Mechanisms of Genetic Variation PDF

Mechanisms of Genetic Variation Chapter 16 Notice: This material is subject to the U.S. Copyright Law; further RM Image © McGraw Hill, LLC reproduction in violation...

Mechanisms of Genetic Variation Chapter 16 Notice: This material is subject to the U.S. Copyright Law; further RM Image © McGraw Hill, LLC reproduction in violation of the law is prohibited. Genetic Change in Bacteria Organisms adapt to changing environments Natural selection favors genotypes with greater fitness in a specific environment Figure obtained from OpenStax Microbiology CC BY 4.0 Antibiotic Resistance Staphylococcus aureus Since 1940s, treated with penicillin-like antibiotics In 2004, over 60% of S. aureus strains from hospitalized patients were resistant to penicillin derivatives Millions of healthy people in U.S. harbor MRSA Healthcare-associated MRSA resistant to other antibiotics, including vancomycin Vancomycin considered drug of last resort Antimicrobial Resistance in Staphylococci at the Human– Animal Interface. Antimicrobial Resistance - An Open Challenge. doi: 10.5772/61785 So how do multi-drug resistant microbes arise. This requires an understanding of bacterial genetic change. How genes… change and are shared Figures from NIAID (CC BY 2.0) Genetic Change in Bacteria Bacteria adjust to new circumstances by two means I. Regulation of gene expression – Ex: operon, sigma factor II. Genetic change – Mutations – Horizontal gene transfer Genetic change in bacteria alters a cell’s genotype Any genomic change can have a significant impact - Bacteria are haploid - Change in genotype may result in an altered phenotype Genetic Change in Bacteria Genetic change occurs by two mechanisms: 1. Mutation - Changes in existing nucleotide sequence of cell’s DNA - Passed onto progeny – vertical gene transfer 2. Horizontal gene transfer - Movement of DNA from one organism to another (usually via a plasmid) - Can be passed onto progeny (need origin of replication) Figures from NIAID (CC BY 2.0)  Genetic Change in Bacteria Mutations can change an organism’s phenotype Ex: Mutation of gene for tryptophan biosynthesis yields mutant that only grows if tryptophan supplied Biosynthesis mutations -> Mutant termed auxotroph -> growth factor required vs. Prototroph -> does NOT require growth factors Geneticists compare mutants to wild type Ex: Wild-type E. coli strain is prototroph Mutant strains designated by three-letter abbreviations – For example, Trp– cannot make tryptophan -> E. coli Trp- Akardoust Wikimedia Commons CC BY-SA 4.0 – Streptomycin resistance tations can be spontaneous or induced Spontaneous mutations are random and due to normal cell processes - Occur very infrequently Mutation rate: probability of mutation each cell division Typically, between 10–4 and 10–12 (1/10,000 and 1/1 trillion) for a given gene per cell division *units can vary Mutations are passed on to progeny - Occasionally change back to original state: reversion ViralZone, SIB Swiss Institute of Bioinformatics CC BY Spontaneous mutations Although mutations occur infrequently, large populations always contain mutants (remember, one colony = at least ~1 million cells) - All cells in a colony never have identical genotypes - This gives the population a chance to adapt to environmental changes The environment selects cells that grow best under its current conditions (natural selection) For example, antibiotics select for resistant bacteria if present - Antibiotics kill sensitive cells leaving only mutants that are resistant Figure from NIAID (CC BY 2.0)  Spontaneous mutations Single mutation that changes phenotype is rare; 2 even rarer Physicians may prescribe ≥ two antibiotics to reduce possible resistant cells developing Mutations rarely cause a “beneficial” phenotype, and if they do, they often eliminate or reduce pre-existing cellular functions “beneficial” is all about context Mcstrother - Own work Wikimedia Commons CC BY 3.0 Mutation Outcomes Wild type Most prevalent form of gene and its associated phenotype. Forward mutation Wild type mutant form Reversion mutation Mutant phenotype