DOCSLIB.ORG

The Development and Application of a Transposon Insertion Sequencing Methodology in Escherichia Coli BW25113

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Development and Application of a Transposon Insertion Sequencing Methodology in Escherichia Coli BW25113 The Development and Application of a Transposon Insertion Sequencing Methodology in Escherichia coli BW25113 by Ashley Robinson A thesis submitted to The University of Birmingham For the degree of Doctor of Philosophy College of Medical and Dental Sciences September 2016 [1] University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Escherichia coli is one of the most studied model organisms in biology. Even with decades of research, there are a substantial number of genes with an as yet unknown function. Previously, to determine the link between gene function and phenotype took significant experimental effort. However, newer methods are capable of providing large amounts of biological data in short timeframes. One such method, transposon insertion sequencing, is a powerful research tool, which couples transposon mutagenesis and next generation sequencing to identify genes that have important or essential functions. Here, three transposon insertion sequencing methods were compared. The techniques were adapted from previously published literature. Based on a number of metrics one technique was shown to be superior for data generation. This method was chosen for application in further transposon-insertion sequencing experiments. Subsequently, the optimised method was used to assess which genes were essential for the viability of the model organism E. coli K12. The results of this work were compared with the literature and other databases of gene essentiality. A high degree of concordance was observed between our datasets and those generated previously through other methods. Indeed, the method described here was shown to have several benefits over previously used approaches. Finally, genes involved with maintenance of the outer membrane were identified by using markers for membrane permeability in tandem with the chosen method. In keeping with previous literature multiple genes involved with many aspects of the cell envelope were reported. Many of the reported genes were shown to be involved with metabolic processes related to the biogenesis and maintenance of the cell envelope. [2] ACKNOWLEDGEMENTS This collection of ordered letters, by one monkey with one (admittedly modern) typewriter, would not have been completed without the friendship, guidance and support of so many people. I fear this will not do you justice, but here goes! Magic Rainbow Five ® - you have made my time in the lab incredible! I may never be a part of such a friendly, hilarious and supportive microcosm again, and for your friendship I am truly grateful. O Hai Chris – the laughs we have had! I am indebted to you for so much - your graffiti skills, taste in cinema and your incisive wit amongst other things. Thank you, and keep on searching for those triangles bro. Jacky B, Caddaby and Dairylea – your astounding knowledge of science, music and arabica alike always made for an entertaining discussion. Thank you for always being downstairs and available for caffeination, contemplation and celebration. James – my musical brother from an unrelated matriarch! Whenever I hear anything even remotely Latin in origin, I will fondly remember our improvs. Thank you so much for the music and your support. Pete – you have helped me for quite a few years now, and I am indebted – thank you so much for your ever helpful knowledge and insight, as well as Christmas festive cheer. Amanda, Faye, Josh, Sara, Rachael, Tamar, and likely many others that have fallen through the sieve between my ears – I have you all to thank for so much! Ian and Cathy – you have been wonderful to me, and really made me feel like part of the lab family. It has been a privilege –I am eternally grateful for the opportunity you provided, and I hope that what I have done will always be of use. If you ever need computer help… Finally, my beautiful family – Kayleigh, Erin, Niamh and Isla. You enrich my life with an unspeakable quality, and make my days joyous beyond description. Thank you for making life so worthwhile. [3] TABLE OF CONTENTS Chapter 1. General Introduction 10 The model