MOLECULAR GENETICS PCB 4522
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Date Created: 09/18/15
Molecular Genetics PCB4522 Spring 2004 Dr Eva Czarnecka Verner Lecture 10 Conjugation Genes VIII Chapter 13 Course web page httpPCB4522IFASUFLEDU or go to Microbiology 8 Cell Science home page and look under course material The F plasmid is transferred by conjugation between bacteria 1 Bacterial conjugation a plasmid genome or host chromosome is trans erred from one bacterium to another in a mating process mediated by F plasmid 2 Fplasmid an example ofan episome in E coli 3 Episome an element that may exist as a free circular plasmid or that may become integrated into the bacterial chromosome as a liner sequence Bacterial conjugation means to exchange genetic material between bacteria Connects replication With the propagation of the netic unit Fplasmid 1 large circular plasmid 100 kb 2 only 60 ca 60 genes has been mapped 3 32 kb is organized as a unit to transfer is genome to another bacteria transfer region or Ira genes 4 two methods of replication a oriV as free plasmid one copy bacterial chromosome b uses E coli chromosomal origin when integrated oriC oriV is suppressed F39Plasmld Discrete region that has or transfer genes Ongm oftmnsfeo tra amp trb ioci 40 genes used to initiate replication for transfer genes IS elements insertion sequences used in transposition Fpiasmid integrated into cnromosomein rare instances two mechanisms oriV 590110 Inmate 1h molog us plasrniol replication recombination 2transposition F 1 d Hfr cell contains 3913 351111 integrated F plasmid F cell contains episornai F plasmid E ml chromosome After integration Fplasmid replicates as part ofhost replicon 0 1C miV is suppressed Chromosome Transfer formation of Hfr strains high frequency recombination In rare instances a plasmid F can integrate into the chromosome There are two mechanisms of integration F CELL llntagratian at F plasmid 1 Homologous r recombination 2 Transposition HtrCELL The Sex Pilus ome E coI39 strains contain l 39 Fertility plasmids 39 g r Carries the information required for its own transfer DNA is NOT transferred through the pilus Single stranded genomes are generated for bacterial conjugation 1 Fplasmid is 100kb Makes about 5 min to transfer 2 Sequences required for transfer are located within a 333kb transfer region IncFl 3 Pius synthesis and assembly Nicking amp initiation of transfer mating pair pore formation transfer of DNA matingaggregate stabilization surface exclusion regulation tra region of the F plasmid regulation 0 traJ amyator traVtral irection Transcription unit mm gt y oriTtraM J YALEKBP VRC WU N trbCDE traF Ill traH G STD IZ nP Ira amp Irb loci 40 genes Expressed coordinater as a part of single transcription unit traVtral Fig 13 20 Gene VIII F 1321 Fplasmid 5 Fpositive Fl bacteria are able to con39ugate mate with Fminus F 39 bacteria 6 In its integrated form the Fplasmid may cause some or all of the bacterial chromosome to transfer to the Fminus recipient 7 Fpositive bacteria possess pili formed from the pilin protein FPilus 1 Extracellular filament that extends from surface 23 pm hairlike 23 pilicell main structural component is a single subunit pilin coded by traA gene tip protein 2 Pilus recognizes various receptors on the host cell lipopolysaccharide protein OmpA mating pair formation may occur differently on solid or liquid media ng pairing h F nili are essential for transport ul are NOT channels for DNA Conjugation 1 tap ofthe Fpi1us makes contact with recipient cell a pilu is composed ofpilin subunits which form a hollow cylinder ofS nm with 39 diameter no NA does NOT transfer through 5 tube b protential recipient is Fpositiye no connection is formed due to surface exclusion proteins coded by traS and traTofFplasmid Conj ugati 2 Pilus retracts bringing recip circle and un 1200bpsec 3 DNA transferred through channel formed by protein coded by traD gene 4 Transfer begins from anTwhich is nicked by TraYTraI complex at arms site 5 TraYTraI multimenc complex migrates around 39 winds DNA from 5 end at ca on lent closer for transfer Overview of Conjugation 1 Conjugative Plasmids are either self transmissible or mobilizable 2 The transfer systems are encoded by the fra genes contained on the plasmids themselves 3 Tra systems are linked to their incompatibility Inc group Ftype a IncF RP4 plasmids 9 Inc 4 Plasmids that have tranfer systems that allow DNA to unrelated species are known as plas transfer of mids Incw plasmids IncP plasmids a The single strand in each cell undergoes replication to form double stranded DNA promiscuous rig 1322 5 only one unltlengthls transferred Gm Vquot 6 IncN Mechanism of self honor nus when Mechanism of plasmid transmissible transfer F e I obilization 39 pie 1 A site on the plasmid known The mob plasmid cannot lineages origin of t WWW transfer without another 1 quot er7 is nicked by a specific an plasmid 1 ndo se one of th a Mkmm m gene pr M K W cm 2 The other plasmid may otrlaeiiinne A 0M 5 quotMd K B 039 aispiocaniane or mu not be a 52m oiusniia between the two cells and 07 5 msmissib39e P39C SVquotid but only ONE and of DNA is masters UST contain fm My passed through e revllcaw functions cell contact m Z m other cell 5 2rld first C A nic ing 3 If the other plasmid hemmed only amine unll lenglh in f actor is sepmuan tnnaenua rt O O F is self transmissi ble it may also transfer Major Funcfions During Transfer gt40 genes 1 Troy binds near oriT and recruits treI