E.coli systems and recombination: Determinants of diversity: Overall aims ML

1. E.coli systems and recombination: Determinants of diversity: Overall aims ML • Nine/ten lectures with Key topics. • Homologous recombinationand 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.

2. 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

3. R100 F tra genes Tn10 found on R100 Cassette model for Plasmid evolution. 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 • 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

4. Resistance Determinants IS1 Tn3 on R1 Tn4 tra Tn2571 IS2 Tn10 Tn903 on R6 IS1 IS10 IS10 R100 as an example of the Cassette model mer amp sul str kan cm

5. P O E T K MUTATION TRANSCRIPTION BLOCKED. NO ENZYME EXPRESSION 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): Ampr in plasmid RP4 gal operon on defective lambda phage ; dgal

6. Melt and self anneal dgal- polar mutant dgal+ + Melt an anneal 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

7. 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

8. 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

9. 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 repeats • 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 ampr 38 5 Tn 3 5.0 ampr 38 5  5.0 NONE38 5 Tn 1721 5.0 tetr and INTEGRON system38 5

10. IS10-R IS10-L 9.3Kb 1057 bps 9bp duplication 9bp duplication Structure of IS10 and composite Tn10 as an example Active in transposition Defective tetR IR-L IR-R Host Tn10 Transposase

11. Resolution site Resolvase/ repressor Transposase -lactamase Structure of Tn3 as an example 5bp duplication 5bp duplication tnpA tnpR bla IR-R IR-L

12. TRANSPOSON Target sequence + RESOLUTION + + Donor may be degraded Transposition Mechanisms CONSERVATIVE VS REPLICATIVE Independent of RecA Donor CONSERVATIVE TRANSPOSITION REPLICATIVE TRANSPOSITION

13. Ligation Tn3 Transposition is replicative Tn3 Transposase cut Replication 5bpTarget cut

14. + Resolution by TnpR Tn3 Transposition is replicative cont…….. Resolution site analogous to cer Donor Intact + Transposed element replicated

15. Double strand cuts 9bpTarget cut Repair of 9bp gap IS10 (Tn10) transposition is conservative Donor DNA lost / degraded IS10 + Transposition complete

16. Melt, mix then reanneal Package into  phage heads. Infect recA, lac deletion, non-permissive host cells Some sectored colonies But 10% sectored and still segregating 90% blue or white Demonstration of IS10 conservative transposition IS10 constructed into  phage int-, replication deficient: needs permissive host lacZ- insert lacZ+ insert OR Plate on tet/Xgal plates for transposants Therefore transposition must be conservative

17. 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 106 shown following packaging and infection of host cells Host factors such as; Hu protein Integration host factor(Ihf) and supercoiled DNA needed

18. 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 37oC) 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)

19. Resolution site analogous to cer Resolvase/ repressor Transposase -lactamase Repressor regulation: Tn3 tnpA tnpR bla IR-R IR-L

20. Pout IR-L IR-R Host Tn10 Pin IR-L IR-R Host Tn10 GATC CTAG In Pin region Antisense RNA and methylation: IS10R fromTn10 180 base overlap from Poutcauses multicopy inhibition Transposition x10 higher in dam mutants No expression when methylated only after replication and hemimethylation Combination leads to ONLY 0.25 molecules (1 per 4 cells) of transposase (measured using cat gene fusions)

21. IR-L IR-R Transposase Transcriptional frameshift control: IS1 IS1 768 bps: Complex internally. Occasionally a transcriptional frameshift to give fused insA/insB protein and full transposase insB insA No full transposase

22. 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

23. Recombinase 3’conserved 5’conserved 7 bps core sites in variable region Target DNA 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 New gene acquired

24. Overview of gene spread The relative role of transposons vs other recombinational and mutational events. A SPECTRUM of activities leads to variation Plasmid transfer Integron action Homologous recombination Point mutation Transposition *10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Low frequency High diversity High frequency Low diversity * As frequency per cell per generation

25. 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