organism Escherichia coli 11 The cell envelope 11 Inner membrane 14 Protein translocation 14 Energy generation and proton motive force 16 Environmental sensing and response 17 Periplasm 19 Peptidoglycan 20 Chaperones 23 Outer membrane 25 Lipopolysaccharide 25 Lipoproteins 25 Outer membrane proteins (OMPs) 28 Envelope integrity and permeability 31 Vancomycin 31 SDS 32 Industrial application 34 Gene essentiality 35 Research methodologies 37 Advances in DNA sequencing and applications 40 Next generation sequencing 41 Transposon insertion sequencing 44 Aims 47 Chapter 2. Materials and methods 49 Bacterial strains and primers 50 Transposon library creation 50 Two-PCR library preparation method 50 [4] Two-PCR method adaptation 54 Two-PCR method protocol 56 Shearing-based library preparation method 57 Adaptation of the shearing based method 58 Shearing method protocol 61 Hybrid shearing based library preparation method 62 Adaptation of the hybrid method 64 Hybrid method protocol 64 Sequence read analysis 65 Preliminary read processing 65 Primary read processing 66 Essential gene prediction 67 Differential representation calculation 67 Chapter 3. A comparison of transposon sequencing library preparation methods 69 Introduction 70 Results 70 A two-PCR based library preparation method 70 A shearing based library preparation method 77 A hybrid two-PCR/shearing based library preparation method 82 Discussion 88 Chapter 4. Escherichia coli BW25113 essential gene analysis 93 [5] Introduction 94 Results 94 Datasets and essential gene prediction 95 Manual inspection of essential genes 98 Supporting evidence for manually inspected genes 112 Cluster of orthologous groups (COG) analysis 115 Discussion 142 Chapter 5. Escherichia coli BW25113 conditional gene analysis in response to markers for outer membrane permeability 146 Introduction 147 Results 148 Sample datasets 148 Differential representation analysis 148 Genes differentially represented after growth in the presence of SDS 154 Genes differentially represented after growth in the presence of vancomycin 168 Comparison with other studies 173 Discussion 176 Chapter 6 Final discussion 178 Chapter 7 Bibliography 184 [6] List of Figures Figure 1.1. The E. coli cell envelope. 13 Figure 1.2. ATP Synthase (Berman et al ., 2000). 18 Figure 1.3. Two component systems. 21 Figure 1.4. Peptidoglycan biosynthesis (Typas et al., 2011). 22 Figure 1.5. Lipoprotein anchoring. 27 Figure 1.6. Schematic of the AcrAB/TolC efflux pump. 30 Figure 1.7. Vancomycin and its mechanism of action. 33 Figure 1.8. The Datsenko and Wanner (2000) gene deletion method. 39 Figure 1.9. A simplified depiction of Illumina sequencing by synthesis. 43 Figure 1.10. A simplified depiction of transposon insertion sequencing. 46 Figure 2.1. Transposome mediated mutagenesis. 53 Figure 2.2. The adapted 2 PCR method used in this work. 55 Figure 2.3. The adapted shearing based method used in this work. 59 Figure 2.4. The hybrid based method used in this work. 63 Figure 3.1. Histograms of insertion indexes calculated from datasets generated using the two-PCR methodology. 75 Figure 3.2. Insertion index correlation scatterplots for the datasets generated using the two-PCR method. 76 Figure 3.3. Histograms of insertion indexes calculated from datasets generated using the shearing methodology. 80 Figure. 3.4. Correlation of LB datasets derived from the shearing method. 83 [7] Figure 3.5. Histograms of insertion indexes calculated from datasets generated using the hybrid methodology. 87 Figure 3.6 Insertion index correlation scatterplots for the hybrid datasets. 89 Figure 3.7. Differences in insertion representation between the two-PCR and hybrid methods. 91 Figure 4.1 Insertion index histograms for the combined NTL and LB datasets. 96 Figure 4.2 Comparison of the KEIO, LB and NTL essential gene lists. 97 Figure 4.3 Insertions throughout genes previously predicted to be essential. 99 Figure 4.4 Essential gene regions of grpE and ftsK . 106 Figure 4.5 Insertions into the 5′ and 3′ regions of ftsH and rnpA . 108 Figure 4.6 Insertions into cydC and secD . 109 Figure 4.7 Lack of insertions in genes not reported to be essential from the KEIO library. 111 Figure 4.8 Distribution of COG categories in the summarised gene lists. 117 Figure 4.9 The overlap of an essential gene region with the transmembrane domain of yejM . 144 Figure 5.1. Insertion index correlation scatterplots for the biological replicates of the S4.8 and V100 samples. 150 Figure 5.2. Insertion index histograms for the combined S4.8 and V100 datasets. 151 Figure 5.3. Insertion profiles for genes known to be involved with response to vancomycin and SDS 153 Figure 5.4. Schematic showing the RssB regulation of RpoS. 160 Figure 5.5. Negatively represented genes in the LPS biosynthetic pathway after growth in SDS. 166 [8] Abbreviations ATP – adenosine triphosphate BAM complex – beta barrel assembly machinery COG – cluster of orthologous groups DNA