relaxuse Iik A protein of qgtgtlt174 TruI has nuclease d helicase ATP activity Function enhanced by TraYdt IHF integration iiost factor I is a trensterese a covalent attachment of the 539 end of the DNA to the protein 3 TruD uciive transport binds DNA ATPGTP binding sites inner membrane protein necessary for DNA trensfer Surface Exclusion Reduces conjugafion among cells carrying closely relafed plasmids 5 exclusion groups idenfified To dafe 1 TraT oufer membrane profein Hiaf blocks mafingpair formafion 2 TraS l3locks DNA fransfer Chromosome Transfer Hfr 9 Recipient cell Part 1 oonerceii Recipllul ceii Fquot rnuocnoireit svnllieslus u ear 7 i is plasmid s ear puns contests no no ntr Lnilun Dill causing1mm GUM dieinrissteassi a a ring Genes inicgiauan t 0i F plasmid mu urnot chromosome rr 39 lrinsiuie ziiingio slun edDNA r at Breakage donor DNAslvand a m Wearled r niesnm is worn ai tuo mining and m inc are at in one arm Chromosome Transfer Hfr 9 Recipient cell port 2 ma aonoi nna iecipiem wiirsu amio usunllv r nacipieni oeiiDNA gt Dquot A are remains nit lmegmllun rt donor cnramasamui mm Chromosome transfer The transfer process uses the rolling cir ern od ofreplicau39on The complement to the transferred strand is synthesized in the recipient It takes 100 minto transfer entire chromosome ofE call 2 The double stranded transferred DNA is integrated into the recipient chromosome by double recombination 3 Fposiuye strains support highlevels of recombination and are described as Hfr strains high frequency ofrecornbinau39on Chromosome transfer 4 The transfer ofthe host chromosome is away from the ra region and Fplasrnid except for a small part around 071739 5 Typically only relatively short stretches of ansferred amp are integrated into the recipient Chromosome transfer Chromosome transfer usually does not result in conversion ofrecipient cell to F In chromosome transfer donor DNA integrates into the host genome by recombination or trans osition in plasmid transfer this does not occur Chromosome transfer 8 Bacterial contact usually broken before DNA transfer complete A gradient oftxansfer frequencies around the chromosome E call chromosome as a map divided into 100 minutes the starting point for the gradient ofuansfer is different for each H strain determined by the F facto integration site 1 Plasmids that quotleavequot the gene e chromosomal DNA are known as grime factor 2 They ea recombination chromosome Formaflon of Pr39lmz Facfor39 Plasmids quota region onhe F plasmid Hfr Cell F Cell renumnn lnlegrated F F plasmid plasmid Chromosome Chromosomal DNA Chromosome urrying vequot the chromosom e by homologous resulting in a deletion in the M Iervecmltstvalto endo 3 39 Iralrcuvalentlyattachestu 2ndutDNA quotmm ampunwlnds it nun tucking ntt unwmttinit traMJ YALEKBPVRC WUN trhCDE traF trbB traH G 5n ll IinP uiiactiun m tienstei Channel tm39r sensis IraTroutevmembvaVlE Iml nintein that blocks matlng paiituiinatiun IraSblucks DNA tianstei Surla exclbs Inn as my arm m Neg1w regulrtor D trrnacn 10mm lino Helm IirP Bacterial replication is connected to 1 the cell cycle chapter 13 14 Genes Vlll two links between replication and bacterial cell growth a Frequency of initiation adjusted to t rate cell is growing 9 T completion ofareplication cycle is connected to cell division Connecting bacteIial replication to the cell cycle 2 Doubling time time needed for cell numbers to double In E can they range from 18 min to slower than 18 min average in lab is 60 m39 Connecting bacterial replication to the cell cycle 3 Constants in replication cycle 2 c xed time 40 min to replicate entire chromosome Replication fork moves at c 50000 bpmin g lxed time 20 minbetween end of replication and cell division 2 c 0 min These values apply for doubling times between 18 and 60 min Connecting bacterial replication to the cell cycle 3 Constants in replication cycle continued d When doubling time is less than 60 min initiation ofreplicatlon must occur before t e end ofprevious div ion cycle Connecting bacterial replication to the cell cycle Example cat aunties sterst nannies rtg1au quotte x AI 1quot quotIn tnutnmn occurs til min origins nnitte Dmully tspttcnen chmnnsnms tn ultirork c hro tn 0 so tn e unvrtenpnnn on naunn mansion anstsun ten How does the cell know When to initiate e replication cycle Rapidly growing cells are larger and possess a greater number of origins multiforked chromosomes Initiation occurs at aconstant ratio ofcell mass to the number ofchromosome origins a Bacterium unit cell an tity 17 m long one origin per unit cell A rapidly growing cell with two origins will be 1734 nmlong b At 10 min atter division the cell mass increased suf ciently to support an initiation at both origins How is cell mass titrated 1 Current model suggests that initiation is contio by the accumulation of a positive acting factor an inhibitor Accumulation ofa critical amount would trigger initiation This factoris dilute in newly divided cells lled not 1 mative model suggests that initiationis e accumulation oranegative acting quot quot rmayhesynthesized no initiation and diluted below an effective level in larger cells initiation Assigned text passages You are responsible for Chapter 1315 Genes VIII Figs 1325 amp 1326 The septum divides a bacterium into progeny each containing a chromosome amp Chapter 1321 Genes VIII Figs 13 36 1337 amp1338 Single copy plasmids have a partitioning system c7279 eanfecture 11
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