wild type phenotype Suppressor mutation Wild-type phenotype is restored by a second mutation at a different site than the original mutation. © McGraw Hill, LLC Spontaneous Mutations Base substitution most common Incorrect nucleotide incorporated during DNA replication Point mutation is change of a single base pair AT-to-GC transition mutation © McGraw Hill, LLC Spontaneous Mutations Transition mutation—lead to stable alteration of the nucleotide sequence. (ex: purine for another purine) Transversion mutation—when a purine is substituted for a pyrimidine, causing steric problems. AT-to-GC transition mutation © McGraw Hill, LLC Point Mutation Outcomes 1. Silent mutation: wild-type amino acid Degenerate code – many codons code for the same amino acid (mutation in wobble position) 2. Missense mutation: different amino acid The resulting protein may only partially function – leaky mutation – Cells may grow slower 3. Nonsense mutation: Stop-gain: codes for stop codon -> Any mutation that inactivates yields shorter & typically non- gene is termed a knockout Figure obtained from OpenStax Microbiology CC BY 4.0  Spontaneous Mutations Base substitutions More common in aerobic environments Reactive oxygen species produced during ETC ROS can oxidize nucleotides Oxidized guanine – DNA polymerase often mispairs oxidized guanine with adenine GC -> GA -> TA By Chaya5260 - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=41807093  Spontaneous Mutations Deletion or addition of nucleotides Occurs during DNA replication at short stretches of repeated nucleotides. Impact depends on number of nucleotides - Three base pairs, adds/deletes one codon ‣ One amino acid more or less ‣ Usually minimal Impact (depends on location) ‣ Could be leaky mutation - One or two bp, yields insertion or deletion frameshift mutation ‣ After mutation, different set of codons translated ‣ Often results in premature stop codon ‣ Shortened, nonfunctional protein -> knockout mutation Figure obtained from OpenStax Microbiology CC BY 4.0  Spontaneous Mutations Transposons (jumping genes) Pieces of DNA that can move within genome - Process called transposition Gene that transposon inserts into is most often inactivated = Insertional inactivation (knockout) Most transposons code for transcriptional terminators (ex: hairpin loop) Blocks transcription of downstream genes in operon Arvid Ågren and Andrew G. Clark Wikimedia Commons CC BY Induced Mutations Induced mutations result from outside influence Agent that induces mutation is called a mutagen Two general types: chemical, radiation  Induced Mutations Chemical mutagens may cause base substitutions or frameshift mutations Some chemicals change base-pairing properties by modify nucleobases Increases chance of base substitution Added alkyl group Nitrosoguanidine (alkylating agent) Guanine Methylguanine (pairs with Cytosine) (pairs with Thymine) By Guanin.svg: NEUROtikerderivative work: Xvazquez (talk)derivative work: Kes47 (?) - Guanin.svg, Public Domain, By NEUROtiker - Own work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=2212115 https://commons.wikimedia.org/w/index.php?curid=8271496  Induced Mutations Alkylating agents add alkyl groups onto nucleobases Nitrosoguanidine adds akyl group to guanine – Base-pairs with thymine instead of cytosine – GC-TA transversion Added alkyl group Nitrosoguanidine (alkylating agent) Guanine Methylguanine (pairs with Cytosine) (pairs with Thymine) By Guanin.svg: NEUROtikerderivative work: Xvazquez (talk)derivative work: Kes47 (?) - Guanin.svg, Public Domain, By NEUROtiker - Own work, Public Domain, https://commons.wikimedia.org/w/index.php?curid=2212115 https://commons.wikimedia.org/w/index.php?curid=8271496  Induced Mutations Base analogs resemble normal nucleobase But have different hydrogen-bonding properties Can be mistakenly incorporated by DNA polymerase 2-amino purine resembles adenine, often pairs with cytosine 5-bromouracil resembles thymine, often base-pairs Figure obtained from OpenStax Microbiology CC BY 4.0 Induced Mutations Intercalating agents cause frameshift mutations Flat molecules that intercalate (insert) between adjacent base pairs in DNA strand Pushes nucleotides apart Causes errors during replication Commonly causes insertions and deletions: DNA polymerase is "fooled" Results in frame shift and likely knockout gene Ex: Ethidium bromide (used to stain DNA), Chloroquine (used to treat malaria) Figure obtained from OpenStax Microbiology CC BY 4.0 Induced Mutations Transposition Transposons can be used intentionally as a mutagen to generate mutations and inactivate genes Used in knockout experiments This work by Olivia Foster Rhoades SITNBoston is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License  Induced Mutations Radiation: two types 1. Non-ionizing: ultraviolet radiation forms thymine dimers Covalent bonds between adjacent thymines – Distorts backbone of DNA – Replication and transcription stall at distortion – Cell will die if damage not repaired – Mutations directly result from cell’s SOS repair mechanism Figure obtained from OpenStax Microbiology CC BY 4.0  Induced Mutations Radiation: two types 2. Ionizing: X rays cause single- and double-strand breaks in DNA – Double-strand breaks often produce lethal deletions X rays can also alter nucleobases by increasing Dsup is a DNA-associating ROSfound only in protein tardigrades that covers DNA & suppresses mutations from radiation Tardigrades can withstand 1,000 times more radiation than other organisms By Schokraie E, Warnken U, Hotz-Wagenblatt A, Grohme MA, Hengherr S, et al. (2012) - Schokraie E, Warnken U, Hotz-Wagenblatt A, Grohme MA, Hengherr S, et al. (2012) Comparative proteome analysis of Milnesium Hashimoto T, Kunieda T. DNA Protection Protein, a Novel Mechanism of Radiation Tolerance: Lessons from Tardigrades. Life (Basel). 2017 Jun 15;7(2):26. doi: 10.3390/life7020026 tardigradum in early embryonic state versus adults in active and anhydrobiotic state. PLoS ONE 7(9): e45682. doi:10.1371/journal.pone.0045682, CC BY 2.5, https://commons.wikimedia.org/w/index.php?curid=130857154 PMID: 28617314; PMCID: PMC5492148. Repair of Damaged DNA An enormous amount of spontaneous and mutagen- induced damage to DNA If not repaired, can lead to cell death; cancer (in animals) Ex: Mutations in tumor suppression genes (limit cell growth) Mutations are rare because they are repaired before being passed to progeny Several different DNA repair mechanisms Eukaryotes and prokaryotes share many repair mechanisms  Repair of Damaged DNA  DNA repair is divided into two types: 1. Error-proof repair pathways, prevent mutations – Methyl mismatch repair, photoreactivation, nucleotide excision repair, base excision repair, and recombinational repair 2. Error-prone repair pathways, risk introducing mutations – activated when damage is severe and the cell has no option but to die  Repair of Errors in Nucleotide Incorporation Base substitution Mispairing slightly distorts DNA helix Recognized by repair enzymes Two repair mechanisms: 1. Proofreading 2. Mismatch repair By Eunice Laurent - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=112545564 Repair of Errors in Nucleotide Incorporation Proofreading by DNA polymerase (during replication) DNA polymerase not only synthesizes, but proofreads DNA Polymerases I, II, & III have proofreading ability Can back up, excise mismatched nucleotide 3’ → 5’ exonuclease activity Incorporate correct nucleotide Very efficient but not flawless © McGraw Hill, LLC  Errors missed by DNA polymerase are fixed by Methyl mismatch repair  Determines incorrect nucleotide (point mutation) by detecting the methylation of the parental strand ▫Parental D N A (template strand) methylated. ▫New D N A temporarily lacks methyl groups. ▫The repair system cuts out the mismatch from the unmethylated strand. © McGraw Hill, LLC Mismatch Repair in E. coli Mismatch repair is initiated by the protein MutS Recognizes mismatch, recruits and forms a complex with two other proteins called MutL and MutH Mut L recognizes unmethylated strand at a GATC sequence (GATC is where DNA is methylated) Newly synthesized DNA strands are not methylated until after replication is completed © McGraw Hill, LLC Mismatch