Conjugation - Bacterial Genome Evolution PDF

II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION Genetics 270 CONJUGATION Introduction/history F plasmid Mechanism & regulation of DNA transfer Types of conjugative plasmids/...

II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION Genetics 270 CONJUGATION Introduction/history F plasmid Mechanism & regulation of DNA transfer Types of conjugative plasmids/elements àself-transmissible àmobilizable àpromiscuous Biological roles of conjugation àantibiotic resistance àevolution *chromosome transfer * F prime elements Use of conjugation in the lab 1 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION – Introduction/History Plasmid transferred from DONOR à RECIPIENT § A donor forms a MATING PAIR using a PILUS to interact with recipient § Two strands of plasmid separate in process similar to rolling circle replication § One strand transferred from donor à recipient § Two single strands replicated http://en.wikipedia.org/wiki/Bacterial_conjugati concurrently with transfer on 2 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION – Introduction/History « first observed in 1947 by Lederberg and Tatum – “gene recombination in E. coli” « mixed together different auxotrophic strains and got strains unlike either of parents « interpreted data as “assortment of genes in new combinations” « possible explanations: cell fusion, transforming factors in media, bacterial sex 3 II) BACTERIAL GENOME EVOLUTION CONJUGATION – Introduction/History HORIZONTAL GENE TRANSFER © 1946 Nature Publishing Gro 4 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION – F Plasmid “Fertility” plasmid First conjugative plasmid discovered Entire 100 kb sequence known Contains sequences originating from many different sources 5 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION – F Plasmid ¡ tra genes à required for transfer ¡ 3 replication origins à RepFIA, RepFIB, RepFIC (only RepFIA/oriV functional) ¡ Partitioning system “SELFISH” DNA ELEMENTS ¡ Toxin/anti-toxin systems for post-segregational killing ¡ Genes to prevent induction of SOS response ¡ Genes that block T7 phage development 6 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION – F Plasmid e s gen t ra 7 II) BACTERIAL GENOME EVOLUTION CONJUGATION - Mechanism & Regulation of DNA Transfer DNA transfer is mediated by tra genes, which encode: 1. Mpf component: Mating pair formation 2. Dtr component: DNA transfer & conjugal replication 8 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION - Mechanism & Regulation of DNA Transfer MATING PAIR FORMATION Mpf Component: holds donor & recipient together channel through which DNA transferred during mating http://biologicalexceptions.blogspot.ca/2012/04/life-outside-chromosome.html 9 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION - Mechanism & Regulation of DNA Transfer MATING PAIR FORMATION Pilus o 10 nm diameter tube made of many copies of PILIN protein o Made by donor à attaches to receptor on recipient o Pilus retraction draws donor & recipient together into MATING PAIR PILUS RETRACTION 10 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION - Mechanism & Regulation of DNA Transfer MATING PAIR FORMATION Type IV Secretion Systems ¥ multi-protein machine that synthesizes pilus ¥ after pilus retraction, it is used as conduit for DNA transfer Structural biology: Translocation chamber's secrets. Peter J. Christie Nature 462, 992-994. 12  II) BACTERIAL GENOME EVOLUTION CONJUGATION - Mechanism & Regulation of DNA Transfer MATING PAIR FORMATION Type IV Secretion Systems ¥ Conserved in many organisms ¥ Used for DNA transfer ¥ Also for delivery of virulence factors into eukaryotic cells: http://igem- victoria.pbworks.com/w/page/7992056/Agrobacterium%20project%2 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION - Mechanism & Regulation of DNA Transfer DNA TRANSFER & CONJUGAL REPLICATION Relaxosome complex of proteins that bind at oriT & prepare plasmid for transfer http://2008.igem.org/Team:Heidelberg/Project/Killing_I II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION - Mechanism & Regulation of DNA Transfer DNA TRANSFER & CONJUGAL REPLICATION Relaxosome TraI RELAXASE protein: site-specific endonuclease nicks & attaches to 5’ end at oriT via transesterification pilots strand into recipient cell recircularizes transferred strand via transesterification in recipient II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION - Mechanism & Regulation of DNA Transfer DNA TRANSFER & CONJUGAL REPLICATION Relaxosome Also requires other proteins: § TraY & host IHF à essential accessory proteins for nicking § TraM & TraD (MPF genes) à coordinate DNA processing with transfer apparatus § TraU – dtr component § helicase 16 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION - Mechanism & Regulation of DNA Transfer DNA TRANSFER & CONJUGAL REPLICATION Coupling Proteins connect Dtr & Mpf components à TraD signal contact with a recipient cell to the Dtr machinery provide specificity à can only transfer some plasmids DNA translocator that pumps DNA into recipient cell à related to FtsK key component that turns type IV protein secreting machine into DNA transfer device 17 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION - Mechanism & Regulation of DNA Transfer DNA TRANSFER & CONJUGAL REPLICATION II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION - Mechanism & Regulation of DNA Transfer REGULATION OF CONJUGATION ² once plasmid transfer initiated, very efficient à almost 100% of cell contacts result in DNA transfer ² plasmid transfer occurs at high