I. PLASMID-MEDIATED CONJUGATION

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1 In this exercise we explore the creation of new genotypes by horizontal gene transfer. There are 3 different genetic elements at play: the E. coli genome, the conjugal plasmid F, and the transposon Tn5. Our experiment should reveal several basic interactions of these three players. I. PLASMID-MEDIATED CONJUGATION Plasmid-mediated conjugation is a powerful mechanism for horizontal gene transfer in prokaryotes, and is now widely recognized as an important contributor to the evolution of bacterial populations. Conjugation, a process which promotes DNA transfer from a donor to a recipient cell mediated by physical contact, has been reported in many bacterial groups. Donor ability is conferred by genes encoding conjugative transfer functions that are coded by a self-transmissible, or conjugative, plasmid. As a means of genetic exchange among individual cells and populations, both within and between bacterial species, conjugation is a phenomenon of fundamental evolutionary and ecological significance. The significance of this process has been further highlighted by evidence that conjugation systems can even facilitate inter-domain transfer of genetic information. Ti plasmid-mediated T-DNA transfer from Agrobacterium species to plants appears to represent a modified form of bacterial conjugation. Transfer of plasmids from Escherichia coli to Saccharomyces cerevisiae (yeast) has also been demonstrated. In this exercise we try to demonstrate transfer of an F plasmid derivative (an F' plasmid). The F (fertility) plasmid of E. coli K-12 was the first plasmid to be described. F' plasmids are modified forms of the native E. coli plasmid F that contain one or more genes that are ordinarily part of the E. coli genome. Structure Of The F Plasmid The F plasmid is a circular DNA molecule, 100 kb in size, and contains about 100 genes. The various functional regions of F and the position of orit (coordinate 66.7F) are indicated on the map shown in Fig. 1. FIGURE 1 Physical and functional map of the F plasmid. Numbers within the map indicate kilobase coordinates based on the 100-kb F

2 map. The transposable elements 2, 1S3, and Tn1000 are represented by solid boxes. The extents of the replication (RepFIA, RepFIB, and RepFIC), transfer, and leading regions are indicated outside the map. The origin of conjugative transfer, and leading regions are indicated outside the map. The origin of conjugative transfer (orit) is denoted by a triangle indicating the direction of single-stranded DNA transfer (leading region transferred first). Leading Region The F DNA sequences located between the origin of conjugal transfer, orit, and RepFIA are presumed to be the first to enter the recipient cell during conjugation and have therefore been designated the leading region. Leading-region gene products are thought to assist in establishing F DNA in the recipient. Autonomous Replication The RepFIA region is responsible for the typical replication of F in the E. coli host. Replication and associated maintenance and partitioning mechanisms act in concert to maintain the plasmid at one to two copies per cell. The secondary replication region, RepFIB, is independently functional and can sustain plasmid replication in the absence of RepFIA. The RepFIC region includes an incomplete remnant of a replication system that is used by some other related plasmids. Transposable Elements The F sequence includes a single copy of Tn1000 and 2 and two copies of 3. Insertional inactivation of the transfer region regulatory gene, fino, by 3 is fortuitously responsible for the constitutively high levels of conjugative transfer exhibited by F. These elements also serve as the substrate for homologous recombination events with the host cell genome leading the genome fusion (Hfr strains). F' plasmids are generated by subsequent imprecise excision of F from the composite genome of an Hfr strain. Conjugative Transfer Including the orit site (map position 66.7F), the transfer (tra) region encodes all of the F genes known to be required for efficient conjugative transfer.

3 Figure 2. Figure 2 depicts the stages of intercellular contact and DNA transfer thought to occur during F- mediated conjugation. These are as follows: Contact between F+ donor and F recipient cells is initiated by interaction between the tip of an F pilus and the recipient cell surface. Susequently, depolymerization of the pilus subunits draws the cells into close physical contact. Conjugating cells are typically aggregated in close, stable wall-wall association. A large number of the F products required for conjugative transfer are involved in F-pilus synthesis and aggregate stabilization. A protein complex (orit complex) assembles on the origin of transfer. One DNA strand in the orit site is nicked by an endonuclease (TraI) that also catalyzes the covalent attachment of the 5' end of the nicked strand DNA to the protein. TraI, is also a helicase and can unwind the nicked strand in the 5' to 3' direction. During transfer, this protein is associated with the site of intercellular connection between donor and recipients cells, and through which this single strand of DNA is passed. TraI may also subsequently catalyze re-circularization of the transferred strand to terminate transfer Replacement strand synthesis in the donor and complementary-strand synthesis in the recipient depend on host cell enzymes. Figure 2 shows DNA synthesis in the donor by a rolling-circle mechanism.

