DOCSLIB.ORG

Bacterial Evolution on Demand

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Bacterial Evolution on Demand INSIGHT ANTIBIOTIC RESISTANCE Bacterial evolution on demand Bacteria carry antibiotic resistant genes on movable sections of DNA that allow them to select the relevant genes on demand. PA˚ L J JOHNSEN, JOA˜ O A GAMA AND KLAUS HARMS genes known as gene cassettes. The ones Related research article Souque C, Escu- located at the start of the integron produce dero JA, MacLean RC. 2021. Integron activ- more proteins than the ones closer to the end ity accelerates the evolution of antibiotic (Collis and Hall, 1995). During stress situations resistance. eLife 10:e62474. doi: 10.7554/ – such as exposure to antibiotics – an enzyme eLife.62474 called integrase is produced, allowing the microbes to shuffle the order of the cassettes in the integron (Guerin et al., 2009). Conse- quently, gene cassettes encoding the appropri- ate adaptive response to an antibiotic will be acteria are the most abundant form of moved closer to the start, where expression lev- life and inhabit virtually every environ- els are higher (Figure 1). It has been proposed B ment on Earth, from the soil to the that this mechanism allows bacteria to adapt to human body. They display remarkable genetic fluctuating environmental conditions ‘on flexibility and can respond rapidly to environ- demand’, but this hypothesis has never been mental changes. Bacteria have developed differ- experimentally tested (Escudero et al., 2015). ent strategies to increase their genetic diversity, Now, in eLife, Ce´lia Souque, Jose´ A. Escu- including the use of mobile genetic elements, dero and Craig MacLean of the University of which can either move around the genome or be Oxford and the Universidade Complutense de transferred to a different bacterium. These Madrid report new insights into how bacteria mobile genetic elements enable bacteria to evolve on demand when exposed to antibiotic acquire new genes that have already run the treatment (Souque et al., 2021). Souque et al. gauntlet of natural selection in other bacterial used two strains of the bacterium Pseudomonas species – exemplified by the rapid emergence aeruginosa, differing only by the presence or and global spread of antibiotic resistance. absence of a functional integrase. Both strains However, producing resistance proteins in contained an integron with three gene cassettes the absence of antibiotics, or acquiring resis- encoding resistance to different antibiotics, with tance genes that are not yet needed, is costly the relevant resistant gene in the last position. and may reduce the viability of a bacterium. One The researchers used an experimental evolu- way to counterbalance this burden is a genetic tion protocol known as the evolutionary ramp element known as the integron. Integrons are design. In this set up, bacteria are challenged genetic platforms that can capture and shuffle with daily doublings of antibiotic concentrations, genes, thus providing instant adaptive benefits which force the bacterial populations to rapidly Copyright Johnsen et al. This in fluctuating environments (Stokes and Hall, increase their antibiotic resistance levels or face article is distributed under the terms 1989). extinction. After 13 concentration ramps with of the Creative Commons Attribution Integrons are not mobile in their own right, the antibiotic gentamicin, significantly more License, which permits unrestricted but they are often embedded within mobile populations containing a functional integrase use and redistribution provided that the original author and source are genetic elements that can facilitate their trans- survived than their integrase-negative counter- credited. fer. They contain an array of antibiotic resistant parts, which largely went extinct. Johnsen et al. eLife 2021;10:e68070. DOI: https://doi.org/10.7554/eLife.68070 1 of 3 Insight Antibiotic Resistance Bacterial evolution on demand Figure 1. Evolutionary dynamics of integrons. (A) Class-1 integrons consist of a 5’-conserved region (black section) and an integrase (intI) that can capture gene cassettes (GC, colored rectangles) and insert them at the attI gene site. They are the most wide-spread class of these genetic elements and contain arrays ranging from one to five gene cassettes. The gene cassettes are separated by attC sites (trapezoids with black borders). Exposure to low doses of antibiotic (yellow tablets) activates the integrase (pacman symbols). (B) The integrase can rearrange the gene cassettes by either cutting them at attC sites (b1), or by inserting previously excised or free gene cassettes at the attI site (b2). The integrase acts by breaking and rejoining the bottom DNA strand (red). After DNA