rate in recent recipients ² However, after initial MPF, plasmid transfer occurs only sporadically WHY? 19 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION - Mechanism & Regulation of DNA Transfer REGULATION OF CONJUGATION « due to regulation of traJ expression by FinP & FinO TraJ transcriptional activator of tra genes FinP sRNA inhibits traJ mRNA translation & stimulates degradation FinO stabilizes & unfolds FinP http://gloverlab.biochem.ualberta.ca/secondaryp ages/research/finop.html 20 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION - Mechanism & Regulation of DNA Transfer REGULATION OF CONJUGATION 21 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION TYPES OF CONJUGATIVE ELEMENTS Self-Transmissible encode all functions needed to move amongst cells Mobilizable § can be transferred by a self-transmissible plasmid residing in same cell § contain own Dtr systems à referred to as mob genes § can usually be mobilized by a variety of tra systems § relaxases of mobilizable plasmids evolved to communicate with many coupling proteins of many self-transmissible plasmids § in practice à only oriT region needed for a plasmid to be mobilized by a compatible self-transmissible plasmid 22 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION TYPES OF CONJUGATIVE ELEMENTS 23 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION TYPES OF CONJUGATIVE ELEMENTS Promiscuous can transfer DNA between unrelated species Cyanobacteria, Gram-positive bacteria, plant cells important in evolution à similar genes in different organisms contribute to spread of antibiotic resistance *bacteria carrying R plasmids selected for in normal flora *can be passed to pathogens 24 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION BIOLOGICAL ROLES OF CONJUGATION: Antibiotic resistance Evolution 1) ANTIBIOTIC RESISTANCE conjugative plasmids are major way that antibiotic resistance determinants are spread 25 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION BIOLOGICAL ROLES OF CONJUGATION: 1) ANTIBIOTIC RESISTANCE 26 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION BIOLOGICAL ROLES OF CONJUGATION: 2) EVOLUTION « conjugative plasmids can transfer chromosomal DNA between bacteria that can lead to acquisition of new genes/mutations « two mechanisms: by integrating into the chromosome à Hfr strains http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit1/HGT/ u4fg24a.html 27 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION BIOLOGICAL ROLES OF CONJUGATION: 2) EVOLUTION à Hfr strains ª Hfr = high frequence of recombination ª many recombinants form when Hfr strain mixed with other bacteria of same strain ª due homologous recombination of F/conjugative plasmid into chromosome (often between insertion sequence/IS elements) 28 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION BIOLOGICAL ROLES OF CONJUGATION: 2) EVOLUTION à Hfr strains ª subsequent initiation of conjugation leads to transfer of plasmid AND chromosome 29 http://www.bio.miami.edu/dana/250/250S11_5print.html II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION BIOLOGICAL ROLES OF CONJUGATION: 2) EVOLUTION à Hfr strains ª what Lederberg & Tatum observed ª transfer of whole chromosome rare since mating pairs break apart before this can happen ª transfer of entire plasmid sequence rare since oriT in Hfr strain is in middle of integrated plasmid http://classes.midlandstech.edu/carterp/Courses/bio225/chap08/Microbial 30 %20Genetics%206.htm II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION BIOLOGICAL ROLES OF CONJUGATION: 2) EVOLUTION à Hfr strains http://www.quia.com/jg/1275308list.html 31 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION BIOLOGICAL ROLES OF CONJUGATION: 2) EVOLUTION « conjugative plasmids can transfer chromosomal DNA between bacteria « two mechanisms: 1. Hfr / chromo DNA à recipient 2. through imperfect excision of Hfr from the chromosome à F’ factors http://www.mun.ca/biology/scarr/4241_mkm_chap7.html 32 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION BIOLOGICAL ROLES OF CONJUGATION: 2) EVOLUTION à F’ factors § created through a homologous recombination event that leads to excision of a conjugative plasmid plus some chromosomal DNA from the chromosome § often through recombination between insertion sequence (IS) elements § can be very large 33 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION BIOLOGICAL ROLES OF CONJUGATION: 2) EVOLUTION à F’ factors proC+ F’ can transfer Pro+ trait to pro- recipient http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/problems/excha nge/conjugation/ 34 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION USES OF CONJUGATION IN THE LAB à TRIPARENTAL MATINGS ¥ ability of conjugative plasmids to transfer plasmid DNA used to move genes ¥ accomplished through highly efficient TRIPARENTAL MATINGS ¥ utilize 3 strains: ① strain carrying self- transmissible plasmid ② strain carrying mobilizable plasmid with gene(s) to be moved ③ desired recipient strain 35 II) BACTERIAL GENOME EVOLUTION HORIZONTAL GENE TRANSFER CONJUGATION USES OF CONJUGATION IN THE LAB à TRIPARENTAL MATINGS I. some self-transmissible plasmid will transfer into strain with mobilizable plasmid II. quickly spread through strain with mobilizable plasmid because conjugation de-repressed immediately after transfer III. then can efficiently move mobilizable plasmid to recipient for same reason 36  TA TA TA TA TA TA TA TA TA TA TA TA PSK – Post-Segregational Killing - Stable Toxin - Unstable Antitoxin, needs to be continually produced TA TA T T from plasmid TA T Also called “Plasmid Addiction”