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MICROBIAL GENETICS

MICROBIAL GENETICS. GENETIC EXCHANGES IN BACTERIA MECHANISMS OF GENE TRANSFER IN BACTERIA. BACTERIA AS GENETIC MODELS. GENETIC RECOMBINATION IN BACTERIA AND GENETIC DIVERSITY

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MICROBIAL GENETICS

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  1. MICROBIAL GENETICS GENETIC EXCHANGES IN BACTERIA MECHANISMS OF GENE TRANSFER IN BACTERIA

  2. BACTERIA AS GENETIC MODELS • GENETIC RECOMBINATION IN BACTERIA AND GENETIC DIVERSITY • Mechanisms of gene transfer occur separately from bacterial reproduction, and in addition to mutation, are another major source of genetic variation in bacterial populations • Vertical Gene Transfer-Occurs when genes are passed from an organism to its offspring (common in plants and animals); bacteria can pass their genes to their offspring • Horizontal Gene Transfer-genes are passed laterally to other microbes of the same generation (not a frequent event ≈ less than 1 % of an entire population will be recombinants)

  3. MECHANISMS OF GENE TRANSFER IN BACTERIA • TRANSFORMATION • Process of gene transfer where bacterial cells assimilate foreign DNA from surroundings • Historical Beginnings-Frederick Griffith - 1928 • Streptococcus pneumoniae

  4. BACTERIAL PLASMIDS PLASMIDS PROVIDE Novel enzymatic (metabolic) activities-dissimilation plasmids Utilization as cloning vehicles in biotechnology

  5. Griffith ExperimentThere Are Two Strains of Streptococcus pneumoniae Smooth colonies (S) Rough colonies (R) S strain is virulent R strain is benign

  6. TRANSFORMATION Griffith Experiment demonstrated Transformation Tortora Ch. 8 Transformation

  7. MECHANISMS OF GENE TRANSFER IN BACTERIA • TRANSFORMATION • Process documented in other genera of bacteria • Staphylococcus • Bacillus • Haemophilus • Neisseria • Acinetobacter • DNA UPTAKE • Competence • Donor/Recipient

  8. TRANSFORMATION

  9. MECHANISMS OF GENE TRANSFER IN BACTERIA TRANSFORMATION • PLASMID DNA TRANSFER BY TRANSFORMATION in vitro • Though lacking such proteins, E. coli can be artificially induced to take up foreign DNA by incubating the bacteria in a culture medium that has a high concentration of calcium ions (competence induced by CaCl2) • This technique of artificially inducing transformation is used by the biotechnology industry to introduce foreign genes (plasmid DNA) into bacterial genomes, so that bacterial cells can produce proteins characteristic of other species (e.g., human insulin and human growth hormone) • Genetic Mapping w/ Transformation • Co-transfer (Co-transformation frequency) • Selection Animation: Ch. 8 Transformation

  10. MECHANISMS OF GENE TRANSFER IN BACTERIA • CONJUGATION • Direct gene transfer between two cells that are temporarily joined (physical contact required) • Discovered by Joshua Lederberg and Edward Tatum • Best-studied examples - conjugation in E. coli • A DNA-donating E. coli cell (male cell) extends external appendages or sex pili • Sex piliattach to a DNA-receiving cell (female cell) and forms a cytoplasmic bridge between the two cells through which DNA transfer occurs • The ability to form sex pili and to transfer DNA is conferred by genes in a plasmid called the F (fertility) plasmid

  11. PHYSICAL CONTACT REQUIRED FOR CONJUGATION

  12. MECHANISMS OF GENETRANSFER: CONJUGATION • F PLASMID & CONJUGATION • F plasmid has ≈ 25 genes, most of which are involved in the production of sex pili • Bacterial cells that contain the F factor can donate DNA ("male") are called F+ cells • The F factor replicates in synchrony with chromosomal DNA, so the F+ factor is heritable; that is, division of an F+ cell results in two F+ daughter cells

  13. MECHANISMS OF GENETRANSFER: CONJUGATION

  14. MECHANISMS OF GENETRANSFER: CONJUGATION

  15. F PLASMID & CONJUGATION CONJUGATIONhttp://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter13/animation_quiz_3.html CONJUGATIONconjugation_f.swf • Cells w/o F factor are designated F– ("female") • During conjugation between an F+ and an F– bacterium: • The F factor replicates by rolling circle replication. The 5' end of the copy peels off the circular plasmid and is transferred in linear form • The F+ cell transfers a copy of its F factor to the F– partner, and the F– cell becomes F Animation: Ch. 8 Conjugation-f Tortora