replication (gray arrows), daughter integrons are parental (derived from the blue top strand) or carry a changed gene cassette array (derived from the bottom strand). (C) Increased antibiotic concentrations exert selective pressure on the diversity of the generated integron. Those carrying the appropriate gene cassette (yellow rectangle) in the first position are favored (+) due to higher expression of resistance, while other gene cassette arrays are counter-selected (–). The ability to move the required gentamicin- antibiotics or only subjected to low concentra- resistance gene cassette forward in the integron tions, did not show any structural variation in the to increase resistance levels may at first resem- cassette arrays. Taken together, this provides ble adaptive evolution with a neo-Lamarckian compelling evidence favoring the ‘evolution on flavor. However, examining the genetic demand hypothesis’. sequence of the earlier evolved strains (that This study represents an excellent example of arose at the start of the ramp experiment) how hypotheses concerning the evolution and revealed a range of cassette arrays, with the maintenance of mobile genetic elements can be three gene cassettes in various positions. This tested using combined approaches of experi- suggests that integrase activity generates ran- mental evolution, population biology and genet- dom genetic diversity upon which natural selec- ics. To that end, Souque et al. also propose new tion could act. mechanistic insights into the recombination In fact, structural variation was found in half dynamics within integron cassette arrays. They of the evolved populations early in the evolu- found integrons with gentamicin-resistance cas- tionary ramp experiments. But at the highest settes both in first and last position, suggesting antibiotic concentrations, most of them con- a ‘copy and paste’ mechanism of the integrase, tained a single variant i.e., they all contained the rather than a ‘cut and paste’ one. same cassette array. However, the evolved con- Duplicated gene cassettes indeed exist in trol populations that were either not exposed to integron-containing bacterial isolates from Johnsen et al. eLife 2021;10:e68070. DOI: https://doi.org/10.7554/eLife.68070 2 of 3 Insight Antibiotic Resistance Bacterial evolution on demand environmental and clinical samples, but they are Klaus Harms is at the Department of Pharmacy, likely rarer than the proposed preferential ‘copy Faculty of Health Sciences, The Arctic University of and paste’ mechanism. This could be due to the Norway, Tromsø, Norway suggested short-read sequencing bias. Another https://orcid.org/0000-0003-1454-0692 possibility could be that gene cassettes are excised from other integrons within the same cell, and then integrated into the first position. Competing interests: The authors declare that no In our opinion, whether integrase activity competing interests exist. mechanistically leads to ‘copy and paste’ versus Published 06 April 2021 ‘cut and paste’ could only be resolved using experimental assays with a single integron, but References we acknowledge that this is difficult to obtain. Collis CM, Hall RM. 1995. Expression of antibiotic Even when integrons are located on the chromo- resistance genes in the integrated cassettes of integrons. Antimicrobial Agents and Chemotherapy some, multiple copies will be present just before 39:155–162. DOI: https://doi.org/10.1128/AAC.39.1. cell division. 155 Finally, Souque et al. also addressed the Escudero JA, Loot C, Nivina A, Mazel D. 2015. The question why the integrase enzyme is costly integron: adaptation on demand. Microbiology (Starikova et al., 2012; Lacotte et al., Spectrum 3. Guerin E, Cambray G, Sanchez-Alberola N, Campoy S, 2017). This has previously been linked to off-tar- Erill I, Da Re S, Gonzalez-Zorn B, Barbe´J, Ploy MC, get integrase activity resulting in deleterious Mazel D. 2009. The SOS response controls integron genetic rearrangements (Harms et al., 2013). recombination. Science 324:1034. DOI: https://doi. The fact that most integrase-containing popula- org/10.1126/science.1172914, PMID: 19460999 tions in this study rarely undergo genetic shuffel- Harms K, Starikova I, Johnsen PJ. 2013. Costly Class-1 integrons and the domestication of the the functional ing suggests that off-target integrase activity is integrase. Mobile Genetic Elements 3:e24774. rare and that other not-mutually exclusive mech- DOI: https://doi.org/10.4161/mge.24774, PMID: 23 anisms could explain the costly nature of active 914313 integrases. Lacotte Y, Ploy MC, Raherison S. 2017. Class 1 Understanding the evolutionary dynamics of integrons are low-cost structures in Escherichia coli. The ISME Journal 11:1535–1544. DOI: https://doi.org/ these fascinating integrons and the benefits they 10.1038/ismej.2017.38, PMID: 28387772 provide to bacteria may help us on the quest of Souque C, Escudero JA, MacLean
