DOCSLIB.ORG

BACTERIAL CONJUGATION 28 253 Bacterial 28 Conjugation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
BACTERIAL CONJUGATION 28 253 Bacterial 28 Conjugation 43038_CH28_0253.qxd 1/3/07 3:56 PM Page 253 BACTERIAL CONJUGATION 28 253 Bacterial 28 Conjugation enetic variability in eukaryotes involves processes occurring during sexual reproduction. Meiosis itself helps “shuffle” the G genes donated to an egg or female gamete, while the process of crossing over (exchange of genetic material between homologous chro- mosomes) during meiosis I further mixes up the genes. However, in prokaryotic organisms, there is no sexual reproduction. Yet genetic vari- ability occurs to maintain the evolutionary success within and between species. In prokaryotes, horizontal (lateral) gene transfer can occur between cells within the same generation. One mechanism driving genetic variability in bacteria is conjuga- tion. This form of horizontal gene transfer involves the one-way transfer of genetic material from a donor cell to a recipient cell. Transfer requires a physical contact between the two cells through the formation of a con- jugation bridge. Cells having an F factor represent donor cells because they have the ability to produce the conjugation bridge. If the F factor is extrachromosomal (a plasmid), the donor cells are designated as F؉. If the F factor becomes incorporated into the bacterial chromosome of the donor, the resulting cells are designated Hfr, for high-frequency recombi- nants. Cells missing the F factor are the recipients of the conjugation process and are designated as F4. In F+ cells, a copy of the plasmid usually is completely transferred to an F4 recipient. The result is that the F4 cell now has an F plasmid, so it has become an F+ cell. In Hfr cells, there is a transfer of chromosomal genes, but the conjugation bridge usually breaks before a complete copy of the donor chromosome can be transferred to an F4 cell. A few genes may be transferred, so the recipient is referred to as an F4 recombinant. In this experiment, you will prepare three cultures of Escherichia coli: one containing an Hfr strain sensitive to the antibiotic streptomycin (Str-s); another containing an F4 strain that is resistant to streptomycin (Str-r) but requires the amino acids threonine (thr) and leucine (leu) to build pro- teins, and the vitamin thiamine (thi); and a third containing a mixture of the Hfr and F− strains. Following a short incubation period, isolation of only the F− recombinants will be performed by plating the mixed culture on a min- imal medium containing streptomycin and thiamine. A genetic map indi- cating the origin and direction of transfer and the sites of the relevant marker genes is provided in Figure 28.1. 43038_CH28_0253.qxd 1/3/07 3:56 PM Page 254 254 28 BACTERIAL CONJUGATION Origin and order of gene transfer THR LEU THI 0 80 HFR 10 STR-R 70 20 60 30 50 40 Time FIGURE 28.1 (minutes) The genetic map for the E. coli HFR for transfer strain S pecial Materials • 12-hour nutrient broth cultures of F− E. coli strain [thr−, leu−, thi−, and Str-r] (ATCC e23724) • 12-hour nutrient broth cultures of Hfr E. coli strain [thr+, leu+, thi+, and Str-s] (ATCC e 23740) • Minimal medium agar plates with streptomycin and thiamine • Beakers with 95% ethyl alcohol • Bent (L-shaped) glass rods • 1-ml sterile pipettes and mechanical pipetting devices • Sterile 13- × 100-mm test tubes • Waterbath at 37°C P rocedure PURPOSE: to demonstrate 1. Obtain two 1-ml sterile pipettes. Use one pipette to aseptically transfer bacterial genetic recombina- 1 ml of the F− E. coli broth culture and the other pipette to aseptically trans- tion through conjugation and the formation of recombi- fer 0.3 ml of the Hfr E. coli broth culture into a sterile 13- × 100-mm test nant cells. tube. 2. Mix by gently rotating the culture between the palms of your hands. 3. Incubate the culture for 30 minutes at 37°C waterbath. 4. Obtain two minimal medium plus streptomycin and thiamine agar plates. Label the bottom of both plates with your name and date. Label one plate “Hfr” and the other “F−.” 