MICROBIAL GENETICS

1. 1 MICROBIAL GENETICS

2. 2 MICROBIAL GENETICS RECOMBINATION Homologous, Site-specific, illegitimate Breaking and Re-joining Parental chromosomes, Recombinant chromosomes TRANSFORMATION Streptococcus pneumoniae: Wild-type, smooth colonies, capsule production, pathogenic Rough mutant, no capsule production, non-pathogenic Transformation of auxotrophic mutants Donor cells, donor DNA, recipient cells, recombinants, transformants Phenotypes, Selection Transformation by plasmid DNA Antibiotic resistance plasmids Penicillin-resistance, penicillinase, beta-lactamase

3. 3 INTRODUCTION TO MICROBIAL GENETICS Genetic information is very stable (in most organisms) because nature has evolved mechanisms to repair damaged DNA and to ensure great fidelity in DNA replication, As a result, mutations occur very randomly. That is very good for each species because they continue to survive and compete successfully in their environment. On the other hand, new kinds of organisms are clearly needed from time to time, for example to take advantage of changing environments, either good or bad changes in the environment. There are two ways that new organisms can arise. Mutation. Some times, mutants are improved organisms and can grow and survive and compete better than the original organism. An example is ability to grow in the presence of some antibiotic. Other times, mutations will create defects and the mutants which suffer those mutations will not be able to survive. Genetic Exchanges. The second way is by combining genes from different organisms to create new organisms. These exchanges move genes or larger fragments of chromosomes from one organism to another and create recombinant organisms with combinations of properties from the original organisms. A. NATURAL EXCHANGES OF CHROMOSOMES OR CHROMOSOME FRAGMENTS IN MICROBES. Genetic exchanges occur in microbes by three natural mechanisms; Transformation, Transduction, and Conjugation. Transformation involves the breakage or lysis of one cell (the donor) with the resulting breakage of the chromosome and release of chromosomal fragments into the environment, the uptake of those chromosomal fragments by another cell (the recipient) and the incorporation of donor DNA fragment into the chromosome of the recipient. Transduction is the movement of chromosomal fragments from one cell (the donor) by growth of some phage on the donor cell, the incorporation of donor DNA into the chromosome of the phage (or packaging of donor DNA into phage heads) generating transducing phages or transducing particles, the subsequent infection of another cell (the recipient) by the transducing phage or particle, injection of donor DNA into the recipient cell, and incorporation of donor DNA into the chromosome of the recipient. Conjugation is the transfer of chromosome fragments (or entire chromosomes, but this is rare) from male cells (donors) into the cytoplasm of female cells (recipients). The transferred fragments are incorporated into the chromosome of the recipients by homologous recombination. B. BACTERIAL PLASMIDSPlasmids are small DNA molecules which are not normally part of the chromosome and which are usually circular and which usually are not essential for the growth of the organism. They often code for only 10 to 50 genes. Entire plasmids can be transferred from cell to cell by all three procedures, transformation, transduction and conjugation. It is not essential that the transferred plasmids be incorporated into the chromosome of the recipient. C. GENETIC RECOMBINATION Breaking and rejoining DNA fragments is sometimes involved in generating chromosome fragments to be transferred and breaking and rejoining is always required to incorporate DNA fragments into the recipient chromosome. This breaking and rejoining is called genetic recombination. Three types of recombination: Homologous recombination- Depends on breaking and rejoining DNA in regions of identical or very similar nucleotide sequence. There is no requirement for any specific nucleotide sequence. That is, any regions of two DNA molecules can be broken and rejoined, so long as the nucleotides in those two molecules have the same or very similar sequences. For example, any region of the entire E.coli chromosome is subject to homologous recombination. Site-specific recombination- Depends on breaking and rejoining two molecules at specific sites, where defined sequences of nucleotides pairs are recognized by an enzyme which catalyzes breaking and rejoining. For example, phage lambda integrates into the E. coli chromosome at one specific, unique site, the prophage attachment site. Illegitimate recombination. Very rarely, breaking and rejoining can occur between two regions of no similarity of nucleotide sequence. This happens by mistake. Although not common, it occurs and in huge populations, as in bacterial and phage populations, it certainly can be found to occur. D. RECOMBINANT DNA TECHNOLOGY. Chromosome fragments (genes) from one organism can be joined to plasmids or virus DNA in the test tube and introduced into another, often unrelated organism to generate recombinant organisms.

