1. Microbial Genetics MICB404, Spring 2008 Lecture #14 Conjugation: Mechanisms of plasmid-mediated gene transfer

2. Summaries, exam and quiz returned today New quiz. Due Friday 03-07-08 Supplemental reading posted on website (URL to be distributed). 1)Lanka and Wilkins (1995) DNA processing reactions in bacterial conjugation. Annu. Rev. Biochem. 64, 141-169. 2)Foley et al., (1998) A short noncoding viral DNA element showing characteristics of a replication origin confers bacteriophage resistance to Streptococcus thermophilus. Virology 250, 377- 387.

3. Review ColE1 plasmids Mutation of RNA I = Mut of RNA II Different secondary structure Altered G + C content or distribution

4. Foley et al., 1998 Article is, or will be, available on MICB404 website Plasmids containing phage ori Acts as an origin of replication driven by phage infection.

5. Could a plasmid contain no genes?

6. Conjugation One bacterium (the "male" or donor cell) transfers DNA to another (the "female" or recipient cell) while the cells are in physical contact. The recipient that has received DNA from a donor is called a “transconjugant”

7. Conjugation Lederberg & Tatum, 1947 –started with two strains of E. coli that had different nutritional deficiencies caused by mutations in genes that normally synthesize biotin, cysteine, leucine, phenylalanine, thiamine, and threonine “... single nutritional requirements were established as single mutational steps under the influence of X-ray or ultra-violet. By successive treatments, strains with several requirements have been obtained” –strains were both triple auxotrophic mutants, as shown below, where the genes are listed alphabetically:

8. Conjugation Lederberg & Tatum, 1947 –Neither auxotrophic strain grows on minimal medium –Mix 2 strains together, plate to minimal medium Isolates recovered with prototrophic phenotype Strain Y10bio + cys + leu - phe + thi - thr - Strain Y24bio-cys - leu+phe-thi + thr +

9. Conjugation Lederberg & Tatum, 1947 Two strains exchanged genetic information; progeny had characteristics of both parents

10. Conjugation Genetic information shared during conjugation is plasmid DNA –Transfer is replicative –Process similar to rolling-circle replication One single DNA strand remains in donor, one transferred to recipient Both ssDNA are replicated

11. Conjugation Plasmids can be –self-transmissible encode all functions required for conjugation –mobilizable encode some functions required for transfer rely on self-transmissible plasmid for remainder

12. Conjugation Transfer occurs via physical contact –Gram-negative bacteria: sex pilus produced from genes on self-transmissible plasmid tra genes –various types of transfer systems –correlate with Inc group to prevent transfer of plasmids to recipients already carrying plasmids incompatible with donor cell plasmid

13. Conjugation tra genes: 11 or more in this large self- transmissible plasmid –oriT, origin required for conjugation

14. Conjugation Transfer between strains of one species Transfer between different bacterial species –Promiscuous plasmids e.g. IncN, IncP, IncW IncP: self-transmissible and capable of mobilization from E. coli to most other Gram- negative bacteria, and to others (g+, plants) –Evolutionary role: similar genes found in evolutionarily distant organisms –Spread of antibiotic resistance

15. Mechanism of Conjugation Overview I –Contact made between donor and recipient pilus formation –Plasmid is nicked –One DNA strand attached to relaxase enzyme through ester bond transesterification plasmid duplex separated by strand displacement

16. Mechanism of Conjugation Overview II –DNA strand attached to relaxase trans- ferred into recipient cell replication of donor cell strand from 3’-OH of transferred strand –ssDNAs are recircularized –complementary strand synthesized in recipient

17. Mechanism of Conjugation tra genes –DNA transfer and replication (Dtr) component: functions required to prepare plasmid DNA for transfer –mating pair formation (Mpf) component: proteins involved in forming transfer structure Membrane associated Sex pilus

18. Mechanism of Conjugation DNA processing (Dtr) component proteins –Relaxase: endonuclease, introducing specific nick in plasmid Protein bound to 5’-PO 4 –Relaxase is then transferred into recipient cell along with DNA

19. Mechanism of Conjugation Relaxase –In recipient cell, enzyme recircularizes plasmid by breaking protein-DNA bond and ligating 5’-PO 4 with 3’-OH

20. Mechanism of Conjugation Relaxosome –Multi-component complex containing relaxase Accessory proteins (binding) Regulatory proteins (coordinating Dtr and Mpf activities) Helicase (strand separation at oriT)

21. Mechanism of Conjugation Primase –Synthesize RNA primers required for complementary strand synthesis of recipient cell strand –Translated in donor cell, transferred into recipient cell

22. Mechanism of Conjugation Mpf component proteins –Cell-cell attachment –Formation of channel through which transfer takes place –Regulate Dtr activity –Akin to virulence protein secretion systems & natural transformation factors

23. Mechanism of Conjugation Pilus –Composed of pilin protein molecules –Attaches donor to recipient –DNA transfer occurs through channel or pore, not pilus

