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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. E.coli systems and recombination: Determinants of diversity: Overall aims ML Nine/ten lectures with Key topics. Homologous recombination and DNA repair Role of methylation and repair. Role of Plasmids; control of replication, transfer and stability. Illegitimate recombination: transposons and integrons Regulation of DNA transposition. You should: Have a basic grounding for further reading and other systems covered in the course (e.g pathogens). Be able to critically read key papers in the area. Critically assess the development of ideas to date.
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Plasmid Evolution and Role of mobile DNA Elements Plasmid structure and evolution: Cassette model Discovery of transposable elements in bacteria Classes of transposable element Distribution of these elements Mechanisms of transposition Negative control of transposition Target site specificity and immunity Integron mechanism for acquisition of genes Overview of gene spread via plasmid / transposon vectors You should be able to discuss the RELATIVE role of moveable or transposable DNA elements and the host factors controlling them in the evolution of diverse microbial genomes
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Cassette model for Plasmid evolution. Many different types of plasmid Three basic units / regions 1.Transfer / 2. Replication / 3. Determinant Antibiotic resistance plasmids Phages replicate as plasmids Catabolic plasmids e.g. Pseudomonas spp and Rhodococcus spp Most are closed circular More large linear mega plasmids / second chromosomes discovered e.g. Borrelia, Streptomyces and Rhodococcus spp Many antibiotic resistance plasmids such as R1, R6 and R100 are closely related to F- plasmids in the Enterobacteriaceae e.g. F and R100 from Shigella flexneri R100 F tra genes Tn10 found on R100
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. R100 as an example of the Cassette model tra IS2 IS10 Tn10 Tn2571 IS1 Tn4 Tn3 on R1 Tn903 on R6 Resistance Determinants mer amp sul str kan cm
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Discovery of Transposable DNA elements in bacteria First noted in 1967 in E.coli as cause of polar mutations in; gal operon (Saedler) lac operon (Shapiro) High frequency of spontaneous reversion to gal or lac + Hedges and Jacob (1974) demonstrated 1st Transposon Tn1 (Tn3 related): Amp r in plasmid RP4 P O ETK MUTATION gal operon on defective lambda phage ; dgal TRANSCRIPTION BLOCKED. NO ENZYME EXPRESSION
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Discovery of Transposable DNA elements in bacteria DNA of dgal phage analysed by density gradient centrifugation and by homology annealing and EM sizing Inserts detected as approx’ 800 bps or 1500 bps Responsible for the POLAR effect on gene expression Looping indicated that there were inverted repeats at the ends Named Insertion Sequences IS1 and IS2 dgal + + dgal - polar mutant Melt and self anneal Melt an anneal
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Classes of transposable DNA in bacteria Many elements discovered since first ones There are four basic types The Insertion sequences and their composite elements TYPE I The Tn3 family of elements TYPE II The transposing bacteriophages (e.g. mu - not covered here) TYPE III The conjugative transposons (e.g. Tn916 carrying tet resistance around a range of host cells in Enterococcus and other bacteria). Large family found in these Gram positive bacteria with broad host range. Carry Integration / excision determinants and plasmid transfer genes. INTEGRATE - EXCISE -TRANSFER ON PLASMID (not covered in detail here). Many features in common but with exceptions MUST have precise end recognition EITHER use terminal inverted repeat sequences OR in some cases integrate at specific sequences to produce a consensus sequence for end recognition Often generate duplications at target sites
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Classes of Insertion sequences in bacteria 19 families based on combinations of the following criteria: 1) similarities in genetic organisation (arrangement of open reading frames) 2) marked identities or similarities in their Transposases (common domains or motifs); DDE Motif conserved 3) similar features of their ends (terminal IRs) 4) fate of the nucleotide sequence of their target sites (generation of a direct target duplication of determined length). IS DATABASE is best reference source http://www-is.biotoul.fr
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Properties of some transposable DNA elements TYPE I Insertion sequences and their composite transposons shown in handout. See IS FINDER WWW SITE http://www- is.biotoul.fr/is.html. Indicates size, duplications and inverted repeatshttp://www- is.biotoul.fr/is.html Composite elements flanked by IS elements Multiple copies in different bacteria WIDELY DISTRIBUTED TYPE II The Tn3 like elements. Many ANTIBIOTIC RESISTANCE DETERMINANTS Type Kbps Marker Inverted repeats Target dup’ Tn 1 5.0 amp r 38 5 Tn 3 5.0 amp r 38 5 5.0 NONE 38 5 Tn 1721 5.0 tet r and INTEGRON system 38 5