Repair in E. coli Mut HSL complex causes DNA to loop Mut H cleaves the unmethylated strand at the upstream GATC sequence UvrD (a helicase) unwinds, and an endonuclease excises the DNA between the strand break and the mismatch Gap filled by DNA polymerase I and ligase © McGraw Hill, LLC Repair of Modified Nucleobases in DNA  Modified nucleobases lead to base substitutions if not repaired before DNA is replicated Ex: Repair of oxidized guanine – Base excision repair Glycosylase removes oxidized guanine nucleobase Endonuclease VI recognizes missing nucleobase and cuts DNA backbone at this site DNA polymerase I removes short section; synthesizes replacement DNA ligase seals gap © McGraw Hill, LLC Repair of Thymine Dimers Several methods to repair damage from UV light Photoreactivation (light repair) Photolyase (PhrB) recognizes distortion and uses energy from visible light Breaks covalent bonds of thymine dimer Nucleotide excision repair (dark repair) Enzymes detect distorted DNA and remove the damaged section Nucleotide Excision Repair UvrAB complex scans DNA until UvrA detects distortion UvrA disassociates, while UvrB melts DNA to form ssDNA bubble UvrA recruits UvrC and UvrC cuts DNA around the primer dimer And with the help of Helicase UvrD removes the cut single- stranded DNA DNA poly I and ligase fill in the gap © McGraw Hill, LLC  Repair of Thymine Dimers SOS repair: “last resort” repair mechanism UV Damage so severe that photoreactivation and excision repair cannot correct all damage DNA and RNA polymerases stall at damaged site → = no replication & transcription → Cell either dies or SOS is activated Algarni S, Ricke SC, Foley SL and Han J (2022) The Dynamics of the Antimicrobial Resistance Mobilome of Salmonella enterica and Related Enteric Bacteria. Front. Microbiol. 13:859854. doi: 10.3389/fmicb.2022.859854 (CC BY). Repair of Thymine Dimers SOS repair: “last resort” repair mechanism Several dozen SOS repair enzymes are induced by severe damage – RecA proteins sense DNA damage; induces transcription of SOS repair operon by interacting with repressor of operon (LexA) – DNA polymerase V (UmuD’2C) synthesizes even in extensively damaged regions → Has NO proofreading ability, so errors made during replication → Result is SOS mutagenesis Algarni S, Ricke SC, Foley SL and Han J (2022) The Dynamics of the Antimicrobial Resistance Mobilome of Salmonella enterica and Related Enteric Bacteria. Front. Microbiol. 13:859854. doi: 10.3389/fmicb.2022.859854 (CC BY). Mutant Selection Mutants are important in determining the functions of genes Chemicals, radiation, used intentionally to cause mutations Specialized techniques used to isolate mutant among wildtype – Difficult because mutants are rare & difficult to isolate Two main approaches 1. Direct selection: cells inoculated onto medium that supports growth of mutant but not wildtype Ex: antibiotic-resistant mutants exposed to antibiotics and grow 2. Indirect selection: Used to isolate auxotroph from prototrophic strain Figure from NIAID (CC BY 2.0)  Mutant Selection Indirect selection Used to isolate mutant auxotroph from wildtype prototrophic strain Ex: Replica plating Utilizes differences in growth of auxotrophs on nutrient-rich and -poor media Remember: Auxotroph requires growth factors © McGraw Hill, LLC Identifying Mutagens Using Bacterial “Guinea Pigs” Screening for Possible Carcinogens Carcinogens are mutagens which may cause cancer - Animal tests expensive (>$100,000), time-consuming (2-3 years) Ames test measures effect of mutation rate on specific gene using bacteria as a model organism  Mutant Selection Ames test - Mutated SalmonellaHis- cells are auxotrophic (require histidine) - Measure mutation rate by examining reversion rate of (auxotroph to prototroph) ‣ If chemical is mutagenic, it will induce mutations, some mutations will result in His- reverting to His+ ‣ Compare reversion rate with potential mutagen (test) vs normal reversion rate (control) By Histidine - Own work, CC BY-SA 3.0 https://commons.wikimedia.org/w/index.php?curid=13155414 Horizontal Gene Transfer as a Mechanism of Genetic Change  Horizontal (lateral) gene transfer Genes from one independent, mature organism to another.  