Load more

Recommended publications
  • A Systems Biology Approach to Identify Adjunctive Drug Targets in Two Major Mycobacterial Pathogens
    A Systems Biology Approach to Identify Adjunctive Drug Targets in Two Major Mycobacterial Pathogens by William M. Matern A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland May, 2019 ⃝c William M. Matern 2019 All rights reserved Abstract This thesis explores novel drug targets to accelerate therapy for infections caused by the important human pathogens Mycobacterium avium (Mav) and Mycobacterium tuberculosis (Mtb). Infections with these bacterial species are notoriously difficult to treat - requiring months to years of intensive antibiotic therapy. Decreasing this length may help reduce expenditures necessary for monitoring therapy, improve patient outcomes, and reduce the significant morbidity and mortality caused by Mav and especially the global pathogen Mtb. Bacterial antibiotic persistence has been defined as the ability of bacteria to survive inhigh concentrations of antibiotics without genetic mutation (ie antibiotic resistance). Thus, infections caused by Mav and Mtb are highly persistent. A major focus of this work is the discovery of mechanisms underlying this persistence phenomenon in hopes that this knowledge can be exploited to improve available therapies. A major portion of the work is carried out using high-throughput genomic screens involving tech- niques such as transposon mutagenesis and transposon sequencing (Tn-seq). Statistical methods are developed and implemented to analyze this dataset with a focus on non-parametric methods. Novel discoveries include identification of the essential genes of Mav as well as particular genes that assist in bacterial survival during antibiotic exposure. Mechanisms underlying antibiotic persistence are discussed and explored in follow-up experiments guided by the high-throughput data.
    [Show full text]
  • 1 Reflecting on a Decade of Transposon-Insertion Sequencing 1 2
    1 Reflecting on a Decade of Transposon-Insertion Sequencing 2 3 Amy K. Cain1*, Lars Barquist2,3, Andrew L. Goodman4, Ian T. Paulsen1, Julian Parkhill5 and 4 Tim van Opijnen6* 5 6 Authors Affiliations: 7 1ARC Centre of Excellence in Synthetic Biology, Department of Molecular Sciences, 8 Macquarie University, Sydney, NSW, Australia 9 2 Helmholtz Institute for RNA-based Infection Research, Helmholtz Centre for Infection 10 Research, Würzburg, BY, Germany 11 3 Faculty of Medicine, University of Würzburg, Würzburg, BY, Germany 12 4 Department of Microbial Pathogenesis and Microbial Sciences Institute, Yale University 13 School of Medicine, New Haven, CT, USA 14 5Department of Veterinary Medicine, University of Cambridge, Cambridge, Cambs, UK 15 6 Department of Biology, Boston College, Boston, MA, USA 16 17 *corresponding authors: [email protected] or [email protected] 18 1 19 Abstract 20 It has been 10 years since the birth of modern transposon-insertion sequencing (TIS) 21 methods, which combine genome-wide transposon mutagenesis with high-throughput 22 sequencing to estimate the fitness contribution or essentiality of each genetic component 23 simultaneously in a bacterial genome. Four TIS variations were published in 2009: 24 transposon sequencing (Tn-Seq), transposon-directed insertion site sequencing (TraDIS), 25 insertion sequencing (INSeq) and high-throughput insertion tracking by deep sequencing 26 (HITS). TIS has since become an important tool for molecular microbiologists, being one of 27 the few genome-wide techniques that directly links phenotype to genotype and ultimately can 28 assign gene function. In this review, we discuss the recent applications of TIS to answer 29 overarching biological questions.
