Microbial Genetics BI 302 Lecture Notes PDF

# Lecture 5: Genetic Exchange ## Microbial Genetics ### BI 302 **Instructor:** Dr. Nalina Nadarajah **Email:** [email protected] ## Agenda for This Week - An introduction to genetic exchange between bacteria - Transduction - Transposition ## Transduction - Transduction...

# Lecture 5: Genetic Exchange ## Microbial Genetics ### BI 302 **Instructor:** Dr. Nalina Nadarajah **Email:** [email protected] ## Agenda for This Week - An introduction to genetic exchange between bacteria - Transduction - Transposition ## Transduction - Transduction occurs when bacteriophages carry DNA from one bacterium to another. - Most bacteriophages have a limited host range: transduction between the same or closely related species only. ### Glossary - **Transducing phage**: a phage that contains a piece of bacterial chromosome inside its capsid - **Transductant**: a bacterial cell that has received genes from another bacterium via transduction - **Transduction**: type of genetic exchange when a virus carries genes from one bacterium to another - **Co-transduction**: 2 or more genes are transferred together from one bacterium to another (only genes located close together). ## Bacteriophage Life Cycle - Lytic Cycle 1. Attachment of T4 to receptors on *E. coli* cell wall 2. Penetration of the cell wall by tail core; inject DNA into host 3. *E. coli* DNA is hydrolyzed. Phage DNA directs biosynthesis of viral parts using the host cell’s machinery 4. The phages mature as the parts are assembled 5. Lysis of *E. coli* and release of the new phages ## Lytic Life Cycle 1. Binding 2. Injection of nucleic acid 3. Reprogramming 4. Production of phage particles 5. Assembly 6. Lysin, lysis and escape ## Requirement for Transduction - The phage degrades the bacterial chromosome - The process of packaging DNA into phage capsid is not highly specific for phage DNA only - The bacterial genes transferred by the phage recombines with the chromosome in the recipient cell ## Generalized Transduction - Discovered by Lederberg & Zinder in 1952 - Mixed 2 strains of *Salmonella typhimurium*: *phe+ trp+ tyrt met his* x *phe trp-tyr met⁺ his+*. - Few prototrophic mutants appeared on minimal media. ### Due to conjugation? ### Does genetic exchange between bacteria always require cell-to-cell contact? ### Conclusion: Genetic exchange did not take place via conjugation; bacteriophage was found to be the agent of transfer ## Steps of Generalized Transduction - Virtually any gene can be transferred (from lytic cycle) - A phage attaches to cell wall of bacterium and injects DNA. - The bacterial chromosome is broken down and biosynthesis of phage DNA and protein occurs. - Host DNA accidentally packaged into viral capsid – defective - Lysate from infected cell contains a mixture of normal phage and transducing (defective) phage particles. - When lysate infects new recipient cells, most are infected with normal infective phage but a small number receive the transducing particles which inject the DNA from previous host. - Injected DNA can't replicate (defective phage) but can undergo genetic recombination with the DNA of the new host. ## Generalized Transduction by Lytic Phage 1. Host chromosome is disrupted: E. *coli* bacterial chromosome 2. lac operon is incorporated into phage 3. Host cell lyses 4. Phage infects a lac cell 5. Functional lac operon can be incorporated ## Consequences of Generalized Transduction - Any bacterial gene can be transferred - host’s chromosome broken down into fragments. - Whatever piece of bacterial DNA get packaged within the phage is the genetic material that will be transferred. - the probability of a transducing phage containing a particular gene is quite low: 1 cell in 106 – 108 transduced for a specific gene - Phages that form transducing phages can be either virulent or temperate – need lytic cycle → package host DNA into capsid - Generalized transduction can result in the transfer of 50-100 new genes and make dramatic changes to the properties of the bacteria. - Transduction plays an important role in the transfer of antibiotic resistance and pathogenicity factors. ## Gene Mapping in Phages - Due to the limited size of phage particle, only about 1% of bacterial chromosome can be transduced. - The overall rate of transduction ranges from 1 in 100,000 to 1,000,000. - Since the chance of a cell being transduced by 2 separate phages is extremely small, any cotransduced genes are usually located close together - Rate of cotransduction can indicate the physical distance between genes. ## Gene Mapping using Phages - Genes located closed to one another are more likely to be cotranduced. ## Steps in a Cotransduction Expt. - Infection, production of new phages, and lysis - An occasional phage will contain a piece of the bacterial chromosome - Occasionally, a recipient met and/or arg from a P1 phage ## Phage Cross - Hershey and Rotman's cross – 1949 - Examined the rate of