a bacterial species and rapid determination of essential gene sets. We have used transposon insertion sequencing to systematically analyze thousands of Pseudomonas protegens Pf-5 genes and gain insights into gene functions and interactions that are not readily available using traditional methods. Comparing Pf-5 essential genes with those of three other pseudomonads highlights how gene essentiality varies between closely related species.

scholarly journals Elucidating essential genes in plant-associated Pseudomonas protegens Pf-5 using transposon insertion sequencing

2020 ◽  
Author(s):  
Belinda K Fabian ◽  
Christie Foster ◽  
Amy J Asher ◽  
Liam DH Elbourne ◽  
Amy K Cain ◽  
...  
Keyword(s):  
Essential Gene ◽  
Essential Genes ◽  
Biological Processes ◽  
Associated Bacteria ◽  
Transposon Insertion ◽  
Gene Sets ◽  
Genome Wide ◽  
Opportunistic Human Pathogen ◽  
Transposon Insertion Sequencing ◽  
Insight Into

AbstractGene essentiality studies have been performed on numerous bacterial pathogens, but essential gene sets have been determined for only a few plant-associated bacteria. Pseudomonas protegens Pf-5 is a plant-commensal, biocontrol bacteria that can control disease-causing pathogens on a wide range of crops. Work on Pf-5 has mostly focused on secondary metabolism and biocontrol genes, but genome-wide approaches such as high-throughput transposon mutagenesis have not yet been used in this species. Here we generated a dense P. protegens Pf-5 transposon mutant library and used transposon-directed insertion site sequencing (TraDIS) to identify 446 genes essential for growth on rich media. Genes required for fundamental cellular machinery were enriched in the essential gene set, while genes related to nutrient biosynthesis, stress responses and transport were under-represented. Comparison of the essential gene sets of Pf-5 and P. aeruginosa PA14, an opportunistic human pathogen, provides insight into the biological processes important for their different lifestyles. Key differences include cytochrome c biogenesis, formation of periplasmic disulfide bonds, lipid biosynthesis, ribonuclease activity, lipopolysaccharides and cell surface structures. Comparison of the Pf-5 in silico predicted and in vitro determined essential gene sets highlighted the essential cellular functions that are over- and underestimated by each method. Expanding essentiality studies into bacteria with a range of lifestyles can improve our understanding of the biological processes important for survival and growth in different environmental niches.ImportanceEssential genes are those crucial for survival or normal growth rates in an organism. Essential gene sets have been identified in numerous bacterial pathogens, but only a few plant-associated bacteria. Employing genome-wide approaches, such as transposon insertion sequencing, allows for the concurrent analysis of all genes of a bacterial species and rapid determination of essential gene sets. We have used transposon insertion sequencing to systematically analyze thousands of Pseudomonas protegens Pf-5 genes and gain insights into gene functions and interactions that are not readily available using traditional methods. Comparing Pf-5 essential genes with those of P. aeruginosa PA14, an opportunistic human pathogen, provides insight into differences in gene essentiality which may be linked to their different lifestyles.

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 TEGS-CN: A Statistical Method for Pathway Analysis of Genome-wide Copy Number Profile

2014 ◽  
Vol 13s4 ◽  
pp. CIN.S13978
Author(s):  
Yen-Tsung Huang ◽  
Thomas Hsu ◽  
David C. Christiani
Keyword(s):  
Copy Number ◽  
Copy Number Data ◽  
Copy Number Profile ◽  
Test Statistic ◽  
Gene Set ◽  
Bonferroni Adjustment ◽  
Gene Sets ◽  
Genome Wide ◽  
A Genome ◽  
Pathway Analyses

The effects of copy number alterations make up a significant part of the tumor genome profile, but pathway analyses of these alterations are still not well established. We proposed a novel method to analyze multiple copy numbers of genes within a pathway, termed Test for the Effect of a Gene Set with Copy Number data (TEGS-CN). TEGS-CN was adapted from TEGS, a method that we previously developed for gene expression data using a variance component score test. With additional development, we extend the method to analyze DNA copy number data, accounting for different sizes and thus various numbers of copy number probes in genes. The test statistic follows a mixture of X 2 distributions that can be obtained using permutation with scaled X 2 approximation. We conducted simulation studies to evaluate the size and the power of TEGS-CN and to compare its performance with TEGS. We analyzed a genome-wide copy number data from 264 patients of non-small-cell lung cancer. With the Molecular Signatures Database (MSigDB) pathway database, the genome-wide copy number data can be classified into 1814 biological pathways or gene sets. We investigated associations of the copy number profile of the 1814 gene sets with pack-years of cigarette smoking. Our analysis revealed five pathways with significant P values after Bonferroni adjustment (<2.8 x 10-5), including the PTEN pathway (7.8 x 10-7), the gene set up-regulated under heat shock (3.6 x 10-6), the gene sets involved in the immune profile for rejection of kidney transplantation (9.2 x 10-6) and for transcriptional control of leukocytes (2.2 x 10-5), and the ganglioside biosynthesis pathway (2.7 x 10-5). In conclusion, we present a new method for pathway analyses of copy number data, and causal mechanisms of the five pathways require further study.