    [Show full text]
  • Essential Genome of Campylobacter Jejuni Rabindra K
    Mandal et al. BMC Genomics (2017) 18:616 DOI 10.1186/s12864-017-4032-8 RESEARCHARTICLE Open Access Essential genome of Campylobacter jejuni Rabindra K. Mandal1,3*, Tieshan Jiang1 and Young Min Kwon1,2 Abstract Background: Campylobacter species are a leading cause of bacterial foodborne illness worldwide. Despite the global efforts to curb them, Campylobacter infections have increased continuously in both developed and developing countries. The development of effective strategies to control the infection by this pathogen is warranted. The essential genes of bacteria are the most prominent targets for this purpose. In this study, we used transposon sequencing (Tn-seq) of a genome-saturating library of Tn5 insertion mutants to define the essential genome of C. jejuni at a high resolution. Result: We constructed a Tn5 mutant library of unprecedented complexity in C. jejuni NCTC 11168 with 95,929 unique insertions throughout the genome and used the genomic DNA of the library for the reconstruction of Tn5 libraries in the same (C. jejuni NCTC 11168) and different strain background (C. jejuni 81–176) through natural transformation. We identified 166 essential protein-coding genes and 20 essential transfer RNAs (tRNA) in C. jejuni NCTC 11168 which were intolerant to Tn5 insertions during in vitro growth. The reconstructed C. jejuni 81–176 library had 384 protein coding genes with no Tn5 insertions. Essential genes in both strain backgrounds were highly enriched in the cluster of orthologous group (COG) categories of ‘Translation, ribosomal structure and biogenesis (J)’, ‘Energy production and conversion (C)’,and‘Coenzyme transport and metabolism (H)’. Conclusion: Comparative analysis among this and previous studies identified 50 core essential genes of C.
    [Show full text]
  • Genome-Wide Determination of Gene Essentiality by Transposon Insertion Sequencing in Yeast Pichia Pastoris
    www.nature.com/scientificreports OPEN Genome-Wide Determination of Gene Essentiality by Transposon Insertion Sequencing in Yeast Pichia Received: 8 December 2017 Accepted: 19 June 2018 pastoris Published: xx xx xxxx Jinxiang Zhu1, Ruiqing Gong1, Qiaoyun Zhu1, Qiulin He1, Ning Xu1, Yichun Xu1, Menghao Cai1, Xiangshan Zhou1, Yuanxing Zhang1,2 & Mian Zhou1 In many prokaryotes but limited eukaryotic species, the combination of transposon mutagenesis and high-throughput sequencing has greatly accelerated the identifcation of essential genes. Here we successfully applied this technique to the methylotrophic yeast Pichia pastoris and classifed its conditionally essential/non-essential gene sets. Firstly, we showed that two DNA transposons, TcBuster and Sleeping beauty, had high transposition activities in P. pastoris. By merging their insertion libraries and performing Tn-seq, we identifed a total of 202,858 unique insertions under glucose supported growth condition. We then developed a machine learning method to classify the 5,040 annotated genes into putatively essential, putatively non-essential, ambig1 and ambig2 groups, and validated the accuracy of this classifcation model. Besides, Tn-seq was also performed under methanol supported growth condition and methanol specifc essential genes were identifed. The comparison of conditionally essential genes between glucose and methanol supported growth conditions helped to reveal potential novel targets involved in methanol metabolism and signaling. Our fndings suggest that transposon mutagenesis and Tn-seq could be applied in the methylotrophic yeast Pichia pastoris to classify conditionally essential/non-essential gene sets. Our work also shows that determining gene essentiality under diferent culture conditions could help to screen for novel functional components specifcally involved in methanol metabolism.
    [Show full text]
  • High-Throughput Parallel Sequencing to Measure Fitness of Leptospira Interrogans Transposon Insertion Mutants During Acute Infection
    UCLA UCLA Previously Published Works Title High-Throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants during Acute Infection. Permalink https://escholarship.org/uc/item/33s941jw Journal PLoS neglected tropical diseases, 10(11) ISSN 1935-2727 Authors Lourdault, Kristel Matsunaga, James Haake, David A Publication Date 2016-11-08 DOI 10.1371/journal.pntd.0005117 Peer reviewed eScholarship.org Powered by the California Digital Library University of California RESEARCH ARTICLE High-Throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants during Acute Infection Kristel Lourdault1,2*, James Matsunaga1,2, David A. Haake1,2,3,4 1 Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, United States of a11111 America, 2 Departments of Medicine, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America, 3 Departments of Urology, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California, United States of America, 4 Departments of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America * [email protected] OPEN ACCESS Citation: Lourdault K, Matsunaga J, Haake DA Abstract (2016) High-Throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Pathogenic species of Leptospira are the causative agents of leptospirosis, a zoonotic dis- Transposon Insertion Mutants during Acute ease that causes mortality and morbidity worldwide. The understanding of the virulence Infection. PLoS Negl Trop Dis 10(11): e0005117. mechanisms of Leptospira spp is still at an early stage due to the limited number of genetic doi:10.1371/journal.pntd.0005117 tools available for this microorganism.