Load more

Recommended publications
  • Piggybac Transposase Tools for Genome Engineering.Pdf
    piggyBac transposase tools for genome engineering PNAS PLUS Xianghong Lia,b, Erin R. Burnightc, Ashley L. Cooneyd, Nirav Malanie, Troy Bradye, Jeffry D. Sanderf,g, Janice Staberd, Sarah J. Wheelanh, J. Keith Joungf,g, Paul B. McCray, Jr.c,d, Frederic D. Bushmane, Patrick L. Sinnd,1, and Nancy L. Craiga,b,1 aHoward Hughes Medical Institute and bDepartment of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; cGenetics Program, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242; dDepartment of Pediatrics, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242; eDepartment of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; fMolecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Boston, MA 02129; gDepartment of Pathology, Harvard Medical School, Boston, MA 02115; and hDivision of Biostatistics and Bioinformatics, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Edited by Richard A. Young, Massachusetts Institute of Technology, Cambridge, MA, and approved May 1, 2013 (received for review December 30, 2012) The transposon piggyBac is being used increasingly for genetic stud- site-directed mutagenesis of the PB catalytic domain and isolated − ies. Here, we describe modified versions of piggyBac transposase an excision competent/integration defective (Exc+Int ) PB. We − that have potentially wide-ranging applications, such as reversible also find that introduction of the Exc+Int mutations into a hy- − transgenesis and modified targeting of insertions. piggyBac is dis- peractive version of PB (5, 8, 15) yields an Exc+Int transposase tinguished by its ability to excise precisely, restoring the donor site whose excision frequency is five- to six-fold higher than that of to its pretransposon state.
    [Show full text]
  • A Homologous Recombination-Based Method of Genetic Engineering
    PROTOCOL Recombineering: a homologous recombination-based method of genetic engineering Shyam K Sharan1, Lynn C Thomason2, Sergey G Kuznetsov1 & Donald L Court3 1Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA. 2SAIC-Frederick Inc., Gene Regulation and Chromosome Biology Laboratory, Basic Research Program, Frederick, Maryland, 21702, USA. 3Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, USA. Correspondence should be addressed to S.K.S. ([email protected]) or D.L.C. ([email protected]). Published online 29 January 2009; doi:10.1038/nprot.2008.227 Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in Escherichia coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-stranded linear DNA substrates, natureprotocols so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene / m tags can be inserted and regions of bacterial artificial chromosomes or the E. coli genome can be subcloned by gene retrieval using o c . recombineering. Most of these constructs can be made within about 1 week’s time. e r u t a n . INTRODUCTION w w Recombineering is an in vivo method of genetic engineering used This protocol will emphasize modification of bacterial artificial w / / 1–5 : primarily in Escherichia coli that uses short 50 base homologies .
    [Show full text]
  • An Optimized Preparation Method for Long Ssdna Donors to Facilitate Quick Knock-In Mouse Generation
    cells Article An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation Yukiko U. Inoue 1,* , Yuki Morimoto 1, Mayumi Yamada 2, Ryosuke Kaneko 3, Kazumi Shimaoka 1, Shinji Oki 4, Mayuko Hotta 1, Junko Asami 1, Eriko Koike 1, Kei Hori 1, Mikio Hoshino 1, Itaru Imayoshi 2,5 and Takayoshi Inoue 1,* 1 Department of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Ogawahigashi, Kodaira, Tokyo 187-8502, Japan; [email protected] (Y.M.); [email protected] (K.S.); [email protected] (M.H.); [email protected] (J.A.); [email protected] (E.K.); [email protected] (K.H.); [email protected] (M.H.) 2 Research Center for Dynamic Living Systems, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan; [email protected] (M.Y.); [email protected] (I.I.) 3 KOKORO-Biology Group, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan; [email protected] 4 Department of Immunology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Ogawahigashi, Kodaira, Tokyo 187-8502, Japan; [email protected] 5 Department of Deconstruction of Stem Cells, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan * Correspondence: [email protected] (Y.U.I.); [email protected] (T.I.) Abstract: Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles Citation: Inoue, Y.U.; Morimoto, Y.; in investigating various molecular functions in vivo.