recombination in T2 bacteriophage. - T2 single stranded DNA phage - 2 strains that differed in plaque appearance and host range - one strain could infect and lyse type B E.coli but not B/2 cells. - wild type, normal host range h+ - only one host → turbid plaques when plated with both hosts. - abnormal plaques – large plaques due to rapid lysis (r¯) - the other strain can infect and lyse both B and B/2 cells - mutant host range, h (clear plaques) - wild type plaques – small plaques (r+) ## How can we determine the position of a gene on a phage chromosome - An *E. coli* was infected with 2 different phages: **h+r**, **h-r+** ## Hershey and Rotman’s Cross - Non-recombinant phage produces cloudy, large plaques - Recombinant phage produces cloudy, small plaques - Some don’t cross over, resulting in nonrecombinant progeny. ## Results of a cross for the h and r genes in phage T2 (*hr+ × h+r*) | Genotype | Plaques | Designation | |---|---|---| | *hr+* | 42 | | | *h+r+* | 34 | Parental progeny 76% | | *h+ r-* | 12 | | | *hr* | 12 | Recombinant 24% | *** RF = (recombinant plaques (***h+r+***) + (***h® r¯***) / total plaques ## Conclusion: The recombination frequency indicates that the distance between h and r genes is 24% ## Practice Problems 1 - Two mutations that affect plaque morphology has been identified (a, b). Phages carrying both mutations (a¯b¯) were mixed with wild type (a+b+) and added to a host bacterial culture. Subsequent to infection & lysis following plaques were observed: - a+b+ = 101 - a-b+ = 58 - a+b¯ = 42 - a-b¯ = 99 - Total Plaques = 300 - Calculate RF? ## Practice Problem 2 - A donor strain of bacteria with genes a+ b+ c+ is infected with phages to map the donor chromosome by generalized transduction. The phage lysate from the bacterial cells is collected and used to infect a second strain of bacteria that are a¯ b¯ c¯. Bacteria with the a⁺ gene are selected and the percentage of cells with cotransduced b+ and c⁺ genes are recorded: - Donor: a+b+c+ - Recipient: a-b-c - Selected gene: a+ 25% - Selected gene: b+ 3% - Selected gene: c+ 3% - Which gene is closer to the gene "a"? ## Transposition - The movement of a transposable element (segments of DNA) from one location to another. - Different from homologous recombination - Consequences: mutations by inserting into genes, increase or decrease gene expression ## Barbara McClintock and Transposable Genetic Elements - Research involved understanding the cytogenetics of corn. - Discovered in 1948 (published in 1953), that the chromosome-breaking locus moved from one chromosomal location to another. - she called it transposition. - Encountered a lot of skepticism and hostility. - Received Nobel Prize in 1983 - 35 years after discovery ## Mechanism of Transposition - Staggered breaks are made in the target DNA. - The transposable element is joined to ss ends of the target DNA. - DNA is replicated at the single stranded gaps. ## Insertion of Transposable Element into DNA - Staggered cuts are made in the target DNA. - A transposable element inserts itself into the DNA. - Gaps filled in by DNA polymerase. - The staggered cuts leave short, ss DNA. - Replication of this ss DNA creates the flanking direct repeats. ## Flanking Direct Repeats - Short, flanking direct repeats of 3-12 bp present on both sides of most transposable elements. - Not part of a transposable element. - do not travel with the transposable element. - Generated in the process of transposition at the point of insertion. - The sequence vary, but the length is constant for each type of transposable element. - Staggered cuts creates flanking direct repeats. ## Types of Transposable Genetic Elements - They vary from simple (insertion sequences) to complex transposons. - Insertion sequences (IS) - Transposons - composite transposons - non-composite transposons ## Insertion sequences (IS) - IS are the simplest transposable genetic elements that carry no known genes except those that are required for transposition. - gene for transposase enzyme - makes staggered cuts in DNA into which IS can insert - Nomenclature – IS1 - Range in size from 750 bp to 2000 bp - IS are small stretches of DNA that have at their ends inverted repeat (IR) sequences, which are involved in transposition: sometimes called inverted terminal repeats (ITRs) ## Terminal Inverted Repeats (IR) - Found at the end - Act as recognition sites for the binding of the transposase enzyme - 9 to 40 bp long - Both inverted and complementary. - Required for transposition to take place. ## Importance of IS - Mutation: the introduction of an IS into a bacterial gene will result in the inactivation of the gene - The sites at which plasmids insert into the bacterial chromosome are at IS in the chromosome. ## Transposons - Transposons are mobile DNA sequences (“jumping genes") found in the genome of all organisms. - found in the main chromosomes of organisms, in plasmids, and in the genetic material of viruses. - naturally