    [Show full text]
  • Translating Genomics Research Into Control of Tuberculosis: Lessons Learned and Future Prospects Digby F Warner1,2,3 and Valerie Mizrahi1,2,3*
    Warner and Mizrahi Genome Biology 2014, 15:514 http://genomebiology.com/2014/15/11/514 REVIEW Translating genomics research into control of tuberculosis: lessons learned and future prospects Digby F Warner1,2,3 and Valerie Mizrahi1,2,3* Abstract spite of recent gains in the battle against TB, the current rate of decline in TB incidence of 2% per annum falls far Genomics research has enabled crucial insights into short of this target [5]. This alarming situation under- the adaptive evolution of Mycobacterium tuberculosis scores the urgent need for new tools to control this dev- as an obligate human pathogen. Here, we highlight astating disease. major recent advances and evaluate the potential for Fundamental TB research poses very specific practical genomics approaches to inform tuberculosis control and financial challenges. As an infectious pathogen, M. efforts in high-burden settings. tuberculosis can only be manipulated in purpose-built biosafety level 3 containment laboratories by specialist personnel. The construction and maintenance of such Introduction facilities requires significant financial investment; more- Tuberculosis (TB) is a leading cause of death as a result over, the running costs necessary to ensure continued of an infectious bacterial agent, claiming 1.4 million lives compliance with the stringent safety regulations are each year [1]. With an estimated global burden of 8.7 high, and are incurred in addition to standard laboratory million incident cases per annum, TB remains a major operating expenses. From a practical perspective, M. tu- public health threat. In high-burden regions such as berculosis is an intractable experimental subject: the ba- sub-Saharan Africa, the TB epidemic is exacerbated by cillus is notorious for its slow growth rate in vitro and co-morbidities, including HIV and diabetes, as well as for its tendency to form aggregates in liquid media.
    [Show full text]
  • The Essential Gene Set of a Photosynthetic Organism PNAS PLUS
    The essential gene set of a photosynthetic organism PNAS PLUS Benjamin E. Rubina, Kelly M. Wetmoreb, Morgan N. Priceb, Spencer Diamonda, Ryan K. Shultzabergerc, Laura C. Lowea, Genevieve Curtina, Adam P. Arkinb,d, Adam Deutschbauerb, and Susan S. Goldena,1 aDivision of Biological Sciences, University of California, San Diego, La Jolla, CA 92093; bPhysical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; cKavli Institute for Brain and Mind, University of California, San Diego, La Jolla, CA 92093; and dDepartment of Bioengineering, University of California, Berkeley, CA 94720 Contributed by Susan S. Golden, September 29, 2015 (sent for review July 16, 2015; reviewed by Caroline S. Harwood and William B. Whitman) Synechococcus elongatus PCC 7942 is a model organism used for Tn-seq–like system in Chlamydomonas reinhardtii; however, the studying photosynthesis and the circadian clock, and it is being mutant library currently lacks sufficient saturation to determine developed for the production of fuel, industrial chemicals, and gene essentiality (12). To date, the essential genes for photo- pharmaceuticals. To identify a comprehensive set of genes and autotrophs have only been estimated by indirect means, such as intergenic regions that impacts fitness in S. elongatus, we created by comparative genomics (13). The absence of experimentally a pooled library of ∼250,000 transposon mutants and used sequencing determined essential gene sets in photosynthetic organisms, de- to identify the insertion locations. By analyzing the distribution and spite their importance to the environment and industrial pro- survival of these mutants, we identified 718 of the organism’s 2,723 duction, is largely because of the difficulty and time required for genes as essential for survival under laboratory conditions.