4. 4 GENETIC RECOMBINATION • BREAKING AND JOINING DNA • CHROMOSOMES / MOLECULES / FRAGMENTS • HOMOLOGOUS- ANY REGIONS OF DNA AS LONG AS THEIR NUCLEOTIDE SEQUENCES ARE IDENTICAL OR VERY SIMILAR • SITE SPECIFIC- TWO FRAGMENTS ONLY IF THEY CONTAIN SPECIFIC SITES (NUCLEOTIDE SEQUENCES) RECOGNIZED BY SPECIFIC ENZYME • ILLEGITIMATE- FRAGMENTS WITH NO SIMILAR SEQUENCES RARE; ERRORS

5. 5 WAYS TO TRANSFER DNA: TRANSFORMATION TRANSDUCTION CONJUGATION ALL DEPEND ON RECOMBINATION (WITH SOME EXCEPTIONS)

6. HOMOLOGOUS RECOMBINATION (REPAIRS DOUBLE STRAND BREAKS A B a b DAMAGE BRANCH MIGRATION REC BCD HELICASE + EXONUCLEASE ENDONUCLEASE + DNA LIGASE c c SITE REC A STRAND INVASION HOLLIDAY JUNCTION GAP FILLING BLUE = NEW SYNTHESIS

7. HOLLIDAY JUNCTION RESOLUTION A B a b NICK VERTICAL ROTATE RIGHT ENDS LIGATE A b a B RECOMBINANT CHROMOSOMES WILD-TYPE SEQUENCE RESTORED FROM UNDAMAGED CHROMOSOME

8. HOLLIDAY JUNCTION RESOLUTION NICK HORIZIONTAL LIGATE A C B A C B c a b c C a b C c c ASSUME PARENTAL CHROMOSOMES: A C B + a c b DIFFER IN THREE MUTATIONS REPLICATON YIELDS a b C A C B a c b c A B A C B A c B a c b a C b PARENTAL RECOMBINANTS

9. APPRECIATE ! HOLLIDAY JUNCTION (BRANCH MIGRATION)

10. HOMOLOGOUS RECOMBINATION – ILLUSTRATED WITH LINEAR CHROMOSOMES. BACTERIA HAVE CIRCULAR CHROMOSOMES? A SINGLE CROSSOVER EVENT (ILLUSTRATED) WOULD INTEGRATE ONE CHROMOSOME INTO THE OTHER? HOW TO DEAL WITH THIS? SECOND HOLLIDAY JUNCTION FORMS TO LEFT AND ITS RESOLUTION SEPARATES TWO CHROMOSOMES ANOTHER BREAK SOMEWHERE AROUND THE CHROMOSOME IS REPAIRED, ALSO BY HOMOLOGOUS RECOMBINATION, RESULTING IN TWO CROSSOVER EVENTS WHICH SEPARATE TWO RECOMBINANT CHROMOSOMES.

11. 11 5' 3' MUTATION = 3' 5' 3' 5' 5' 5' 3' 5' SINGLE STRAND FRAGMENTS CAN RECOMBINE INTO A CHROMOSOME WILD-TYPE FRAGMENT INVASION NICK AND DISPLACE ONE RECIPIENT STRAND FRAGMENT; LIGASE + DEGRADED REPLICATION [ = NEW SYNTHESIS] MUTANT CHROMOSOME WILD-TYPE CHROMOSOME

12. 12 TRANSFORMATION - LINEAR FRAGMENTS OF CHROMOSOME STREPTOCOCCUS PNEUMONIAE

13. 13 NEGATIVE STAIN CELLS SURROUNDED BY LARGE CAPSULE

14. 14 STREPTOCOCCUS PNEUMONIAE ROUGH MUTANT = MUTATION IN CAPSULE BIOSYNTHESIS GENE DNA FROM WILD-TYPE CELL WILD-TYPE CAPSULE BIOSYNTHESIS GENE HOMOLOGOUS RECOMBINATION

15. 15 [ = NEW SYNTHESIS] FRAGMENT DEGRADED HETERODUPLEX DNA AT THAT POSITION BINARY FISSION WILD-TYPE CELL ROUGH MUTANT WILD-TYPE GENE EXPRESSED

16. 16 STREPTOCOCCUS HEMOPHILUS BACILLUS DONOR DNA: WILD-TYPE LEU+ LEUCINE RECIPIENT CELLS: LEUCINE REQUIRING MUTANT LEU- • EXTRACT DNA FROM DONOR CELLS • MIX DONOR DNA AND RECIPIENT CELLS • RECIPIENT CELLS TAKE UP DONOR DNA • HOMOLOGOUS RECOMBINATION • SELECT WILD-TYPE RECOMBINANTS (TRANSFORMANTS)

17. 17 SELECTING LEUCINE+ TRANSFORMANTS DONOR DNA (LEU+) 2-3 µg GLUCOSE MEDIUM [NO LEUCINE] RECIPIENT CELLS (LEU- MUTANT) ~1 x 108 LEU+TRANSFORMANTS (~102 -103) DONOR DNA AND RECIPIENT CELLS

18. 18 TRANSFORMATION BY PLASMID DNA PLASMID PENICILLINASE GENE FOR PENICILLINASE • BACTERIUM RESISTANT TO PENICILLIN • ISOLATION OF PLASMID DNA • BREAK CELLS • SEPARATE PLASMID DNA FROM CHROMOSOME FRAGMENTS • PREP OF PLASMID DNA

19. TRANSFORMATION BY PLASMID DNA 19 RICH MEDIUM AND PENICILLIN RICH MEDIUM • CONTROL - PLATE • PLASMID DNA • ~1 - 10µg • [PENICILLIN • RESISTANCE] • CONTROL - PLATE • PENICILLIN-SENSITIVE CULTURE • ~108 CELLS • MIX – PLASMID • DNA (1 - 10µg) • AND • PENICILLIN-SENSITIVE CULTURE • [RECIPIENT] PENICILLIN-RESISTANT

