Plasmids and Plasmid Biology By KonradT. Juszkiewicz, MD,

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Plasmids and Plasmid Biology By Konrad. T. Juszkiewicz, MD, MPH Plasmids and Plasmid Biology By Konrad. T. Juszkiewicz, MD, MPH

 • Plasmidstructure • Plasmidreplicationandcopynumber control • Plasmidtransfer • Plasmidsastools • Fplasmids. Plasmids and Plasmid Biology • Plasmidstructure • Plasmidreplicationandcopynumber control • Plasmidtransfer • Plasmidsastools • Fplasmids. Plasmids and Plasmid Biology

Plasmids • Extrachromosomal DNA, usually circular-parasite?  • Usually encode ancillary functions for in vitro growthPlasmids • Extrachromosomal DNA, usually circular-parasite? • Usually encode ancillary functions for in vitro growth • Can be essential for specific environments: virulence, antibiotics resistance, use of unusual nutrients, production of bacteriocins (colicins) • Must be a replicon — self-replicating genetic unit

Plasmids • Plasmid DNA must replicate every time host cell divides or it will be lostPlasmids • Plasmid DNA must replicate every time host cell divides or it will be lost a. DNA replication b. partitioning (making sure each progeny cells receives a plasmid) • High copy plasmids are usually small; low copy plasmids can be large • Partitioning is strictly controlled for low copy, but loose for high cop

Plasmids • Plasmidreplicationrequireshostcellfunctions • Copynumberisregulatedbyinitiationofplasmid replication • Plasmidsareincompatiblewhentheycannotbe stablymaintainedinthesamecellbecausethey interferewitheachother’sreplication. Plasmids • Plasmidreplicationrequireshostcellfunctions • Copynumberisregulatedbyinitiationofplasmid replication • Plasmidsareincompatiblewhentheycannotbe stablymaintainedinthesamecellbecausethey interferewitheachother’sreplication.

Cs. Cl gradient with ethidium bromide and UV light. Three forms of plasmid DNA “ OldCs. Cl gradient with ethidium bromide and UV light. Three forms of plasmid DNA “ Old School method of purifying plasmid ”

Virulence plasmids from Salmonella, Shigella,  Yersinia, B. anthracis, E. coli,  and others. Virulence plasmids from Salmonella, Shigella, Yersinia, B. anthracis, E. coli, and others.

Plasmid replication • Plasmid replication requires host DNA replication machinery. • Most wild plasmids carry genesPlasmid replication • Plasmid replication requires host DNA replication machinery. • Most wild plasmids carry genes needed for transfer and copy number control. • All self replication plasmids have a ori. V : origin of replication • Some plasmids carry and ori. T : origin of transfer. These plasmids will also carry functions needed to be mobilized or mob genes.

Plasmid replication • Plasmid segregation is maintained by a par  locus-a partition locus that ensuresPlasmid replication • Plasmid segregation is maintained by a par locus-a partition locus that ensures each daughter cells gets on plasmid. Not all plasmids have such sequences. • There are 5 main “incompatibility” groups of plasmid replication. Not all plasmids can live with each other. • Agents that disrupt DNA replication destabilize or cure plasmids from cells

Antisense. RNAgenecontrol. the. RNARNAhybridisverystable blocksmosttranslationandtanscription requires. RNAasestodegrade commonthemeinbacterialgeneregulationaswearelearning Antisense. RNAgenecontrol. the. RNARNAhybridisverystable blocksmosttranslationandtanscription requires. RNAasestodegrade commonthemeinbacterialgeneregulationaswearelearning

Antisense RNA: RNA-RNA hybrid blocks replication GGCUAAUUCC Antisense RNA is also used in euks called CCGAUUAAGGAntisense RNA: RNA-RNA hybrid blocks replication GGCUAAUUCC Antisense RNA is also used in euks called CCGAUUAAGG si. RNA Blocking RNA priming for DNA Pol. I prevents replication RNA I-small inhibitory RNA that binds to RNAII will act as a primer for DNA replication Rop: plasmid encoded proteins which stabilizes the RNAI-RNAII complex. Anti-sense RNA replication control

Col. E 1 Replication Controlanexampleof primercontrolof replication • RNAIIwillserveasa primerforthe replicationfork.  • The 3’endisprocessed byhost.Col. E 1 Replication Controlanexampleof primercontrolof replication • RNAIIwillserveasa primerforthe replicationfork. • The 3’endisprocessed byhost. Rnase. Htoallow efficient. RNADNA hybridtoform • Thehybridactsasa primerforhost. Pol 1 • Astheconcentrationof plasmidincreases, Rop doesalso • Ropstabilizesthe RNA 1 IIcomplex • No. RNAforreplication priming. Col. E 1 replicationdoesnotneed plasmidencodedrepproteins

The events upon entry into a cell • Rep. A m. RNA is made from PrepThe events upon entry into a cell • Rep. A m. RNA is made from Prep until copy number becomes high • Cop. B expression increase an Cop represses Rep. A expression at Prep. A • Cop. A now is made-a 90 base antisense RNA • Cop. A binds to 5 -end of the Rep. A m. RNA, forming ds. RNA • This is recognized by host RNAase. III and degraded. Thus concentration of Rep. A protein is maintained by rate of RNA-RNA hybrid formation.