    [Show full text]
  • Transposon Based Mutagenesis and Mapping of Transposon Insertion Sites Within the Ehrlichia Chaffeensis Genome Using Semi Random Two-Step Pcr
    TRANSPOSON BASED MUTAGENESIS AND MAPPING OF TRANSPOSON INSERTION SITES WITHIN THE EHRLICHIA CHAFFEENSIS GENOME USING SEMI RANDOM TWO-STEP PCR by VIJAYA VARMA INDUKURI B.S., ANDHRA UNIVERSITY, India, 2008 A THESIS Submitted in partial fulfillment of the requirements for the degree MASTER OF SCIENCE Department of Diagnostic Medicine/Pathobiology College of Veterinary Medicine KANSAS STATE UNIVERSITY Manhattan, Kansas 2013 Approved by: Major Professor Roman Reddy Ganta Copyright VIJAYA VARMA INDUKURI 2013 Abstract Ehrlichia chaffeensis a tick transmitted Anaplasmataceae family pathogen responsible for human monocytic ehrlichiosis. Differential gene expression appears to be an important pathogen adaptation mechanism for its survival in dual hosts. One of the ways to test this hypothesis is by performing mutational analysis that aids in altering the expression of genes. Mutagenesis is also a useful tool to study the effects of a gene function in an organism. Focus of my research has been to prepare several modified Himar transposon mutagenesis constructs for their value in introducing mutations in E. chaffeensis genome. While the work is in progress, research team from our group used existing Himar transposon mutagenesis plasmids and was able to create mutations in E. chaffeensis. Multiple mutations were identified by Southern blot analysis. I redirected my research efforts towards mapping the genomic insertion sites by performing the semi-random two step PCR (ST-PCR) method, followed by DNA sequence analysis. In this method, the first PCR is performed with genomic DNA as the template with a primer specific to the insertion segment and the second primer containing an anchored degenerate sequence segment. The product from the first PCR is used in the second PCR with nested transposon insertion primer and a primer designed to bind to the known sequence portion of degenerate primer segment.
    [Show full text]
  • The Evolutionary History of Chromosomal Super-Integrons Provides an Ancestry for Multiresistant Integrons
    The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons Dean A. Rowe-Magnus*, Anne-Marie Guerout*, Pascaline Ploncard*, Broderick Dychinco†, Julian Davies†, and Didier Mazel*‡ *Unite´de Programmation Mole´culaire et Toxicologie Ge´ne´ tique, Centre National de la Recherche Scientifique Unite´de Recherche Associe´e 1444, De´partement des Biotechnologies, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris, France; and †Department of Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, BC, Canada V6T 1Z3 Communicated by Allan Campbell, Stanford University, Stanford, CA, November 2, 2000 (received for review August 1, 2000) Integrons are genetic elements that acquire and exchange exog- located downstream of a resident promoter internal to the intI enous DNA, known as gene cassettes, by a site-specific recombi- gene, drives expression of the encoded proteins (13). Most of the nation mechanism. Characterized gene cassettes consist of a target attC sites of the integron cassettes identified to date are unique. recombination sequence (attC site) usually associated with a single Their length and sequence vary considerably (from 57 to 141 bp) open reading frame coding for an antibiotic resistance determi- and their similarities are primarily restricted to their boundaries, nant. The affiliation of multiresistant integrons (MRIs), which which correspond to the inverse core site (RYYYAAC) and the contain various combinations of antibiotic resistance gene cas- core site [G2TTRRRY; 2, recombination point (11, 14)]. settes, with transferable elements underlies the rapid evolution of More than 60 different antibiotic-resistance genes, covering multidrug resistance among diverse Gram-negative bacteria. Yet most antimicrobials presently in use, have been characterized in the origin of MRIs remains unknown.