20. A DNA HELICASE WITH SS NUCLEASE ACTIVITY (3’ 5’ AND 5’ 3’) GENERATING A SS REGION WITH A 3’ END- REC BCD ENZYME + CHI SITE C B D CHROMOSOME FRAGMENT (RESULT OF IONIZING RADIATION; OTHER DAMAGE) UNWINDS DS ~1000 BP/SEC CLEAVES 3’ END (PREFERENTIALLY) SSB

21. 2. ENCOUNTERS  SITE  = 5’ GCTGGTGG3’ CHANGES REC BCD SPECIFICITY REC BCD 5’- 3’ NUCLEASE HYDROLYZES 5’ END (PREFERENTIALLY)

22. GENERATES A 3’ END WITH  SITE LOADS REC A PROTEIN WHICH COATS SS AND CATALYZES HOMOLOGOUS PAIRING AND STRAND EXCHANGE  REC A PROTEIN

23. 14 TRANSDUCTION - GENERALIZED A GROW PI ON DONOR PHAGE P1 ~108 HOST E. COLI ~108/ML WILD-TYPE LEUCINE+ [DONOR] ADSORPTION PENETRATION SYNTHESIS TAILS P1 DNA HEADS LEU+ ASSEMBLY LEU+

24. 14 TRANSDUCTION - GENERALIZED A GROW PI ON DONOR PHAGE P1 ~108 HOST E. COLI ~108/ML WILD-TYPE LEUCINE+ [DONOR] ADSORPTION PENETRATION SYNTHESIS TAILS P1 DNA HEADS LEU+ ASSEMBLY LEU+

25. 15 LYSIS PRODUCES P1 PHAGE PREP LEUCINE GENES OF HOST (DONOR) PROGENY P1 PHAGES ~1010/ ML TRANSDUCING PARTICLE ~107/ ML

26. 16 INFECT RECIPIENT WITH P1 (GROWN ON WILD-TYPE LEU +DONOR) RECIPIENT IS LEUCINE-REQUIRING MUTANT P1 PHAGE PREP CONTAINS P1 AND TRANSDUCING PARTICLES P1 + RECIPIENT LYTIC GROWTH TRANSDUCING PARTICLE + RECIPIENT TRANSDUCTION WILD-TYPE LEUCINE GENE LEUCINE REQUIRING MUTANT [RECIPIENT] = MUTATION IN LEUCINE GENE

27. 17 HOMOLOGOUS RECOMBINATION NEAR LEFT AND RIGHT ENDS FRAGMENT DEGRADED WILD-TYPE LEUCINE+ TRANSDUCTANT- BINARY FISSION PRODUCES ONLY LEU+ PROGENY

28. 18 • B. SPECIALIZED TRANSDUCTION (E.G., LAMBDA PHAGE) • – ONLY HOST GENES NEAR PROPHAGE CAN BE MOVED • PHAGE LYSOGENIZES DONOR, FORMS LYSOGEN • INDUCTION WITH PRODUCTION OF PROGENY PHAGES AND • SOME PHAGES PICK UP HOST (DONOR) DNA; BECOME • TRANSDUCING PHAGES • PHAGE PREP USED TO INFECT SECOND HOST (RECIPIENT) • INFECTED CELLS – SOME WILL • 1. PRODUCE PROGENY PHAGES • 2. BECOME LAMBDA LYSOGENS • 3. BE INFECTED BY TRANSDUCING PHAGES • AND WILL RECOMBINE THE INCOMING • DNA INTO THE RECIPIENT CHROMOSOME • THOSE CELLS ARE PARTIAL DIPLOIDS -

29. 19 SPECIALIZED TRANSDUCTION LYSOGEN FORMATION MIX l PREP AND GAL+ HOST [WILD-TYPE] LYSIS - SOME CELLS LYSOGENY - SOME CELLS [ISOLATE LYSOGENS FOR NEXT STEP] GAL = GALACTOSE LYSOGEN = E. COLI (l +) - STRUCTURE

30. 20 PROPHAGE INDUCTION DNA DAMAGE REPRESSOR CLEAVAGE LYTIC GENES NO LONGER INHIBITED EXCISION, LYTIC GROWTH PROGENY PHAGES, LYSIS EXCISION REPRESSOR FRAGMENT EXCISIONASE REPLICATED

31. 21 2. INDUCTION OF LYSOGEN l PREP l+AND lGAL+ TRANSDUCING PARTICLES l+~1010 lGAL+~106 GAL+E. COLI (l+) ~108/ml INDUCTION INDUCTION - PROPHAGE LOOPS OUT

32. 22 ILLEGITIMATE RECOMBINATION =lGAL+ HOST CHROMOSOME DELETED FOR GAL = PACKAGING lGAL+ INTO l HEAD CUTTING COS SITE TO LINEARIZE =lGAL+ HEAD PACKAGES NOTE: LYSIS OF INDUCED CULTURE = l +lGAL+ =lGAL+