    [Show full text]
  • Using Transposon Sequencing to Identify Vulnerabilities in Staphylococcus Aureus
    Using Transposon Sequencing to Identify Vulnerabilities in Staphylococcus aureus The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Coe, Kathryn Ann. 2019. Using Transposon Sequencing to Identify Vulnerabilities in Staphylococcus aureus. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42013046 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Using Transposon Sequencing to Identify Vulnerabilities in Staphylococcus aureus A dissertation presented by Kathryn Ann Coe to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biological and Biomedical Sciences Harvard University, Cambridge, Massachusetts June 2019 © 2019 Kathryn Ann Coe All rights reserved. Dissertation Advisors: Professor Suzanne Walker Kathryn Ann Coe Professor Yonatan Grad Using Transposon Sequencing to Identify Vulnerabilities in Staphylococcus aureus Abstract Antibiotic resistant infections cost thousands of lives in the United States every year. Resistance exists for every known antibiotic, making the development new antibiotics crucial to human health. One technique that has become instrumental in prioritizing targets for antibiotic development is transposon sequencing (Tn-Seq). Tn-Seq is a powerful high-throughput technology that connects bacterial genes with phenotypes and can be used to identify genes that are essential for bacterial survival. In the early years of Tn-Seq, results from a single representative strain were largely assumed to reflect the entire species.
    [Show full text]
  • Peptidoglycan Synthesis in Mycobacterium Tuberculosis Is Organized Into Networks with Varying Drug Susceptibility
    Peptidoglycan synthesis in Mycobacterium tuberculosis is organized into networks with varying drug susceptibility Karen J. Kiesera, Catherine Baranowskia, Michael C. Chaob, Jarukit E. Longc, Christopher M. Sassettic,d, Matthew K. Waldorb,d,e, James C. Sacchettinif, Thomas R. Ioergerg, and Eric J. Rubina,e,1 aDepartment of Immunology and Infectious Disease, Harvard T. H. Chan School of Public Health, Boston, MA 02115; bDivision of Infectious Diseases, Brigham & Women’s Hospital, Boston, MA 02115; cDepartment of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester MA 01655; dHoward Hughes Medical Institute, Chevy Chase, MD 20815; eDepartment of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115; fDepartment of Biochemistry and Biophysics and Department of Chemistry, Texas A&M University, College Station, TX 77843; and gDepartment of Computer Science, Texas A&M University, College Station, TX 77843 Edited by Carolyn R. Bertozzi, University of California, Berkeley, CA, and approved September 8, 2015 (received for review July 17, 2015) Peptidoglycan (PG), a complex polymer composed of saccharide proper cell-wall synthesis. One method of coordination is the use chains cross-linked by short peptides, is a critical component of the of large protein complexes, the elongation complex and divisome, bacterial cell wall. PG synthesis has been extensively studied in which mediate cell-wall biogenesis during cell elongation or di- model organisms but remains poorly understood in mycobacteria, vision, respectively (2). The essential activity of these enzymes a genus that includes the important human pathogen Mycobacte- makes them prime drug targets; indeed, PBPs and Ldts are inhibited rium tuberculosis (Mtb). The principle PG synthetic enzymes have by carbapenems and penicillin (6, 7), which remains one of the most similar and, at times, overlapping functions.
    [Show full text]
  • A Decade of Advances in Transposon-Insertion Sequencing
    REVIEWS A decade of advances in transposon- insertion sequencing Amy K. Cain 1 ✉ , Lars Barquist 2,3, Andrew L. Goodman 4,5, Ian T. Paulsen1, Julian Parkhill 6 and Tim van Opijnen 7 ✉ Abstract | It has been 10 years since the introduction of modern transposon-insertion sequencing (TIS) methods, which combine genome- wide transposon mutagenesis with high- throughput sequencing to estimate the fitness contribution or essentiality of each genetic component in a bacterial genome. Four TIS variations were published in 2009: transposon sequencing (Tn-Seq), transposon- directed insertion site sequencing (TraDIS), insertion sequencing (INSeq) and high- throughput insertion tracking by deep sequencing (HITS). TIS has since become an important tool for molecular microbiologists, being one of the few genome-wide techniques that directly links phenotype to genotype and ultimately can assign gene function. In this Review, we discuss the recent applications of TIS to answer overarching biological questions. We explore emerging and multidisciplinary methods that build on TIS, with an eye towards future applications. Transposon- insertion sequencing (TIS) methods different sites. By direct sequencing of the transposon- combine large- scale transposon mutagenesis with next- flanking regions of the initial library, potential essential generation sequencing to estimate the essentiality and/or features can be identified as those that do not tolerate fitness contribution of each genetic feature in a bacterial insertions. Alternatively, the library can be subjected to a genome simultaneously. A strength of TIS is that exper- selective condition, for instance antibiotic stress (Fig. 1B), 1ARC Centre of Excellence in Synthetic Biology, iments are performed with pooled transposon libraries, to query non- essential features involved in survival and Department of Molecular which allows direct linkage of phenotype to genotype growth within that environment.