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Structure of IS10 and composite Tn10 as an example 9.3Kb IS10-L IS10-R tetR Defective Active in transposition 1057 bps IR-L IR-R Tn10 Host 9bp duplication Transposase
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Structure of Tn3 as an example 5bp duplication IR-L IR-RtnpA Resolution site tnpRbla -lactamase Transposase Resolvase/ repressor
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Transposition Mechanisms CONSERVATIVE VS REPLICATIVE Independent of RecA + Donor Target sequence REPLICATIVE TRANSPOSITION RESOLUTION CONSERVATIVE TRANSPOSITION + + TRANSPOSON Donor may be degraded
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Tn3 Transposition is replicative Tn3 Transposase cut 5bpTarget cut Ligation Replication
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Tn3 Transposition is replicative cont…….. Resolution site analogous to cer Resolution by TnpR + Donor Intact + Transposed element replicated
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. IS10 (Tn10) transposition is conservative IS10 Double strand cuts 9bpTarget cut + Donor DNA lost / degraded Repair of 9bp gap Transposition complete
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Demonstration of IS10 conservative transposition OR lacZ + insert lacZ - insert Melt, mix then reanneal Package into phage heads. Infect recA, lac deletion, non-permissive host cells IS10 constructed into phage int -, replication deficient : needs permissive host Plate on tet/Xgal plates for transposants Some sectored colonies 90% blue or white Therefore transposition must be conservative 10% sectored and still segregating But
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Transposition demonstrated in vitro IS10 transposase makes double stranded cuts And can form circles via single stranded ligation Only Mg+ needed in reaction In vitro transposition shown using vectors Rates of about 1 in 10 6 shown following packaging and infection of host cells Host factors such as; Hu protein Integration host factor(Ihf) and supercoiled DNA needed
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Negative control of transposition All transposons appear to be under negative regulation This brings transposition recombinational frequencies down to around 10 -3 to 10 -6 In E. coli the growth temperature greatly affects many transposition events. Higher frequencies at lower temperatures (below 37 o C) Especially IS1 and Tn3. Basis not known. Negative control due to: A. Repressor molecule Tn3 (earlier) B. Antisense RNA (Tn10) C. Methylation (Tn10 and many IS elements) D. Transcriptional frameshift (IS1 specifically)
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Repressor regulation: Tn3 IR-L IR-RtnpA Resolution site analogous to cer tnpRbla -lactamase Transposase Resolvase/ repressor
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Antisense RNA and methylation: IS10R fromTn10 IR-L IR-R Tn10 Host Pin Pout 180 base overlap from Pout causes multicopy inhibition IR-L IR-R Tn10 Host Transposition x10 higher in dam mutants No expression when methylated only after replication and hemimethylation GATC CTAG In Pin region Combination leads to ONLY 0.25 molecules (1 per 4 cells) of transposase (measured using cat gene fusions)
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Transcriptional frameshift control: IS1 IR-L IR-R IS1 768 bps: Complex internally. insA insB No full transposase Occasionally a transcriptional frameshift to give fused insA/insB protein and full transposase Transposase
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Target site specificity and “immunity” Many relatively NON specific in target preference Often NO common features Tn5 and IS1 prefer hot spot AT rich DNA Tn7 has specific target Tn10 shows some preference for a consensus NGCTNAGCN but not clear cut. “IMMUNITY” shown by Type II elements (Tn3) Low probability of second transposition in a plasmid E. coli chromosome shows strong “immunity” Basis is not known
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Integron mechanism for acquisition of genes Discovered in some Tn3 like elements such as Tn21 They are found WITHIN these elements They explain the acquisition of new genes/markers 5’conserved 3’conserved Recombinase 7 bps core sites in variable region Target DNA New gene acquired
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Overview of gene spread The relative role of transposons vs other recombinational and mutational events. A SPECTRUM of activities leads to variation High frequency Low diversity Low frequency High diversity Homologous recombination Plasmid transfer Transposition Point mutation *10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 * As frequency per cell per generation Integron action
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©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. The END for NOW The force that through the green fuse drives the flower Drives my green age; that blasts the roots of trees Is my destroyer. And I am dumb to tell the crooked rose My youth is bent by the same wintry fever The force that drives the water through the rocks Drives my red blood; that dries the mouthing streams Turns mine to wax. And I am dumb to mouth unto my veins How at the mountain spring the same mouth sucks Dylan Thomas 1914 - 1953
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