5. Using the spread-plate technique illustrated in Figure 28.2, aseptically add 0.1 ml of each E. coli strain to its appropriately labeled agar plate. With an alcohol-dipped and flamed glass rod, spread the inoculum over the entire surface of the agar plate. Only place the glass rod in 6. Following the 30-minute incubation, vigorously agitate the mixed culture the Bunsen burner flame to terminate the genetic transfer by breaking the conjugation bridge. long enough to catch the alcohol on fire. Longer 7. Obtain another minimal medium plus streptomycin and thiamine agar exposure to the flame will melt the glass or cause the plate. Label the bottom of the plate with your name and date. Label this plate rod to break. ! “Hfr × F−.” 43038_CH28_0253.qxd 1/3/07 3:56 PM Page 255 BACTERIAL CONJUGATION 28 255 (a) Dip the bent glass rod into the (b) Sterilize the glass rod by flaming beaker of 95% ethyl alcohol. with a Bunsen burner. (c) Remove from Bunsen burner, allow (d) Spread the inoculum over the agar flame to extinguish, and cool the surface by rotating the plate. glass rod. FIGURE 28.2 The Spread-Plate Technique. 8. Aseptically add 0.1 ml of the mixed culture. Spread the inoculum over the entire surface with a sterile glass rod as described in step 5. 9. Incubate all three plates in an inverted position for 48 hours at 37°C; then refrigerate. 10. Observe the three plates for the presence of (+) or absence of (−) bacte- rial colonies. Enter your data in the Results section (Table 28.1). In your conclusions, write why you would expect to see colonies or see no colonies on the appropriate minimal medium plus streptomycin and thiamine agar plates. 43038_CH28_0253.qxd 1/3/07 3:56 PM Page 256 256 28 BACTERIAL CONJUGATION Q uestions 1. Explain why you would not expect any growth on the agar plates con- taining only donor or recipient cells. 2. What specific genes were transferred from donor to recipient cells? 3. Assess the importance of streptomycin as a marker in the donor and recip- ient cells. 4. Which plate or plates represented the controls in the conjugation experi- ment? 5. Describe how conjugation can be a mechanism for genetic variability in bacteria. 43038_CH28_0253.qxd 1/3/07 3:56 PM Page 257 BACTERIAL CONJUGATION 28 257 Name Date Section Exercise28 Results Bacterial Conjugation Table 28.1. Conjugation in Bacteria Minimal Medium Plus Streptomycin and Thiamine Agar Plate _ _ ” Hfr” “F ” “Hfr ؋ F“ Colonies present (+) or absent (-) Observations and Conclusions: 43038_CH28_0253.qxd 1/3/07 3:56 PM Page 258.
Load more

Recommended publications
  • Comprehensive Analysis of Mobile Genetic Elements in the Gut Microbiome Reveals Phylum-Level Niche-Adaptive Gene Pools
    bioRxiv preprint doi: https://doi.org/10.1101/214213; this version posted December 22, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Comprehensive analysis of mobile genetic elements in the gut microbiome 2 reveals phylum-level niche-adaptive gene pools 3 Xiaofang Jiang1,2,†, Andrew Brantley Hall2,3,†, Ramnik J. Xavier1,2,3,4, and Eric Alm1,2,5,* 4 1 Center for Microbiome Informatics and Therapeutics, Massachusetts Institute of Technology, 5 Cambridge, MA 02139, USA 6 2 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA 7 3 Center for Computational and Integrative Biology, Massachusetts General Hospital and Harvard 8 Medical School, Boston, MA 02114, USA 9 4 Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General 10 Hospital and Harvard Medical School, Boston, MA 02114, USA 11 5 MIT Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 12 02142, USA 13 † Co-first Authors 14 * Corresponding Author bioRxiv preprint doi: https://doi.org/10.1101/214213; this version posted December 22, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 15 Abstract 16 Mobile genetic elements (MGEs) drive extensive horizontal transfer in the gut microbiome. This transfer 17 could benefit human health by conferring new metabolic capabilities to commensal microbes, or it could 18 threaten human health by spreading antibiotic resistance genes to pathogens. Despite their biological 19 importance and medical relevance, MGEs from the gut microbiome have not been systematically 20 characterized.