  16. MECHANISMS OF GENE TRANSFER: CONJUGATION

  17. THE Hfr CELL Animation Tortora Ch. 8 Conjugation HFR

  18. MECHANISMS OF GENE TRANSFER: CONJUGATION • F PLASMID AND CONJUGATION • The donor cell remains F+, with its original DNA intact • The F factor is an episome and occasionally inserts into the bacterial chromosome • Integrated F factor genes are still expressed • Cells with integrated F factors are called Hfr cells (high frequency of recombination). Conjugation can still occur between an Hfr and an F- bacterium • As the integrated F factor of the Hfr cell transfers to the F– cell, it pulls the bacterial chromosome behind its leading end • The F factor always opens up at the same point for a particular Hfr strain. As rolling circle replication proceeds, the sequence of chromosomal genes behind the 5’ end is always the same

  19. TRANSFER OF CHROMOSOMAL GENES BY AN Hfr STRAIN

  20. MECHANISMS OF GENE TRANSFER: CONJUGATION CONJUGATIONconjugation_hfr.swf • F PLASMID AND CONJUGATION • The conjugation bridge usually breaks before the entire chromosome and tail end of the F factor can be transferred • Result: Only some bacterial genes are donated • The recipient F– cell does not become an F+ cell, because only part of the F factor is transferred • The recipient cell becomes a partial diploid or merodiploid • Recombination occurs between the Hfr chromosomal fragment and the F– cell. Homologous strand exchange results in recombinant F– cell • Asexual reproduction of the recombinant F– cell produces a bacterial colony that is genetically different from both original parental cells

  21. MECHANISMS OF GENE TRANSFER: CONJUGATIONR PLASMIDS • ANTIBIOTIC RESISTANCE • The R plasmids (for resistance), carry genes that confer resistance to certain antibiotics • Some carry up to ten genes for antibiotic-resistance • During conjugation, some mobilize their own transfer to nonresistant cells

  22. R PLASMIDS

  23. MECHANISMS OF GENE TRANSFER: CONJUGATIONR PLASMIDS • ANTIBIOTIC RESISTANCE • Increased antibiotic use has selected for antibiotic resistant bacterial strains carrying the R plasmid • R plasmids can transfer resistance genes to bacteria of different species including patho-genic strains • Result: Resistant strains of pathogens are now more common

  24. SUMMARY OF GENE TRANSACTIONS MEDIATED VIA CONJUGATION

  25. Hfr is sensitive to streptomycin. This convenient as we could eliminate the donor and select for only the Strr F- recombinant cells as the selective medium contains streptomycin

  26. SELECTION REPLICA PLATING

  27. PROTOTROPHS/AUXOTROPHS

  28. MECHANISMS OF GENE TRANSFER • TRANSDUCTION • Gene transfer from one bacterium to another via bacteriophage • Generalized transduction • Occurs when random pieces of host cell DNA are packaged within a phage capsid during the lytic cycle • This process can transfer almost any host gene and little or no phage genes • When the phage particle infects new host cell, the donor cell DNA can recombine with the recipient cell DNA • Typical of bacteriophages P1 in E. coli and P22 in Salmonella

  29. MECHANISMS OF GENE TRANSFER: TRANSDUCTION BACTERIOPHAGE LIFE CYCLEhttp://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter17/animation_quiz_1.html • PRODUCTION OF A GENERALIZED TRANSDUCING PHAGE

  30. GENERALIZED TRANSDUCTION GENERALIZED TRANSDUCTIONhttp://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter13/animation_quiz_2.html Animation Tortora Ch. 8 transduction_generalized.swf

  31. MECHANISMS OF GENE TRANSFER: TRANSDUCTION PHAGE LAMBDA REPLICATION CYCLEhttp://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter17/animation_quiz_2.html SPECIALIZED TRANSDUCTIONhttp://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter17/animation_quiz_3.html SPECIALIZED TRANSDUCTIONtransduction_specialized.swf • SPECIALIZED TRANSDUCTION • Occurs when a prophage excises from bacterial chromosome and carries w/ it only certain host genes adjacent to the excision site • Also known as restricted transduction • Carried out only by temperate phages (Lambda) • Specialized transduction differs from general transduction in that: • Specific host genes and most phage genes are packed into the same virion • Transduced bacterial genes are restricted to specific genes adjacent to prophage insertion site • In generalized transduction, host genes are randomly selected and almost any host gene can be transferred

  32. MECHANISMS OF GENE TRANSFER SPECIALIZED TRANSDUCTION • INSERTION SITE • Site-specific recombination event between bacterial gal & bio genes • Excision event results in phage particles carrying either the gal or bio genes