    [Show full text]
  • Electroporation-Mediated and EBV LMP1-Regulated Gene Therapy in A
    Cancer Gene Therapy (2003) 10, 626–636 r 2003 Nature Publishing Group All rights reserved 0929-1903/03 $25.00 www.nature.com/cgt Electroporation-mediated and EBV LMP1-regulated gene therapy in a syngenic mouse tumor model Yu-hua Hsieh,1 Chang-jer Wu,2 Kai-ping Chow,3 Chia-lung Tsai,1 and Yu-sun Chang3 1Institute of Microbiology and Immunology, National Yang-Ming University, Shih-pai, Taipei 112, Republic of China; 2Institute of Preventive Medicine, National Defense Medical Center, Taipei 114, Republic of China; and 3Graduate Institute of Basic Medical Sciences, Chang-Gung University, Kwei-shan, Taoyuan 333, Taiwan, Republic of China. Latent membrane protein 1 (LMP1) is an Epstein–Barr virus (EBV)-encoded oncogene expressed in EBV-associated nasopharyngeal carcinoma (NPC). Previous studies indicate that a strategy combining LMP1-mediated NF-kB activation and the HSV thymidine kinase/Ganciclovir (HSVtk/GCV) prodrug system leads to regression of tumor growth in nude mice. To improve the efficacy of this strategy in immunocompetent hosts, we developed a therapeutic cassette, p6kB-EDL1E-tk, containing six copies of the NF-kB binding motif and an epithelial-specific EBV promoter, ED-L1E. The cassette was tested in a murine CT-26 carcinoma model in syngenic Balb/c mice. Coinjection of an LMP1-expressing vector and p6kB-EDL1E-tk by in vivo electroporation in mouse muscle revealed at least two-fold higher TK enzymatic activity than that of previously tested pLTR-tk. Furthermore, growth was attenuated in a group of mice containing LMP1-positive tumors that were intratumorally injected with the p6kB-EDL1E-tk cassette and GCV via in vivo electroporation, but not in mice treated with p6kB-EDL1E-tk or GCV alone.
    [Show full text]
  • Evaluation on Reprogramed Biological Processes in Transgenic Maize
    www.nature.com/scientificreports OPEN Evaluation on reprogramed biological processes in transgenic maize varieties using transcriptomics and metabolomics Wei Fu1, Pengyu Zhu1, Mingnan Qu2, Wang Zhi1, Yongjiang Zhang1, Feiwu Li3* & Shuifang Zhu1* Genetic engineering (GM) has great potential to improve maize productivity, but rises some concerns on unintended efects, and equivalent as their comparators. There are some limitations through targeted analysis to detect the UE in genetically modifed organisms in many previous studies. We here reported a case-study on the efects of introducing herbicides and insect resistance (HIR) gene cassette on molecular profling (transcripts and metabolites) in a popular maize variety Zhengdan958 (ZD958) in China. We found that introducing HIR gene cassette bring a limited numbers of diferential abundant genes (DAGs) or diferential abundant metabolites (DAMs) between transgenic events and non-transgenic control. In contrast, averaged 10 times more DAGs and DAMs were observed when performed comparison under diferent growing environments in three diferent ecological regions of China than the numbers induced by gene efects. Major biological pathways relating to stress response or signaling transduction could explain somehow the efects of growing environments. We further compared two transgenic events mediated ZD958 (GM-ZD958) with either transgenic parent GM-Z58, and other genetic background nonGM-Z58, nonGM-ZD958, and Chang7-2. We found that the numbers of DAGs and DAMs between GM-ZD958 and its one parent maize variety, Z58 or GM-Z58 is equivalent, but not Chang7-2. These fndings suggest that greater efects due to diferent genetic background on altered molecular profling than gene modifcation itself. This study provides a case evidence indicating marginal efects of gene pleiotropic efects, and environmental efects should be emphasized.