    [Show full text]
  • Section 4. Guidance Document on Horizontal Gene Transfer Between Bacteria
    306 - PART 2. DOCUMENTS ON MICRO-ORGANISMS Section 4. Guidance document on horizontal gene transfer between bacteria 1. Introduction Horizontal gene transfer (HGT) 1 refers to the stable transfer of genetic material from one organism to another without reproduction. The significance of horizontal gene transfer was first recognised when evidence was found for ‘infectious heredity’ of multiple antibiotic resistance to pathogens (Watanabe, 1963). The assumed importance of HGT has changed several times (Doolittle et al., 2003) but there is general agreement now that HGT is a major, if not the dominant, force in bacterial evolution. Massive gene exchanges in completely sequenced genomes were discovered by deviant composition, anomalous phylogenetic distribution, great similarity of genes from distantly related species, and incongruent phylogenetic trees (Ochman et al., 2000; Koonin et al., 2001; Jain et al., 2002; Doolittle et al., 2003; Kurland et al., 2003; Philippe and Douady, 2003). There is also much evidence now for HGT by mobile genetic elements (MGEs) being an ongoing process that plays a primary role in the ecological adaptation of prokaryotes. Well documented is the example of the dissemination of antibiotic resistance genes by HGT that allowed bacterial populations to rapidly adapt to a strong selective pressure by agronomically and medically used antibiotics (Tschäpe, 1994; Witte, 1998; Mazel and Davies, 1999). MGEs shape bacterial genomes, promote intra-species variability and distribute genes between distantly related bacterial genera. Horizontal gene transfer (HGT) between bacteria is driven by three major processes: transformation (the uptake of free DNA), transduction (gene transfer mediated by bacteriophages) and conjugation (gene transfer by means of plasmids or conjugative and integrated elements).
    [Show full text]
  • The Obscure World of Integrative and Mobilizable Elements Gérard Guédon, Virginie Libante, Charles Coluzzi, Sophie Payot-Lacroix, Nathalie Leblond-Bourget
    The obscure world of integrative and mobilizable elements Gérard Guédon, Virginie Libante, Charles Coluzzi, Sophie Payot-Lacroix, Nathalie Leblond-Bourget To cite this version: Gérard Guédon, Virginie Libante, Charles Coluzzi, Sophie Payot-Lacroix, Nathalie Leblond-Bourget. The obscure world of integrative and mobilizable elements: Highly widespread elements that pirate bacterial conjugative systems. Genes, MDPI, 2017, 8 (11), pp.337. 10.3390/genes8110337. hal- 01686871 HAL Id: hal-01686871 https://hal.archives-ouvertes.fr/hal-01686871 Submitted on 26 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License G C A T T A C G G C A T genes Review The Obscure World of Integrative and Mobilizable Elements, Highly Widespread Elements that Pirate Bacterial Conjugative Systems Gérard Guédon *, Virginie Libante, Charles Coluzzi, Sophie Payot and Nathalie Leblond-Bourget * ID DynAMic, Université de Lorraine, INRA, 54506 Vandœuvre-lès-Nancy, France; [email protected] (V.L.); [email protected] (C.C.); [email protected] (S.P.) * Correspondence: [email protected] (G.G.); [email protected] (N.L.-B.); Tel.: +33-037-274-5142 (G.G.); +33-037-274-5146 (N.L.-B.) Received: 12 October 2017; Accepted: 15 November 2017; Published: 22 November 2017 Abstract: Conjugation is a key mechanism of bacterial evolution that involves mobile genetic elements.