4 II. Transposable Elements in Bacteria Transposable Elements are DNA sequences that are capable of mediating their own movement (transposition) to new locations within the genome they inhabit. Barabara McClintock was awarded a Nobel Prize for her pioneering discovery of transposable elements in the genome of maize. Transposable elements of various types are widespread in genomes of eukaryotes and bacteria. In bacteria, transposable elements can generally be assigned to one of two major types, "Insertion Sequences ()" and "Composite Transposons". In practice, composite transposons are typically referred to simply as "transposons". Insertion sequences ('s) are transposable elements whose only genes are directly related to promotion and regulation of their transposition, typically the gene for the so-called transposase enzyme. elements are between 700-2,000 bp in length and are characterized by short, terminal, inverted repeat sequences with the ORF or ORF's in between. They are normal constituents of many bacterial chromosomes and plasmids. Composite transposons generally consist of two copies of the same element flanking variable amounts of other DNA sequences coding for one or several genes with diverse functions. The best known transposons are those which were discovered as parts of antibiotic resistance plasmids.s The diagram below compares the typical structure of an element with the transposon Tn5. Tn5 carries 3 antibiotic resistance genes sandwiched between 2 copies of 50. Only elements will be discussed below. elements participate in rather bewildering array of molecular events that alter the genomes which they inhabit. The most important of these are: Transposition movement and insertion at a different location in the same DNA molecule, or in different molecule in the cell. The transposition process is often accompanied by replication of the (replicative transposition), leading to an increase in the copy # of the. The diagram below shows replicative transposition of an.

5 Insertional Inactivation Insertion of an within a coding sequence generally leads to the loss of gene function (null mutation). Homologous Recombination Multiple copies of the same in the same cell are substrates for homologous recombination events that may lead to DNA deletions, sequence inversions, or fusion of separate DNA molecules. For example, homologous recombination between copies of the same element in a conjugal plasmid and the bacterial chromosome leads to formation of Hfr strains, as shown below. F + Hfr The multiplicity of transpositional and recombinational events associated with elements allows them to unlock the Pandora's box of genome plasticity for bacterial chromosomes and plasmids in which they are found. In fact, the K-12 laboratory strains of E. coli show considerable variability in the number and location of elements in their genomes due to transposition events that have occurred since the parent strain was first isolated in 1922.

6 III. PROCEDURE We provide overnight liquid suspension cultures of the donor (F') strain SC138, and the recipient (F - ) strain SC109 grown in LB medium to a concentration of 2 X 10 8 cells/ml (approx.). See the previous exercise for a description of these strains. The most crucial aspect of strain growth is to ensure that the F' strain has functional F-pili to initiate conjugation with the F -. This is ensured by growing them in fresh medium at 37 C with very gentle agitation. Vigorous agitation will break off the pili. NOTE: Pay careful attention to aseptic transfer technique in all steps. 1. Set up 3 tubes as follows: these tubes as follows: Sterile LB Medium 0.5 ml 0.5 ml - F - Culture ml 0.5 ml F' Culture 0.5 ml ml 2. Very gently swirl the tubes to mix the contents, then put them in a 37 C water bath for one hour with very gentle agitation. You can work out the details of the dilution and plating (steps 4 and 5) while the tubes are incubating. 3. After 1 hr. turn up the shaker speed and incubate the tubes for another 30 minutes with more vigorous agitation. 4. Remove the tubes from the water bath and perform a dilution series of tube #3 ONLY to give 10-1, 10-2, 10-3, and 10-4 dilutions. Work the details of this out yourselves and write it down clearly in your notebook. 5. Spread plate 50 ul samples as follows: Minimal Glucose Kan Strep X-Gal Minimal Glucose Strep X-Gal X X X X X X X X X The " X " indicates plates that you spread. You spread plate directly (10 0 ) from all three tubes on both types of plate (hence X X X). i.e. You will be making 4 Kan Strep X-Gal plates and 5 Strep X-Gal plates. 6. Be sure the surface of the agar plates is dry and then incubate them upside down at 37 C.

7 RESULTS and CALCULATIONS 1. Examine all your plates qualitatively and describe in your notebook what you observe. Set yourselves the goal of recording your observations in an organized and coherent way that will serve as a finished product to submit with the assignment, so stop and think before you begin scribbling things down. You may use diagrams to complement your text. At this point do not attempt to interpret or to explain the observations. 3. For any plates that have discrete (i.e. physically separate) colonies, count them. You can use the colony counter devices if there are plates with more than a few dozen colonies. Use the colony counts to calculate cell concentration values (cells/ml) for the experimental tubes at the end of the conjugation procedure. 3. Reconciliation: Share your observations and results with the other lab groups. Write a summary of the results based on this comparison.