occurring mutagen - Can move from one region of a chromosome to another region of the same chromosome or to a different chromosome (eukaryotes) or a plasmid ## Transposons Cont. - Transposons are transposable genetic elements. - carry one or more other genes in addition to those which are essential for transposition. - Nomenclature – Tn - The structure of a transposon is similar to that of an insertion sequence, but larger. - The extra genes are located between the terminal repeated sequences. - Some Tn elements have a gene (e.g. drug resistance) flanked by two IS in opposite orientations. ## Composite Transposons - Contain 2 IS elements at either end and a gene in the middle - E.g. Tn 10 consists of ~ 9300 bp: carries a gene for tet between two IS 10 insertion sequence at the ends - Composite transposon also ends in IR - transposase produced by one of the IS catalyzes the transposition of both to move together with the gene in between. - Also generate flanking direct repeats at the site of insertion ## Noncomposite Transposons - Transposable elements found in bacteria that lack IS - e.g Tn3 carries a genes for transposase, resolvase and ẞ-lactamase (ampR) - resolvase is an enzyme required for some transposition; it aids in crossing over between sites located within the transposable element. - A few bacteriophage reproduce by transposition ## Bacteriophage Mu - Phage Mu is a temperate phage that reproduces by transposition - A simplified cartoon of the Mu genetic map is shown below ## Life Cycle of Mu - When Mu infects a sensitive host, the linear DNA enters the cell and the Mu DNA is inserted into the recipient genome. - Mu DNA is incorporated into the host DNA. - When the repressor is inactivated, the A and B proteins are expressed and Mu transposes by a replicative mechanism to 50 - 100 new sites on the chromosome. - late phage gene products are made (including phage heads, tails, lysis proteins, etc) - the length of Mu DNA is about 37 Kb and about 39 Kb are packaged into each head, so about approximately 2 Kb of host DNA is packaged into each phage. - after assembly of the phage, the host is lysed, releasing 50-100 phage particles ## Properties of Transposable Genetic Elements - **Random movement**: can move from any DNA to any other DNA molecule or to another location on the same molecule; the movement is not totally random; there are preferred sites. - **Not capable of self replication**: the transposable genetic elements do not exist autonomously and thus, to be replicated they must be a part of some other replicon - **Transposition mediated by site-specific recombination**: transposition requires little or no homology between the current location and the new site - **Transposition can be accompanied by duplication**: in many instances transposition of the transposable genetic element results in removal of the element from the original site and insertion at a new site; in some cases accompanied by the duplication of the transposable genetic element - one copy remains at the original site and the other is transposed to the new site. ## Research Use of Transposons - As mutagens - As cloning tags - as vehicles for introducing foreign DNA into model organisms ## Importance of Transposons - Many antibiotic resistance genes are located on transposons. - Since transposons can jump from one DNA molecule to another, these antibiotic resistance transposons are a major factor in the development of ab₨ plasmids: confer multiple drug resistance on a bacterium harboring such a plasmid. - These multi-drug resistance plasmids have become a major medical problem → “superbug" ## Types of Transposition Reactions 1. Non-replicative or "cut and paste" transposition 2. Replicative or "copy and paste" transposition 3. Transposition through an RNA intermediate ## Nonreplicative Transposition - This allows a transposon to move as a physical entity from a donor to a recipient site - This leaves a break at the donor site, which is lethal unless it can be repaired ## Replicative Transposition - Can be between two different DNA or two parts of the same DNA. - Produces an intermediate cointegrate structure. ## Transposition through an RNA Intermediate - Retrotransposons transpose through RNA intermediate. - Via reverse transcription ## References - Text Book - Chapter 6, 13 - iGenetics – A Molecular Approach - 3rd edition Ch. 15 - University of South Carolina School of Medicine - Do bacteria have sex? by Rosemary J. Redfield in NATURE REVIEWS, Genetics, VOLUME 2, AUGUST 2001 - DNA uptake during bacterial transformation by Ines Chen and David Dubnau in NATURE REVIEWS, MICROBIOLOGY, VOLUME 2, MARCH 2004 - Dubnau, D. 1999. DNA uptake in bacteria, Annu. Rev. Microbiol. 53:217-44 - An Introduction to Genetic Analysis. 7th edition. Griffiths AJF, Miller JH, Suzuki DT, et al. New York: W. H. Freeman; 2000 - Chapter 8 from Genetics: A Conceptual Approach, 3rd edition © 2008 by B.A. Pierce, New York: W. H. Freeman