    [Show full text]
  • Genetic Exchange in Bacteria
    Systems Microbiology Monday Oct 16 - Ch 10 -Brock Genetic Exchange in Bacteria •• HomologousHomologous recombinationrecombination •• TransformationTransformation •• PlasmidsPlasmids andand conjugationconjugation •• TransposableTransposable elementselements •• TransductionTransduction (virus(virus mediatedmediated xchangexchange)) Gene exchange in bacteria • Transfer of DNA from one bacterium to another is a common means of gene dispersal. It has a big effect on bacterial evolution, and tremendous practical implications. For example, lateral transfer is responsible for the spread drug resistance determinants between bacterial species. • Three common mechanisms of lateral gene exchange : – Transformation (extracellular DNA uptake) – Conjugation (bacterial mating systems) – Transduction (viral mediated gene exchange) RecA mediated Homologous recombination Images removed due to copyright restrictions. See Figures 10-9 and 10-10 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291. Gene exchange in bacteriaTransformation The Griffith Experiment S Injection Dead mouse; • Discovered by Griffith in yields S1 cells 1928 during the course of his Live "smooth" (encapsulated) studies of virulence in type 1 pneumococci (S1) Streptococcus pneumoniae. Dead S Live mouse Heat-killed S1 • S=smooth colony Live mouse morphotype Live "rough" (unencapsulated) pneumococci (R1 or R2) derived by subculture from S1 or S2, respectively • R=rough colony Dead mouse; yields S cells morphotype R1 + Dead S + 1 Live R1 Killed S1 R2 + Dead S + Dead mouse; yields S1 cells Live R2 Killed S2 Figure by MIT OCW. Gene exchange mechanisms in bacteria Transformation The Griffith Experiment Injection Dead mouse; S yields S cells Avery, MacLeod, and McCarthy (1944) 1 fractionation studies led to conclusion that Live "smooth" (encapsulated) type 1 pneumococci (S ) transformation principle is DNA.
    [Show full text]
  • Lab Exercise 15: Bacterial Conjugation
    Microbiology BIOL 275 BACTERIAL CONJUGATION I. OBJECTIVES • To demonstrate the technical procedure to monitor the conjugational transfer of genetic material from one cell to another. • To learn about the various genetic elements involved in bacterial sex and recombination events. • To recognize and discuss the advantages and limitations of the conjugational procedure in genetic recombination experiments. II. INTRODUCTION There are three different mechanisms for genetic exchange between bacteria: conjugation, transformation and transduction. Conjugation has been shown to occur in many bacterial species especially the Gram negative organisms. In Enterobacteriaceae (Escherichia, Salmonella, Shigella, Vibrio, etc.), the transfer has been shown to cross the taxonomic lines with varying rates of efficiency. Thus, conjugation has become a tool to study the location and function of many genes, including genes responsible for virulence, antibiotic resistance, metabolic functions, regulatory proteins, phage attachment sites, etc. E. coli has been the model organism in conjugation studies. It may become infected with an extrachromosomal genetic element (a plasmid called the F factor) which mediates certain events such as the synthesis of a pilus (the F or sex pilus), and the rapid infection of all susceptible bacteria with this F factor. The pilus has been shown to facilitate attachment of the male (or F+ cells) to the female (or F- cells) which do not contain the F factor. It was previously thought that the hollow center of the pilus constituted a passageway for the F factor to be transferred to the female. However, recent evidence suggests that the pilus may be used as a "hook" to bring the two cells close together and that the DNA transfer actually occurs outside the pilus.
