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Isolation and Characterization of Recombination-Deficient Mutants of Escherichia coli K-12

Isolation and Characterization of Recombination-Deficient Mutants of Escherichia coli K-12. A.J. Clark and A.D. Margulies PNAS 1965. What was known in 1965?. Lederberg and Tatum described conjugation (1942)

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Isolation and Characterization of Recombination-Deficient Mutants of Escherichia coli K-12

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  1. Isolation and Characterization of Recombination-Deficient Mutants of Escherichia coli K-12 A.J. Clark and A.D. Margulies PNAS 1965

  2. What was known in 1965? Lederberg and Tatum described conjugation (1942) 1. Recombination in bacteria and viruses involves the physical interaction of dsDNA from two parents

  3. + What was known about recombination? 2. Unreplicated DNA may contain two strands of DNA derived from two different parents 3. May involve the removal and synthesis of DNA -30nt

  4. Howard-Flanders model for recombination Breakage of parental DNA Degradation of ssDNA Polymerization/ DNA synthesis Restoration of dsDNA

  5. Important points about the Howard-Flanders Model • Prediction: ~A mutation in a common enzyme could affect both recombination and DNA repair • These mutants would posses two phenotypes: 1. Inability to form recombinants by conjugation ~Appear infertile on crosses with Hfr strains 2. Inability to repair photodamaged DNA ~Appear very sensitive to the lethal effects UV

  6. Research objective: • To isolate E. coli mutants that were unable to carry out recombination If their hypothesis regarding a common enzyme was correct, then mutants defective in recombination would also be UV-sensitive!

  7. Experimental Procedure: O/N culture of JC-411 (Leu- Ade+ F-)* Incubated for 1hr at 37 in 1-methyl-3-nitro-1-nitroguanidine (MNNG) buffer (0.1% survival) Mutagen that alkylates DNA *Mutant for synthesis of Leu, wild type for synthesis of Ade and lacks the F-plasmid

  8. What does MNNG do? • Covalently links an alkyl group at position 6 of guanines Guanine O6-methylguanine Pre-mutagenic lesion

  9. How does O6MG affect base pairing? O6MG causes transition mutations A transition mutation is the replacement of a base by another base of the same chemical category ~e.g Pur  Pur (G A) A transversion mutation is the replacement of a base of one chemical category by a base of the other category ~e.g PurPyr (G T)

  10. Experimental Procedure: O/N culture of JC-411 (Leu- Ade+ F-)* Incubated for 1hr at 37 in 1-methyl-3-nitro-1-nitroguanidine buffer (0.1% survival) Mutation Diluted and plated on minimal glucose media Screened for ability to undergo recombination Mutants *Mutant for Leu, wild type for Ade and lacks the F-plasmid

  11. Can they undergo recombination?? Replica plated onto MM covered with a lawn of JC-182 (Leu+ Ade- Hfr) Surviving mutated JC-411 (Leu- Ade+ F-) Screened for colonies that were unable to undergo recombination Able to under go recombination: ~ Leu+ Ade+ colonies (growth on MM) ~ fertile when crossed with Hfr Unable to under go recombination: ~ no colonies (no growth on MM) ~ infertile when crossed with Hfr

  12. Are the mutants truly unable to under go recombination (infertile)? Streaked onto nutrient medium (Purification) Mutated JC-411 Re-streaked onto nutrient medium Tested several colonies for ability to recombine with JC-182 Re-checking phenotype (Hfr strain) None were able too!! (True infertiles)

  13. Results • Screened approximately 2000 survivors: • Isolated two strains that were unable to perform recombination • JC-1553 • JC-1554

  14. Characterizing JC-1553 and JC-1554 • Examined mutants under a microscope • Ensuring that morphology of daughters was similar to mother • Wanted to establish their relationship to JC-411 • Inability to mate with Hfr typical of other bacteria within same genus (is this just contamination?) • Mutants were phenotypically similar to JC-411 in regard to growth factor requirements, inability to ferment sugar and their response to streptomycin and certain phages (contamination not likely then) Why would these mutants appear infertile?

  15. Four possible explanations for infertility with JC-182 • Contact between the mutants (JC-1553 and JC-1554) and Hfr cells (JC-182) did not occur • There was cell contact but chromosomal DNA could not be transferred • Genetic material wasdestroyedas it entered JC-1553 or 1554 • JC-1553 and 1554 were unable to catalyze recombination between chromosomal DNA and DNA taken up into the cell.

  16. What was causing infertility? • Experiment 1 • used a F-plasmid to address if: a) there was proper contact between the bacteria b) genetic material was being transferred c) genetic material was being destroyed • used a chromosomal marker (Leu+) to determine if: a) if recombination was occurring

  17. Experiment 1: Rule out lack of contact between cells Strains used: JC-411 JC-1020 X Looked for: 1) Lac+ SmR merodiploids 2) Leu+ SmR recombinants JC-1553 JC-1020 X JC-1020 JC-1554 X

  18. Results of experiment 1: The number of Lac+ SmR merodiploids similar for JC-411, JC-1553, and JC-1554. The kinetics of formation (how long it took JC-411, JC-1553 and JC-1554 to take up F-lac) were similar.