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 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.

scholarly journals Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering

Microbiology ◽  
2014 ◽  
Vol 160 (11) ◽  
pp. 2341-2351 ◽  
Author(s):  
Mario Juhas ◽  
Daniel R. Reuß ◽  
Bingyao Zhu ◽  
Fabian M. Commichau
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Essential Gene ◽  
Building Blocks ◽  
Essential Genes ◽  
Minimal Cell ◽  
E Coli ◽  
Cell Factories ◽  
Gene Sets ◽  
K 12

Investigation of essential genes, besides contributing to understanding the fundamental principles of life, has numerous practical applications. Essential genes can be exploited as building blocks of a tightly controlled cell ‘chassis’. Bacillus subtilis and Escherichia coli K-12 are both well-characterized model bacteria used as hosts for a plethora of biotechnological applications. Determination of the essential genes that constitute the B. subtilis and E. coli minimal genomes is therefore of the highest importance. Recent advances have led to the modification of the original B. subtilis and E. coli essential gene sets identified 10 years ago. Furthermore, significant progress has been made in the area of genome minimization of both model bacteria. This review provides an update, with particular emphasis on the current essential gene sets and their comparison with the original gene sets identified 10 years ago. Special attention is focused on the genome reduction analyses in B. subtilis and E. coli and the construction of minimal cell factories for industrial applications.

scholarly journals Network properties of cancer prognostic genes and their gene sets in the human protein interactome

2019 ◽  
Author(s):  
Jifeng Zhang ◽  
Cheng Jiang ◽  
Zhicheng Ji ◽  
Chenrun Wang
Keyword(s):  
Essential Gene ◽  
Housekeeping Gene ◽  
Human Protein ◽  
Random Sets ◽  
Gene Set ◽  
Gene Sets ◽  
Interactome Network ◽  
Protein Interactome ◽  
Network Modules ◽  
String Networks

Abstract Background Identifying prognostic genes (PG) is crucial for estimating survival time and providing pinpoint treatments for patients with cancer. However, prognostic genes sets (PGS) reported in most existing research have low reproducibility and overlap ever between the same cancers or their subtypes. Their common characteristic as well as the molecular mechanism of action is still elusive. Methods Here, we obtained nine prognostic gene sets (including 1,439 prognostic genes) of different types of cancer from 23 high quality literatures, and systemically investigated eight network topological properties for PG and PGS compared with background and four other gene sets (cancer gene set CA, essential gene set ES, housekeeping gene set HK, and metastasis-angiogenesis gene set MA) based on the HPRD and String networks. Results The results showed that PG did not occupy key positions in the human protein interactome network, and were more similar to ES rather than CA. Also, PGS had significantly small intraset distance (IAD) and interset distance (IED) in comparison with random sets. Further, we also found that PGS tended to have be distributed within network modules rather than between modules, the functional intersection of the modules enriched with PGS was closely related to cancer. Conclusions Our research reveals the common properties of cancer PG and PGS in the human protein interactome network, and can help us understand and discover cancer prognostic biomarkers.

scholarly journals In vitro exploration of the Xanthomonas hortorum pv. vitians genome using transposon insertion sequencing and comparative genomics to discriminate between core and contextual essential genes

2021 ◽  
Author(s):  
Lucas Morinière ◽  
Solène Lecomte ◽  
Erwan Gueguen ◽  
Franck Bertolla
Keyword(s):  
Comparative Genomics ◽  
Plant Pathogen ◽  
Type Species ◽  
Essential Genes ◽  
Content Type ◽  
Link Type ◽  
Transposon Insertion ◽  
Ecological Features ◽  
Genomic Study ◽  
Transposon Insertion Sequencing

The essential genome of a bacterium encompasses core genes associated with basic cellular processes and conditionally essential genes dependent upon environmental conditions or the genetic context. Comprehensive knowledge of those gene sets allows for a better understanding of fundamental bacterial biology and offers new perspectives for antimicrobial drug research against detrimental bacteria such as pathogens. We investigated the essential genome ofXanthomonas hortorumpv.vitians, a gammaproteobacterial plant pathogen of lettuce (Lactuca sativaL.) which belongs to the plant-pathogen reservoir genusXanthomonasand is affiliated to the familyXanthomonadaceae. No practical means of disease control or prevention against this pathogen is currently available, and its molecular biology is virtually unknown. To reach a comprehensive overview of the essential genome ofX. hortorumpv.vitiansLM16734, we developed a mixed approach combining high-quality full genome sequencing, saturated transposon insertion sequencing (Tn-Seq) in optimal growth conditions, and coupled computational analyses such as comparative genomics, synteny assessment and phylogenomics. Among the 370 essential loci identified by Tn-Seq, a majority was bound to critical cell processes conserved across bacteria. The remaining genes were either related to specific ecological features ofXanthomonasorXanthomonadaceaespecies, or acquired through horizontal gene transfer of mobile genetic elements and associated with ancestral parasitic gene behaviour and bacterial defence systems. Our study sheds new light on our usual concepts about gene essentiality and is pioneering in the molecular and genomic study ofX. hortorumpv.vitians.

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