    [Show full text]
  • Bacterial Conjugation
    16 Mechanisms of Genetic Variation 1 16.1 Mutations 1. Distinguish spontaneous from induced mutations, and list the most common ways each arises 2. Construct a table, concept map, or picture to summarize how base analogues, DNA-modifying agents, and intercalating agents cause mutations 3. Discuss the possible effects of mutations 2 Mutations: Their Chemical Basis and Effects • Stable, heritable changes in sequence of bases in DNA – point mutations most common • from alteration of single pairs of nucleotide • from the addition or deletion of nucleotide pairs – larger mutations are less common • insertions, deletions, inversions, duplication, and translocations of nucleotide sequences • Mutations can be spontaneous or induced 3 16.4 Creating Additional Genetic Variability 1. Describe in general terms how recombinant eukaryotic organisms arise 2. Distinguish vertical gene transfer from horizontal gene transfer 3. Summarize the four possible outcomes of horizontal gene transfer 4. Compare and contrast homologous recombination and site-specific recombination 4 Creating Additional Genetic Variability • Mutations are subject to selective pressure – each mutant form that survives becomes an allele, an alternate form of a gene • Recombination is the process in which one or more nucleic acids are rearranged or combined to produce a new nucleotide sequence (recombinants) 5 Sexual Reproduction and Genetic Variability • Vertical gene transfer = transfer of genes from parents to progeny • In eukaryotes – sexual reproduction is accompanied by genetic
    [Show full text]
  • Mechanism of Genetic Recombination During Bacterial Conjugation of Escherzchza Coli K-12
    MECHANISM OF GENETIC RECOMBINATION DURING BACTERIAL CONJUGATION OF ESCHERZCHZA COLI K-12. 11. INCORPORATION OF THE DONOR DNA FRAGMENT INTO THE RECOMBINANT CHROMOSOME S. E. BRESLER AND V. A. LANZOV Institute of High Molecular Weight Compounds, Academy of Sciences of USSR, Leningrad Received November 30, 1966 INVESTIGATION of genetic recombination in bacteriophages and bacteria (KELLENBERGER.ZICHICHI and WEIGLE1961 ; MESELSONand WEIGLE1961 ; BODMERand GANESAN1964; FOXand ALLEN1965; OPPENHEIMand RILEY1966) has already shown that of the two recombination mechanisms, ILcopychoice” and “breakage-reunion”, the latter is the more plausible. Nevertheless, the model of “copy choice” acquired wide popularity. Therefore it is important to perform new experiments giving direct proofs of physical integration of parental material into recombinants. One experiment of this type fulfilled with a conjugation system is that of SIDDIQI( 1963). Using radioactively labeled F- cells and selecting true recombi- nants (according to a recessive marker T6‘) SIDDIQIsucceeded in demonstrating that the main part of the recipient cell genome (+ 80%) is inherited by the recombinant. Our aim was to design a complementary experiment to show that a part of the labeled donor genome can be detected in the cells which are true recombinants after conjugation. Since it was expected that the recombinant inherits only a small fragment of the donor DNA, the method has to be sufficiently sensitive. The experimental system satisfying these conditions is as follows: 1. The experiment is performed using a population of mainly mononuclear E. coti cells. The possibility of obtaining such a population was shown in the papers of WITKIN( 1951) and MCFALL,PARDEE and STENT(1958). 2.
    [Show full text]
  • Bacteria Evolving: Tracing the Origins of a MRSA Epidemic
    STUDENT VERSION Bacteria Evolving: Tracing the Origins of a MRSA Epidemic PASSAGE TWO How Did MRSA Evolve? def initions DNA: Deoxyribonucleic What allowed USA300 to thrive outside a hospital set- acid is the organic ting, and with such virulence? Scientists knew the answer molecule that forms the genetic material of an lay in the bacteria’s genome, the sum of all its genetic organism. Chromosomes material. Within the bacteria’s genome were the clues are made of DNA. that could explain how the USA300 strain of MRSA had evolved. gene: a section of DNA on a chromosome that Different Types of Mutations encodes, individually Like all organisms, bacteria can acquire new traits through or as part of a group mutations. Mutations are any change in the sequence of of genes, for a specific DNA nucleotides within an organism’s genome. The main hereditary trait. cause of mutations are exposure to foreign chemicals or genome: the complete radiation, errors during DNA replication, and from inser- genetic material or base tion or deletion of DNA segments. If a mutation is benefi- sequence of an organism cial, it gives the organism an evolutionary advantage by or species. helping it and its descendants to survive in a new envi- genotype: the genetic ronment. This is the process of natural selection. makeup of an individual These types of mutations can happen in any organism. In or species. bacteria, DNA can also be acquired through the process phenotype: the of DNA transfer. That means that bacteria can get whole observable characteristics new sets of DNA from other bacteria, creating sudden of an organism or species, and dramatic changes in their genome.