Repproteincontrol. R 1 familyofplamsids.  • Rep-protein expression controlled by antisense Cop. A • Pcop. B-encodesRepproteincontrol. R 1 familyofplamsids. • Rep-protein expression controlled by antisense Cop. A • Pcop. B-encodes Rep and Cop. B • Pcop. A-encodes antisense RNA • plasmid replicates to high level • Cop. B levels rise, shutting off Rep. A production • antisense RNA from Pcop. A made • complexes with rep. A m. RNA • Host RNase. III will cleave the complex. Plasmid copy control balanced by host RNase. III activity and transcription from the plasmid

Iteron. Plasmids: Handcuffing RK 2 andotherbroadhostrange plasmids Rep. Aisabletobindtherepeat sequencesupstreamofthe promoterregionfor rep. A.  • bindingcausestwoplasmidIteron. Plasmids: Handcuffing RK 2 andotherbroadhostrange plasmids Rep. Aisabletobindtherepeat sequencesupstreamofthe promoterregionfor rep. A. • bindingcausestwoplasmid moleculestocouple“handcuff” • preventsreplication. “ copyup”mutants: mutationsin. Rep. Athat arelessabletobindto eachother.

Incompatibility. Groups • Notallplasmidscanlivetogether.  • Plasmidsthatareabletocoexistinthesamecelldonot interferewitheachother’sreplication • Asinglecellcanhaveasmany. Incgroupplasmidsasit cantolerateandreplicate! Partion. Locus : aregiononbroadhostrangeplasmidsthatbindsIncompatibility. Groups • Notallplasmidscanlivetogether. • Plasmidsthatareabletocoexistinthesamecelldonot interferewitheachother’sreplication • Asinglecellcanhaveasmany. Incgroupplasmidsasit cantolerateandreplicate! Partion. Locus : aregiononbroadhostrangeplasmidsthatbinds toastructureontheinnermembraneofthecelltoensureproper segregation. Plasmidslabeledwithfluorescentprotein movetoeachdaughtercellduringdivision. Pogliano, Joe et al. (2001) Proc. Natl. Acad. Sci. USA 98, 4486 —

Figure 4. 18 Par locus • think of this as a primitive centromere • the growingFigure 4. 18 Par locus • think of this as a primitive centromere • the growing filaments push the plasmids to the opposite poles of the cells

Site-directed mutation: Suicide plasmds 1. Plasmid must be unable to replicate without essential replication proteins provideSite-directed mutation: Suicide plasmds 1. Plasmid must be unable to replicate without essential replication proteins provide in trans. 2. It helps if the plasmid can be mobilized- ori. T required 3. Need a selectable marker 4. Large or small region of homologous DNA cloned that will integrate into the chromosomal target. 5. Need a counter selection method to kill the donor cells 6. Screen for what you think is correct. Also, merodiploid reporter strains can be constructed in this manner 1. Make a lac. Z fusion to your promoter of interest 2. Clone into a suicide plasmid 3. Mate into recipient. 4. Resulting strain will harbor a duplication of the promoter region: lac. Z and still have a functional copy of the gene. Why would this be important? Plasmids as genetic tools: Construction of Mutants

R 6 K: broad host plasmid. -Pir is the essential replication protein - pir mutants cannotR 6 K: broad host plasmid. -Pir is the essential replication protein — pir mutants cannot replicate unless supplied in trans. -integration into the chromosome is selected for by growth on ampicillin Howcouldyoumake targetedmutant usingthismethod?

1. large (100 kb) 2. low copy (1 -2 copies/cell) 3. self transmissible 4. requires protein1. large (100 kb) 2. low copy (1 -2 copies/cell) 3. self transmissible 4. requires protein synthesis (chloramphenicol-sensitive) 5. rep. E gene encodes Rep. E protein 6. Rep. E protein binds to origin of replication ( ori. S ) and initiates DNA replication 7. Rep. E binds to the rep. E promoter and activates transcription 8. Rep. E binds to the cop. A/inc. C locus binding copies of F together via Rep. E – inhibiting replication (coupling) F-plasmid

FPilusassembly FPilusassembly

replication IS elements. Primitive transposon 30+ genes needed For transfer Geneticorganizationof. F replication IS elements. Primitive transposon 30+ genes needed For transfer Geneticorganizationof.

F-transfer at fine detail F-transfer at fine detail