    [Show full text]
  • An Efficient Recombination System for Chromosome Engineering in Escherichia Coli
    An efficient recombination system for chromosome engineering in Escherichia coli Daiguan Yu*, Hilary M. Ellis*, E-Chiang Lee†, Nancy A. Jenkins†, Neal G. Copeland†, and Donald L. Court*‡ *Gene Regulation and Chromosome Biology Laboratory and †Mouse Cancer Genetics Program, National Cancer Institute, Division of Basic Science, National Cancer Institute͞Frederick Cancer Research and Development Center, Frederick, MD 21702 Communicated by Allan Campbell, Stanford University, Stanford, CA, March 22, 2000 (received for review January 19, 2000) A recombination system has been developed for efficient chromo- frequencies of recombination with linear DNA containing long some engineering in Escherichia coli by using electroporated linear homologies by transforming in the presence of the bacteriophage DNA. A defective ␭ prophage supplies functions that protect and ␭ recombination functions (Exo and Beta) in a bacterial recBCD recombine an electroporated linear DNA substrate in the bacterial mutant background. This ␭ recombination system, like yeast, cell. The use of recombination eliminates the requirement for uses double strand break repair (10, 11). In a recBC sbcA strain, standard cloning as all novel joints are engineered by chemical short regions of homology can function in recombination of synthesis in vitro and the linear DNA is efficiently recombined into linear DNA; however, recombination is inefficient (12). In recϩ place in vivo. The technology and manipulations required are backgrounds, gene replacement on the chromosome has been simple and straightforward. A temperature-dependent repressor accomplished by integration and excision of episomes carrying tightly controls prophage expression, and, thus, recombination bacterial homology (13, 14). functions can be transiently supplied by shifting cultures to 42°C To obtain efficient recombination of linear donor DNA in for 15 min.
    [Show full text]
  • Simplified Generation of Targeting Constructs Using ET Recombination Pierre-Olivier Angrand, Nathalie Daigle1,Frankvanderhoeven,Hansr.Schöler1 and A
    © 1999 Oxford University Press Nucleic Acids Research, 1999, Vol. 27, No. 17 e16 Simplified generation of targeting constructs using ET recombination Pierre-Olivier Angrand, Nathalie Daigle1,FrankvanderHoeven,HansR.Schöler1 and A. Francis Stewart* Gene Expression Program and 1Germ Cell Biology Laboratory, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69177 Heidelberg, Germany Received June 29, 1999; Revised and Accepted July 19, 1999 ABSTRACT large intact DNA molecules regardless of the disposition of ET recombination is a way to engineer DNA in convenient restriction sites, it presents new possibilities for complex engineering. We developed this potential to create, in Escherichia coli using homologous recombination. one step, a knock-out targeting construct for the Ssrp1 gene Here we develop the potential of ET recombination in (5,6) and to insert an IRES-lacZ-selectable gene cassette (7) two ways relevant to complex engineering exercises into a gene coding for a PHD fingers and SET domain-containing such as building gene targeting constructs. First, a protein, called Nsd2 (P.-O.Angrand et al., submitted). targeting construct was made in a single step. Second, ET recombination was used to place two unique restric- tion sites at precise positions in a large genomic MATERIALS AND METHODS clone. Subsequently a complex targeting construct DNA techniques was created by ligation with a multifunctional cassette. Large-scale plasmid DNA preparation was performed with the Qiagen Plasmid Kit (Qiagen). Restriction endonucleases and INTRODUCTION T4 DNA ligase were purchased from New England Biolabs. Assembling DNA constructs for amplification in Escherichia Plasmids were grown in E.coli strain XL1-blue [F'::Tn10 proA+B+ lacIq ∆(lacZ)M15/recA1 endA1 gyrA96 (Nalr)thi coli is the starting point for many experiments in molecular – + biology.
    [Show full text]
  • Staphylococcus Aureus Isolates from Milk of Lactating Dairy Cattle in Northwestern China Longping Li1,2,3 and Xin Zhao1,4*
    Li and Zhao BMC Veterinary Research (2018) 14:59 https://doi.org/10.1186/s12917-018-1376-5 RESEARCH ARTICLE Open Access Characterization of the resistance class 1 integrons in Staphylococcus aureus isolates from milk of lactating dairy cattle in Northwestern China Longping Li1,2,3 and Xin Zhao1,4* Abstract Background: Integrons are mobile DNA elements and they have an important role in acquisition and dissemination of antimicrobial resistance genes. However, there are limited data available on integrons of Staphylococcus aureus (S. aureus) from bovine mastitis, especially from Chinese dairy cows. To address this knowledge gap, bovine mastitis-inducing S. aureus isolates were investigated for the presence of integrons as well as characterization of gene cassettes. Integrons were detected using PCR reactions and then further characterized by a restriction fragment-length polymorphism analysis and amplicon sequencing. Results: All 121 S. aureus isolates carried the class 1 integrase gene intI1, with no intI2andintI3 genes detected. One hundred and three isolates were positive for the presence of 12 resistance genes, either alone or in combination with other gene cassettes. These resistance genes encoded resistance to trimethoprim (dhfrV, dfrA1, dfrA12), aminoglycosides (aadA1, aadA5, aadA4, aadA24, aacA4, aadA2, aadB), chloramphenicol (cmlA6) and quaternary ammonium compound (qacH) and were organized into 11 different gene cassettes arrangements (A-K). The gene cassette arrays dfrA1-aadA1 (D, 44.6%), aadA2 (K, 31.4%), dfrA12-orfX2-aadA2 (G, 27.3%) and aadA1 (A, 25.6%) were most prevalent. Furthermore, 74 isolates contained combinations of 2 to 4 gene cassette arrays. Finally, all of the integron/cassettes-positive isolates were resistant to aminoglycoside antibiotics.