    [Show full text]
  • A First Approach to Individual-Based Modeling of the Bacterial Conjugation Dynamics
    Facultad Informatica´ Universidad Politecnica´ de Madrid TESIS DE MASTER´ Master´ de Investigacion´ en Tecnolog´ıas para el Desarrollo de Sistemas Software Complejos A first approach to individual-based modeling of the bacterial conjugation dynamics Autor: Antonio P. Garc´ıa Tutor: Alfonso Rodr´ıguez-Paton´ Aradas Septiembre, 2011 FACULTAD DE INFORMÁTICA PRESENTACIÓN DE TESIS DE MÁSTER Alumno: Máster: Título de la Tesis: Tutor: VºBº del Tutor, El alumno, Fecha: A` minha av´o,Dora que se foi este ano. Resumen El objetivo fundamental de esta tesis es emplear el modelado basado en individuos pa- ra el estudio de la din´amica de invasi´onde los pl´asmidosconjugativos en poblaciones bacterianas sobre superficies s´olidas.Los pl´asmidosson peque~nossegmentos de ADN, normalmente circulares que poseen su propio mecanismo de replicaci´on,independiente del ADN cromos´omicoprincipal de las c´elulasbacterianas y que sobreviven como par´asi- tos utilizando la maquinaria celular de su hospedero en su propio beneficio. Adem´asde la capacidad de replicaci´onlos pl´asmidosson capaces de auto-transferirse a otras c´elulas bacterianas en un proceso conocido como conjugaci´onbacteriana. La persistencia como elemento gen´eticom´ovilde los pl´asmidosen escala evolutiva no est´acompletamente ca- racterizada, puesto que en muchas situaciones donde no exista una presi´onselectiva que favorezca las c´elulasportadoras del pl´asmido,el coste metab´olicoque representa deber´ıa paulatinamente eliminarlos de la poblaci´on.Empleando un modelo basado en la ley de acci´onde masas[SL77] se postula que las condiciones requeridas para que un pl´asmido conjugativo se mantenga de forma estable en una poblaci´onson bastante amplias, siendo suficiente que la cantidad de c´elulasinfectadas inicialmente, sea elevada para compensar la p´erdidasegregativa y el coste metab´olicoque acarrea el hecho de portar el pl´asmi- do.
    [Show full text]
  • Genetic Cargo and Bacterial Species Set the Rate of Vesicle-Mediated Horizontal Gene Transfer Received: 31 March 2017 Frances Tran1 & James Q
    www.nature.com/scientificreports OPEN Genetic cargo and bacterial species set the rate of vesicle-mediated horizontal gene transfer Received: 31 March 2017 Frances Tran1 & James Q. Boedicker1,2 Accepted: 27 June 2017 Most bacteria release extracellular vesicles (EVs). Recent studies have found these vesicles are Published: xx xx xxxx capable of gene delivery, however the consequences of vesicle-mediated transfer on the patterns and rates of gene fow within microbial communities remains unclear. Previous studies have not determined the impact of both the genetic cargo and the donor and recipient species on the rate of vesicle-mediated gene exchange. This report examines the potential for EVs as a mechanism of gene transfer within heterogeneous microbial populations. EVs were harvested from three species of Gram- negative microbes carrying diferent plasmids. The dynamics of gene transfer into recipient species was measured. This study demonstrates that vesicles enable gene exchange between fve species of Gram-negative bacteria, and that the identity of the genetic cargo, donor strain, and recipient strain all infuence gene transfer rates. Each species released and acquired vesicles containing genetic material to a variable degree, and the transfer rate did not correlate with the relatedness of the donor and recipient species. The results suggest that EVs may be a general mechanism to exchange non-specialized genetic cargo between bacterial species. Microorganisms possess complex abilities to transfer genetic material through horizontal gene transfer (HGT), fundamentally shaping genetic landscapes and afecting biological functions1–4. Te capacity for DNA exchange in bacteria and the plasticity of their genetic material has amplifed the rate of adaptation and evolu- tion across species and has allowed for stability and growth of complex microbial ecosystems in a multitude of environments5–8.