HGT is important in the adaptation of many species Prokaryotes = Asexual reproduction only HGT increases genetic variation Expansion of ecological niche Genes can be transferred to the same or different species © McGraw Hill, LLC  Horizontal Gene Transfer as a Mechanism of Genetic Change  Genes naturally transferred by three mechanisms 1. Transformation: environmental DNA taken up by bacterial cell 2. Conjugation: bacterial DNA transfer during cell-to-cell contact 3. Transduction: bacterial DNA transferred from one bacteria to another by bacteriophage (virus of bacteria) Figure obtained from OpenStax Microbiology CC BY 4.0 Fate of DNA in Recipient Integration Donor DNA pairs with recipient DNA and recombine. Separate existence of DNA DNA persists separate from recipient chromosome if donor DNA is able to replicate (that is, plasmid). Remain in cytoplasm When donor DNA is unable to replicate. Degradation Led by CRISPR/Cas, preventing the formation of a recombinant cell. © McGraw Hill, LLC Horizontal Gene Transfer Transferred DNA only passed on to all progeny if it is a replicon = Has origin of replication - Chromosomes & most plasmids do - DNA fragments usually do not DNA fragments must be added to chromosome via homologous recombination - Replaces genomic DNA that has similar nucleotide sequence - Only occurs, IF donor DNA is positioned next to complementary region of the recipient’s genome - RecA proteins carry out the process. - Double-strand break occurs and reunion leads to crossing-over. © McGraw Hill, LLC  Bacterial Transformation “For The First Time, Scientists Have Caught Bacteria "Fishing" For DNA From Their Dead Friends” – Science Alert (2018) Vibrio cholerae using harpoon-like pilus (green) to capture free DNA (red) The bacterium extends a Type IV competence pilus through a pore in the outer membrane to nab a piece of DNA on the tip of the pilus and retract in back through the pore Bacterial Transformation Uptake of DNA from the environment outside the cell and maintenance of the DNA in the recipient cell in a heritable form. Natural Transformation Bacteria lyse and release DNA into the environment. These large fragments contain multiple genes. DNA that comes in contact with a competent cell is imported. © McGraw Hill, LLC The process of transformation Model for the role of the type IV pilus in transformation for V. cholerae 1. dsDNA binds to type IV Pilus 2. ssDNA enters cell (nucleases degrade the other strand) 3. RecA recruits ssDNA to homologous chromosomal DNA site and integrates via homologous recombination and the extracted chromosomal DNA strand is degraded 4. Since only ssDNA integrates into chromosome only one daughter cell will inherit new DNA © McGraw Hill, LLC  DNA-Mediated Transformation Transformation Recipient cell must be competent - Physiological state allowing cell to take up DNA Some cells always competent others need special conditions - Ex: Bacillus subtilis uses cell surface receptor-mediated processes to “turn on” genes for competence 1. Two-component regulatory system ‣ Recognizes low nitrogen and/or carbon sources = High concentration of bacteria = ↑ chance of naked DNA 2. Quorum sensing ‣ High concentration of bacteria via signaling molecules = ↑ chance of naked DNA  Transduction Transduction: Transfer of DNA from one cell to another via bacteriophage Two types: 1. Generalized transduction: DNA from random portion of the host genome is accidently packaged inside a phage protein coat (transducing particle) ‣ Transfer of DNA to new bacterial host during infection ‣ DNA inserted into host chromosome by homologous recombination 2. Specialized transduction: DNA from a specific region of the host genome is integrated directly into the phage genome and transferred to new bacterial host © McGraw Hill, LLC Conjugation Conjugation: mechanism of genetic transfer that involves cell- to-cell contact Requires contact between - Donor cell: - contains genes on a conjugative plasmid to make F pilus - Recipient cell: - receives plasmid RM Image © McGraw