    [Show full text]
  • Gene Cassettes Encoding Resistance to Quaternary Ammonium Compounds: a Role in the Origin of Clinical Class 1 Integrons?
    The ISME Journal (2009) 3, 209–215 & 2009 International Society for Microbial Ecology All rights reserved 1751-7362/09 $32.00 www.nature.com/ismej ORIGINAL ARTICLE Gene cassettes encoding resistance to quaternary ammonium compounds: a role in the origin of clinical class 1 integrons? Michael R Gillings1, Duan Xuejun1,2, Simon A Hardwick1, Marita P Holley1 and HW Stokes3 1Department of Biological Sciences, Macquarie University, Sydney, New South Wales, Australia; 2School of Energy and Environmental Engineering, Zhongyuan University of Technology, Zhengzhou, China and 3Department of Chemistry and Biomolecular Science, Macquarie University, Sydney, New South Wales, Australia DNA sequencing, phylogenetic and mapping studies suggest that the class 1 integron found in pathogens arose when one member of the diverse family of environmental class 1 integrons became embedded into a Tn402 transposon. However, the timing of this event and the selective forces that first fixed the newly formed element in a bacterial lineage are still unknown. Biocides have a longer use in clinical practice than antibiotics, and a qac (quaternary ammonium compound) resistance gene, or remnant thereof, is a normal feature of class 1 integrons recovered from clinical isolates. Consequently, it is possible that the initial selective advantage was conferred by resistance to biocides, mediated by qac. Here, we show that diverse qac gene cassettes are a dominant feature of cassette arrays from environmental class 1 integrons, and that they occur in the absence of any antibiotic resistance gene cassettes. They are present in arrays that are dynamic, acquiring and rearranging gene cassettes within the arrays. The abundance of qac gene cassettes makes them a likely candidate for participation in the original insertion into Tn402, and as a source of a readily selectable phenotype.
    [Show full text]
  • Minimal Piggybac Vectors for Chromatin Integration
    Gene Therapy (2014) 21,1–9 & 2014 Macmillan Publishers Limited All rights reserved 0969-7128/14 www.nature.com/gt ENABLING TECHNOLOGIES Minimal piggyBac vectors for chromatin integration V Solodushko1,2, V Bitko3 and B Fouty1,2,4 We describe novel transposon piggyBac vectors engineered to deliver transgenes as efficiently as currently available piggyBac systems, but with significantly less helper DNA co-delivered into the host genome. To generate these plasmids, we identified a previously unreported aspect of transposon biology, that the full-length terminal domains required for successful plasmid- to-chromatin transgene delivery can be removed from the transgene delivery cassette to other parts of the plasmid without significantly impairing transposition efficiency. This is achieved by including in the same plasmid, an additional helper piggyBac sequence that contains both long terminal domains, but is modified to prevent its transposition into the host genome. This design decreases the size of the required terminal domains within the delivered gene cassette of the piggyBac vector from about 1500 to just 98 base pairs. By removing these sequences from the delivered gene cassette, they are no longer incorporated into the host genome which may reduce the risk of target cell transformation. Gene Therapy (2014) 21, 1–9; doi:10.1038/gt.2013.52; published online 17 October 2013 Keywords: piggyBac; mutagenesis; stable gene delivery INTRODUCTION integrated into the host cell genome, they perform no useful Transposon vectors are a proven and viable alternative to viral function. They may potentially increase the risk of cell vectors for stable gene delivery.1–4 Like integrated viruses, transformation due to their retained promoter and enhancer transposons deliver transgenes to target cells in vitro and in vivo activity, however.
    [Show full text]