  19. Conclusions of experiment 1: Facts indicate that JC-1553 and JC-1554: 1) Form effective contacts 2) Genetic material was being transferred 3) Circular genetic material is not destroyed.

  20. Results for Experiment 1: The number of recombinants produced by JC-1553 and -1554 as compared to JC-411 is fewer than 1/1000 indicating: 1) either chromosomal transfer to the mutants is prevented OR 2) the mutants suffer from an inability to catalyze recombination

  21. What is the effect of mutations carried by JC-1553 and 1554? The authors took advantage of known phage phenotypes. Requires the ability to replicate but not recombination (DNA needs to be transferred and not exchanged) Requires recombination (integration into host chromosome)

  22. Experiment 2: Determine what is happening with recombination • Performed a cross with JC-1164 and either JC-1166 or JC-1167 ~JC-1164 ~ lambda lysogenic donor ~JC-1166 ~ T6R mutant of JC-411 ~JC-1167 ~ T6R mutant of JC-1553 Parental derivative Derivative of infertile mutant strain

  23. Experimental Procedure Derived from JC-411 T6R JC-1164 Leu + JC-1166 X Looked for: 1) Infective centers (clear plaques) 2) Leu+ SmR T6R recombinants JC-1164 Leu + JC-1167 X Derived from JC-1553 T6R

  24. What the results would mean: • Appearance of infective centers (plaques) indicates: • DNA is transferred and not exchanged (no phage integration) • Lysis occurred • Appearance of Leu+ SmR T6R recombinants indicates: • Recombination took place • Bad. Why? Because we’re looking for mutants defective in recombination! • Phage entered lysogenic lifecycle

  25. Experiment 2 results: JC-1166 (JC-411 derivative) produced recombinants and infective centers when crossed with a lysogenic donor. JC-1167(JC-1553 derivative) did not produce recombinants, however there were plaques observed. Replication but NOT recombination was occurring.

  26. Experiment 2 Conclusions: • Derivatives of JC-1553 could transfer linear DNA but not recombine it. • Derivatives of JC-1553 are unable to catalyze recombination between the bacterial chromosome and the exogenous DNA. • Similar results were seen with JC-1554 and derivatives.

  27. Next question • Having obtained mutants in which recombination was blocked: • the next question was to ask if JC-1553 and JC-1554 are able to repair damage to DNA • DNA damage was a process that was known to require recombination

  28. Experiment 3: Inducing DNA damage JC-411 JC-1553 Irradiated with UV for different periods of time Survivors were determined by plating onto complex medium JC-1554

  29. Experiment 3 Results After 10 seconds of UV exposure: 40% of JC-411 cells had survived 0.003% of JC-1553 and JC-1554 have survived

  30. Experiment 3 Conclusions: JC-1553 and 1554 are more sensitive to UV light than JC-411. JC-1553 and 1554 differ from JC-411 in two characteristics 1) recombination-deficient 2) UV- sensitive

  31. Now What??? • Need to determine if the two characteristics are due to one or two mutations • Two different methods could be used: • Back-mutants selected for reversion to one of the parental phenotypic characteristics • Conjugational or transductional recombinants formed by crossing a wild-type donor with the mutant strains Which one of these methods is not feasible? Why?

  32. Reversion of JC-1553 and 1554 • Determines the number of mutations present in JC-1554 and JC-1553 because reversions are assumed to be due to a single point mutation • Two independent revertants were obtained from JC-1553 surviving UV irradiation from experiment 3 • Independent experiments obtained a UVRrevertant for both JC-1553 and JC-1554

  33. Characterization of revertants: All four revertants have regained either fully or partially the wild-type ability to form conjugational recombinants, as well as resistanceto UV light Indicating that the there was one mutation in each of JC-1553 and JC-1554 that made the cells deficient in recombination and DNA repair.

  34. Conclusions • Isolated two recombination-deficient (Rec-) mutants, JC-1553 and JC-1554 • Both mutants were able to form F-lac merodiploids and infective centers (plaques) • rules out the possibility of mutant infertility • rules out the idea of destruction of exogenous DNA • mutants were unable to catalyze recombination • Mutants were sensitive to UV light • Revertants regained UVR and conjugational recombination • All these observations support the idea that one gene encodes both UVR and Rec functions

  35. Conclusions Other hypotheses that might support the facts presented: • A single suppressor could cause a phenotypic reversion of two independent mutations (intergenic or intragenic?) 2. A mutant protein may modifyDNA such that neither recombination nor repair of DNA damage may occur 3. A polar mutation may affect the expression of more than one gene • mutation affecting a regulatory gene • i.e. Insertion of a transposon

  36. RecA LexA ssDNA RecA LexA DNA damage proteins Significance • The authors were the first to discover a recombination enzyme and DNA repair enzyme, RecA Recombination DNA repair

  37. Medical Significance • RecA is a homologue to RAD51 in humans • RAD51 has been implicated in certain cancers • Recombination repair is an important aspect in cancer research

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