Hill, LLC Conjugation ‣Conjugative plasmids are small circular pieces of DNA that have all genes required oriV for transfer: F pilus, origin of transfer, origin of replication vs. ‣Mobilizable plasmids contain an origin of replication & transfer but no F pilus genes ▫ Can only be transferred at the same time as conjugative plasmid ‣Conjugation can occur in G+ and G- bacteria but process is quite different. We oriT = origin of transfer will cover the G- process oriV = origin of replication tra operon = genes for transfer (F pilus) © McGraw Hill, LLC  Conjugation Conjugative plasmids Ex: F (fertility) plasmid of E. coli oriV F+ cells have plasmid, F– do not F plasmid codes for F pilus – allows conjugation to occur oriT = origin of transfer oriV = origin of replication tra operon = genes for transfer (F pilus) © McGraw Hill, LLC  Conjugation Plasmid transfer F pilus of donor (F+ cell) binds to specific receptor of recipient (F- cell) F pilus retracts bringing cells close to one another forming link of cytoplasms Relaxase in donor cell cuts and unwinds one strand of plasmid at origin of transfer A second relaxase brings the ssDNA to the T4SS exporter (part of F pilus), which pumps it into F- cell the ssDNA is replicated by DNA poly III to form dsDNA plasmid in both cells Both cells now have a functional F plasmid © McGraw Hill, LLC  Conjugation Plasmid transfer F pilus of donor (F+ cell) binds to specific receptor of recipient (F- cell) F pilus retracts bringing cells close to one another forming link of cytoplasms Relaxase in donor cell cuts and unwinds one strand of plasmid at origin of transfer A second relaxase brings the ssDNA to the T4SS exporter (part of F pilus), which pumps it into F- cell the ssDNA is replicated by DNA poly III to form dsDNA plasmid in both cells Both cells now have a functional F plasmid © McGraw Hill, LLC  Figure obtained from Conjugation OpenStax Microbiology CC BY 4.0 Chromosomal DNA transfer (rare) Involves a Hfr cell (high frequency of recombination) Cell where F plasmid has integrated into chromosome via homologous recombination - episome Entire chromosome becomes one large F plasmid Ex: IS3 (insertion sequence) Serves as a sequence of homology between plasmid and chromosome. Recombination proteins perform integration © McGraw Hill, LLC  Chromosomal DNA transfer 1. Hfr cell produces F pilus and contacts recipient cell 2. Transfer begins with genes on one side of origin of transfer (in chromosome). 3. Connection between cells usually disrupted before complete transfer Full transfer would take ~100 minutes (context: E. coli binary fission 20 minutes) Recipient cell remains F– since incomplete F plasmid transferred 4. Donor DNA can integrate with recipient's chromosome via homologous recombination; if not it will be degraded (not a replicon) © McGraw Hill, LLC Chromosomal DNA transfer – F’ cell Chromosomal genes can accidentally be inserted into a plasmid Occurs when Hfr cell reverses into an F plasmid The process creates a F’ cell F’ plasmid results when small piece of chromosome is accidently removed with F plasmid DNA – F’ cells can form an F pilus and transfer plasmid (containing portion of chromosome) to recipient cell © McGraw Hill, LLC  Summary of DNA Transfer Mechanisms Conjugation, transduction, and transformation are most common But there are many other mechanisms: - Nanotubes: tubular extensions bridge cytoplasm of neighboring cells - Outer-membrane vesicles: secreted vesicles that contain DNA Emamalipour M, Seidi K, Zununi Vahed S, Jahanban-Esfahlan A, Jaymand M, Majdi H, Amoozgar Z, Chitkushev LT, Javaheri T, Jahanban-Esfahlan R and Zare P (2020) Horizontal Gene Transfer: From Evolutionary Flexibility to Disease Progression. Front. Cell Dev. Biol. 8:229. doi:  The Mobile Gene Pool  Genomics reveals surprising variation in the gene pool (pangenome) = sum of all genes of a single species ‣ Ex: less than half of the E. coli pangenome found in an individual strain Core genome – Genes shared between all strains of a species vs. Mobile gene pool – Genes that vary between strains of a species ‣ Often on genetic elements