  33. THE INSTABILITY OF THE GENOME • MECHANISMS OF GENE EXCHANGE TRANSPOSITION (Animation Ch. 8 Transposons) • Transposons • Or transposable genetic elements (mobile genetic elements), can move from one location to another in a cell's genome • Occur as natural agents of genetic change in both prokaryotic and eukaryotic organisms • First proposed in the 1940s by Barbara McClintock, who deduced their existence in maize • Decades later, the importance of her discovery was recognized as the process was described in bacteria • In 1983, at the age of 81, she received the Nobel Prize for her work

  34. BARBARA MCCLINTOCK

  35. TRANSPOSITION

  36. MECHANISMS OF GENE TRANSFER TRANSPOSITIONhttp://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter13/animation_quiz_5.html • TRANSPOSITION • Two patterns of transposition: • Conservative transposition • Movement of preexisting genes from one genomic location to another; the transposon's genes are not replicated before the move, so the number of gene copies is conserved • Replicative transposition • Movement of gene copies from their original site of replication to another location in the genome, so the transposon's genes are inserted at some new site without being lost from the original site

  37. MECHANISMS OF GENE TRASFER • TRANSPOSITION • Transposition is different from all other mechanisms of genetic recombination, b/c transposons may scatter certain genes throughout the genome with no apparent single, specific target • All other mechanisms of genetic recombination depend upon homologous strand exchange (meiotic crossing over in eukaryotes; and transformation, transduction, and conjugation in prokaryotes) • Insertion of episomic plasmids into chromosomes is also site specific, even when it does not require an extensive stretch of DNA homologous to the plasmid

  38. TRANSPOSITION TRANSPOSITION transposons_insertion.swf transposons complex • INSERTION SEQUENCES • Simplest transposons • Contain only the genes necessary for the process of transposition • Include two essential types of nucleotide sequences: • Transposase gene (enzyme that catalyzes transposition event or insertion of transposons into new chromosomal sites) • Binds to inverted repeats & hold them close together • Cuts and reseals DNA required for insertion

  39. MECHANISMS OF GENE TRANSFER • TRANSPOSITION • Inverted repeats (IRs) • Short noncoding nucleotide sequences of DNA that are repeated in reverse order on opposite ends of a transposon • Note that each base sequence (IR) is repeated in reverse at each end of the IS • Inverted repeats: • Contain only 20 to 40 nucleotide pairs • Are recognition sites for transposase • Insertion of transposons also requires other enzymes, such as DNA polymerase

  40. INVERTED REPEATS

  41. MECHANISMS OF GENETRANSFERMECHANISM OFTRANSPOSITION • TRANSPOSITION • At the target site, transposase makes staggered cuts in the two DNA strands, leaving short segments of unpaired DNA at each end of the cut • Transposase inserts the transposon into the open target site • DNA polymerase helps form direct repeats, which flank trans-posons in their target site

  42. MECHANISMS OF GENETRANSFERMECHANISMS OFTRANSPOSITION • TRANSPOSITION • Gaps in the two DNA strands fill in when nucleotides base pair with the exposed single-stranded regions • Direct repeats form as Two or more identical DNA sequences in the same molecule • The transposition process creates direct repeats that flank transposons in their target site

  43. MECHANISMS OFGENE TRANSFER TRANSPOSITION • TRANSPOSITION • Transposed insertion sequences are likely to alter cell's phenotype

  44. TRANSPOSON MUTAGENESIS

  45. MECHANISMS OF GENE TRANSFER • TRANSPOSITION • Though insertion sequences only rarely cause mutations (about 1 in every 106 generations), the mutation rate from transpositions is about the same as the mutation rate from other extrinsic causes, such as radiation and chemical mutagens • Composite (Complex) Transposons • Transposons which include additional genetic material besides that required for transposition; consist of one or more genes flanked by insertion sequences • The additional DNA may have any nucleotide sequence • Can insert into almost any stretch of DNA since insertion is not dependent upon DNA sequence homology Animation: Ch. 8 transposons complex

  46. MECHANISMS OF GENE TRANSFER TRANSPOSITIONtransposons_complex.swf • COMPOSITE (COMPLEX) TRANSPOSONS • Generate genetic diversity in bacteria by moving genes from one chromosome, or even one species, to another which may help bacteria adapt to new environments • An example is transposon that carries a bacterial gene for antibiotic resistance • Examples of genetic elements that contain one or more complex transposons include: F factor; DNA version of the retrovirus genome

  47. COMPOSITE (COMPLEX) TRANSPOSONS

  48. Effects of Transposon Activity Play significant role in bacterial evolution as a source of genetic variation: • Cause mutations by interrupting coding sequences for proteins • Increase or decrease a protein's production by inserting within regulatory regions that control transcription rates (promoters/operators) • Inactivate stop codons and terminator sequences • Transfer antibiotic resistance . • Influence transcription or translation (on/off) • Promoters activate • Stop codons and terminator sequences inactivate • Useful tool in gene purification and function studies

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