24. Mechanism of Conjugation Coupling proteins –Sense contact with recipient cell –Switch on Dtr activity (signals relaxase) –Up-regulate what proteins are transferred to recipient

25. Mechanism of Conjugation tra gene regulation –Expressed immediately after transfer to recipient is completed Subsequently repressed, with sporadic periods of expression –prevents exploitation of pilus as bacteriophage infection site Despite low frequency of tra gene expression in individual cells, at population level conjugation leads rapidly to spread of plasmid among potential recipients –Transfer is nearly 100% efficient

26. Mobilizable plasmids Plasmids requiring tra gene functions from self-transmissible plasmids for cell-cell transfer –Minimum requirement for mobilizable plasmid is oriT sequence –In nature, encode Dtr component functions mob genes allow mobilizable plasmids to exploit tra functions without common oriT sequence Dtr system of mobilizable plasmid must be able to respond to coupling protein of coresident self-transmissible plasmid

27. Donor cell has mobilizable and self- transmissible plasmids Coupling protein of self- transmissible plasmids signals cell contact made mob relaxase initiates transfer of mobilizable plasmid DNA strand Mobilized plasmid replicated in recipient cell Self-transmissible plasmid can be transferred too Mobilizable plasmids

28. Chromosomal transfer During conjugation, transfer of chromosomal DNA can sometimes occur –When plasmid has integrated into chromosome –When chromosome contain oriT sequence, e.g. after transposon insertion –When plasmid contains chromosomal DNA Homologous recombination transposition

29. Hfr strains (High frequency recombination) –Plasmid integrates into chromosome, then during conjugation, chromosomal DNA carried along in transfer –Integration recombination –Insertion Element (IS) transposons provide sequence homology transposition Chromosomal transfer

30. Recombinational integration –One IS2 site in plasmid, 20 in E. coli chromosome 20 different Hfr strains possible –Other Insertion Element sites permit integration as well IS3  Chromosomal transfer

31. Hfr strain DNA transfer Expression of tra genes –Cell-cell contact –Relaxase expression Chromosome nicked at oriT Plasmid DNA strand transferred into recipient –with replication in donor

32. Hfr strain DNA transfer Plasmid DNA transfer followed by chromosomal DNA –Entire chromosome transferred in 100 minutes at 37C –Usually only fragment of chromosome transferred –Fragment can be incor- porated into recipient chromosome by recombination otherwise lost: not all of plasmid transferred

33. Hfr and recipient of different genotypes –Transfer of alleles can result in recombinant transconjugant Genes close to oriT transferred at higher frequency than genes further away –gradient of transfer Hfr strain DNA transfer

34. Hfr gene mapping Genetic markers –Alleles, mutations –Transposons –Phenotypic consequences allow position of marker to be mapped in recipient (transconjugant) cell –Select for recombinant with one marker test for other markers

35. Transfer is linear and unidirectional –So order of markers on chromosome with respect to oriT can be determined from frequency of transfer –By selecting for one marker, then scoring frequency of recombinants for other markers, gene positions can be deduced Most matings interrupted before entire chromosome transferred Hfr gene mapping

36. To map rif marker, cross donor with F - at 42 minutes –donor is proC and rif-8 (sensitive to rifampicin) Hfr gene mapping

37. E. coli chromosome genetic map F in this strain

38. Recipient is hisG1, argH5, and trpA3 –and rif (resistant to rifampicin) Donor & recipient allowed to mate Plated for selection –No proline or (histidine, arginine, or tryptophan) Hfr gene mapping

39. Recombinants will be recipients that have received wildtype hisG, argH, or trpA from donor Replica-plate to tester medium for unselected markers –e.g. His + recombinant: plate to –Arg, -Trp, and Rif plates Hfr gene mapping

40. Plot frequency of marker recombinants versus E. coli genetic map position –In this case, relative to hisG Frequency of unmapped marker indicates its location on chromosome Hfr gene mapping

41. Prime Factors Plasmids that contain chromosomal genes –F plasmid with chromosomal genes: F’ –R plasmid with chromosomal genes: R’ Products of recombination or transposition –e.g. from Hfr –Recombinational excision can carry chromosomal fragment with it

42. Complementation testing –Allelism: are 2 mutations in same or different gene? –Dominance/recessivity –Molecular nature of mutation (point, indel…) –Function acting in cis or in trans (cis –Prime Factors create partial diploids (merodiploids) Prime Factors

43. Selection –Early transfer of distal markers –Replicons Prime Factors

44. Selection –Early transfer of distal markers Markers that were far from oriT in chromosome of Hfr will be close in F’, and transferred early Recipient is merodiploid and transconjugant, not recombinant Capable of conjugation Prime Factors

45. Selection –Replicons Capable of replication independent of chromosome If recipient is defective in recombination, prime factor transconjugants can acquire and transmit selected marker but no recombinants from Hfr individuals form Prime Factors