    [Show full text]
  • Bacterial Conjugation and Its Inhibition
    Bacterial Conjugation and its Inhibition: The Hows and Whys of Conjugation and What Can be Done to Control It Scott A. Lujan A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the department of Biochemistry and Biophysics. Chapel Hill, NC 2008 Approved by Professor Matthew Redinbo, Ph.D. Professor Richard Wolfenden, Ph.D. Professor Steve Matson, Ph.D. Professor Gary Pielak, Ph.D. Professor Brenda Temple, Ph.D. © 2008 Scott A. Lujan ALL RIGHTS RESERVED ii Abstract SCOTT A. LUJAN: Bacterial Conjugation and its Inhibition: The Hows and Whys of Conjugation and What Can be Done to Control It (Under the direction of Matthew R. Redinbo, Ph.D.) Conjugation is the primary vehicle for the horizontal transfer virulence factor genes, such as antibiotic resistance, within and between bacterial strains. In certain epicenters, such as hospitals in less developed parts of the world, immune-compromised patients and misuse of antibiotics combine to select for the development and dissemination of these pathogenicity factors via conjugation. Inhibition of conjugation would prove a boon for curbing the creation and spread of new virulent or multi-drug resistant strains. DNA relaxases are the keystone proteins of each conjugative system. TraI is the relaxase of the F plasmid, the archetypal model system for conjugation. Toward revelation and inhibition of relaxase mechanisms, I used bioinformatics and limited proteolysis to find and identify new domains on the F TraI enzyme. I then solved TraI/DNA co-crystal structures that showed a novel DNA binding mode.
    [Show full text]
  • Integrative and Conjugative Elements (ICE) and Associated Cargo Genes Within and Across Hundreds of Bacterial Genera
    bioRxiv preprint doi: https://doi.org/10.1101/2020.04.07.030320; this version posted April 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Journal of XYZ, 20xx, 1–21 doi: xx.xxxx/xxxx Manuscript in Preparation Paper P APER Integrative and Conjugative Elements (ICE) and Associated Cargo Genes within and across Hundreds of Bacterial Genera J. H. Kaufman1,*, I. Terrizzano1,*, G. Nayar1,*, E. Seabolt1,*, A. Agarwal1,*, I. B. Slizovskiy2,† and N. R. Noyes2,† 1IBM Research - Almaden, 650 Harry Rd., San Jose, CA 95120-6099 and 2Department of Veterinary Population Medicine, University of Minnesota, 1988 Fitch Ave, St. Paul, MN 55108 *[email protected], [email protected], [email protected], [email protected], [email protected] †[email protected] Abstract Horizontal gene transfer mediated by integrative and conjugative elements (ICE) is considered an important evolutionary mechanism of bacteria. It allows organisms to quickly evolve new phenotypic properties including antimicrobial resistance (AMR) and virulence. The rate of ICE-mediated cargo gene exchange has not yet been comprehensively studied within and between bacterial taxa. In this paper we report a big data analysis of ICE and associated cargo genes across over 200,000 bacterial genomes representing 1,345 genera. Our results reveal that half of bacterial genomes contain one or more known ICE features ("ICE genomes"), and that the associated genetic cargo may play an important role in the spread of AMR genes within and between bacterial genera.
    [Show full text]