    [Show full text]
  • Changes in the Genetic Requirements for Microbial Interactions with Increasing Community Complexity Manon Morin1, Emily C Pierce1, Rachel J Dutton1,2*
    RESEARCH ARTICLE Changes in the genetic requirements for microbial interactions with increasing community complexity Manon Morin1, Emily C Pierce1, Rachel J Dutton1,2* 1Division of Biological Sciences, University of California, San Diego, La Jolla, United States; 2Center for Microbiome Innovation, Jacobs School of Engineering, University of California San Diego, La Jolla, United States Abstract Microbial community structure and function rely on complex interactions whose underlying molecular mechanisms are poorly understood. To investigate these interactions in a simple microbiome, we introduced E. coli into an experimental community based on a cheese rind and identified the differences in E. coli’s genetic requirements for growth in interactive and non- interactive contexts using Random Barcode Transposon Sequencing (RB-TnSeq) and RNASeq. Genetic requirements varied among pairwise growth conditions and between pairwise and community conditions. Our analysis points to mechanisms by which growth conditions change as a result of increasing community complexity and suggests that growth within a community relies on a combination of pairwise and higher-order interactions. Our work provides a framework for using the model organism E. coli as a readout to investigate microbial interactions regardless of the genetic tractability of members of the studied ecosystem. DOI: https://doi.org/10.7554/eLife.37072.001 Introduction Microorganisms rarely grow as single isolated species but rather as part of diverse microbial commu- nities. In these communities, bacteria, archaea, protists, viruses and fungi can coexist and perform complex functions impacting biogeochemical cycles and human health (Falkowski et al., 2008; Flint et al., 2012). Deciphering microbial growth principles within a community is challenging due to *For correspondence: the intricate interactions between microorganisms, and between microorganisms and their environ- [email protected] ment.
    [Show full text]
  • Q2 Report Tnseq
    DOE Joint Genome Institute FY15 Q2 Progress Report Q2. Report on progress developing new experimental capabilities to analyze complex genomic or metagenomic datasets. Background The functional annotation of genomes remains an extremely difficult problem that is exacerbated by the continuing accumulation of new genomes encoding a large number of new genes of unknown function. Computational prediction of gene function based on DNA sequence similarity to known genes is successful when closely related, well-annotated model organisms are available. However, a scarcity of functionally well-characterized microbes means that the genomes of many organisms, including those with important roles in bioenergy, the environment and human health, remain poorly annotated. There is therefore a great demand for novel high- throughput approaches to determine gene function from phylogenetically diverse microbes. Transposon mutagenesis and sequencing (TnSeq) is a promising approach for high-throughput annotation of gene function (Figure 1). In this approach, mutagenesis of an bacterium of interest gives rise to a population (or pool) in which each cell has a single random transposon insertion, and which cumulatively contains at least one insertion for every gene in the genome. Transposons used in this strategy contain ‘tags’, which can be identified by DNA sequencing, and used to track changes in the relative abundance of thousands of mutant strains in a single tube reaction. By repeating the experiment under a variety of growth conditions, the combined patterns of mutant fitness across conditions can be used to infer a first-order functional annotation for many genes in the genome. We previously demonstrated that transposon-based mutant fitness provides specific functional annotations for a substantial number of genes with previously unknown function (Deutschbauer et al., 2011).
    [Show full text]