that move between organisms ▫ Plasmids, transposons, genomic islands, phage DNA ‣ May vastly outnumber the sequences in the core genome Hendrickson H (2009) Order and Disorder during Escherichia (~50% of the average E. coli genome) coli Divergence. PLoS Genet 5(1): e1000335. https://doi.org/10.1371/journal.pgen.1000335 The Mobile Gene Pool Plasmids found in most bacteria and archaea oriV Circular dsDNA with origin of replication Usually encode genes nonessential for life Can have many or few genes Can exist as two forms: Low-copy-number - One or a few per cell OR High-copy-number - Many up to 500 Copy number depends on: Origin of replication sequence The size of the plasmid – Larger plasmids = a larger metabolic burden to replicate © McGraw Hill, LLC Plasmids Ensure Inheritance  Plasmids use strategies to maintain themselves in daughter cells. High-copy-number plasmids flood the host cell cytoplasm Low-copy-number plasmids have partitioning systems involving polymerized filaments that move plamid copies into both daughter cells By User:Spaully - Own work, CC BY-SA 2.5, https://commons.wikimedia.org/w/index.php?curid=2081648 The Mobile Gene Pool Resistance plasmids (R plasmids) Carry genes that provide resistance to antibiotics, heavy metals Often two parts: 1. R (resistance) genes (one or many) 2. RTF (resistance transfer factor) = Pilus-synthesis, OriT genes Often broad host range Normal microbiota can carry and transfer to pathogens (ex: MRSA – vancomycin resistance) © McGraw Hill, LLC  The Mobile Gene Pool  Transposons provide mechanism for moving DNA Chromosomal DNA can move between cells if transposon jumps into plasmid Simplest form is called an insertion sequence The gene that encodes transposase flanked by inverted repeats Transposase catalyzes the movement of the transposon to another part of the genome by a cut/copy and paste mechanism by recognizing the inverted repeats © McGraw Hill, LLC The Mobile Gene Pool Composite transposons include one or more genes flanked by insertion sequences Integrate into plasmids and/or chromosome via Non-homologous recombination - Does not require a similar nucleotide sequence for insertion; it just inserts itself © McGraw Hill, LLC The Mobile Gene Pool Simple Transposition Also called cut-and-paste transposition. Transposase catalyzes excision of the mobile genetic element, followed by cleavage of new insertion site and ligation into site. © McGraw Hill, LLC  The Mobile Gene Pool conjugative transposon, carry genes for F pilus and origin of transfer; can be transferred to other cells – The transposon is excised from the chromosome Then circularizes and is eventually transferred as ssDNA via conjugation from the donor to a recipient bacterium in close contact And lastly, integrated into host’s chromosome Commonly carries resistance genes Lambertsen et al. 2017 Molecular Microbiology Published by John Wiley & Sons Ltd https://doi.org/10.1111/mmi.13905  The Mobile Gene Pool Genomic islands Large DNA segments in the genome that originated from another species (via HGT) – Identified by nucleobase composition (%GC) » very different from core genome » often found inserted adjacent to tRNA genes. Plasmids, phage DNA, and pathogenicity use tRNA genes as targets for integration. da Silva Filho AC, Raittz RT, Guizelini D, De Pierri CR, Augusto DW, dos Santos-Weiss ICR and Marchaukoski JN (2018) Comparative Analysis of Genomic Island Prediction Tools. Front. Genet. 9:619. Genomic islands Blocks of genes may provide characteristics that increase fitness - Environmental adaptation ‣ Ecological islands ‣ Utilization of energy sources ‣ pH/temp/salt tolerance - Ability to cause disease ‣ Pathogenicity islands ‣ Adhesions (fimbriae, capsule) ‣ Toxins (hemolysin) da Silva Filho AC, Raittz RT, Guizelini D, De Pierri CR, Augusto DW, dos Santos-Weiss ICR and Marchaukoski JN (2018) Comparative Analysis of Genomic Island Prediction Tools. Front. Genet. 9:619. The Mobile Gene Pool Investigating the mobile gene pool of microbes has advance recombinant technology – genetic engineering Two major techniques (Chapter 17) ‣ Cloning ‣ CRISPR-Cas9