BACTERIAL CONJUGATION I. OBJECTIVES To demonstrate the technical procedure to monitor the conjugational transfer of genetic material from one cell to another. To learn about the various genetic elements involved in bacterial sex and recombination events. To recognize and discuss the advantages and limitations of the conjugational procedure in genetic recombination experiments. II. INTRODUCTION There are three different mechanisms for genetic exchange between bacteria: conjugation, transformation and transduction. Conjugation has been shown to occur in many bacterial species especially the Gram negative organisms. In Enterobacteriaceae (Escherichia, Salmonella, Shigella, Vibrio, etc.), the transfer has been shown to cross the taxonomic lines with varying rates of efficiency. Thus, conjugation has become a tool to study the location and function of many genes, including genes responsible for virulence, antibiotic resistance, metabolic functions, regulatory proteins, phage attachment sites, etc. E. coli has been the model organism in conjugation studies. It may become infected with an extrachromosomal genetic element (a plasmid called the F factor) which mediates certain events such as the synthesis of a pilus (the F or sex pilus), and the rapid infection of all susceptible bacteria with this F factor. The pilus has been shown to facilitate attachment of the male (or F + cells) to the female (or F - cells) which do not contain the F factor. It was previously thought that the hollow center of the pilus constituted a passageway for the F factor to be transferred to the female. However, recent evidence suggests that the pilus may be used as a "hook" to bring the two cells close together and that the DNA transfer actually occurs outside the pilus. The genetic transfer is mediated by the F factor itself, and requires that the F factor be semi-conservatively replicated. One of these strands passes into the female and the female now assumes the characteristics of a male cell. Note that all these functions are caused by genes present on the F plasmid. Scientists found another kind of male bacteria called the Hfr strain in which the F factor integrates into the chromosome of its host. When an Hfr mates with an F - cell, the F factor genes mediate the transfer of the genetic material into the F - cell. Such a conjugational event yields a recombinant F - cell because the integrated F factor is rarely completely transferred. Hfr stands for "high frequency of recombination". The F factor 1
appears permanently "anchored" within a specific site in the bacterial genome and is not excised. These strains may be used for genetic studies to map the location of genes. A bacterial chromosome is considerably longer than an F factor, thus more time is required to replicate and transfer the complete length of the chromosome. Transfer commences from a point within the integrated F factor and proceeds in a linear fashion. The F factor is thus split in this process with some of the F associated genes being transferred first and the rest at the very end of the conjugation process. Since the conjugational juncture is relatively fragile, the conjugation may be mechanically interrupted at different times by vigorous agitation of the mixture (male and female). Under optimal conditions, the entire E. coli chromosome is transferred in 100 min. The recombinants formed as a result of a mating experiment are observed by plating the mating mixture on a medium that selects for the growth of only the recombinants. For example, an Hfr (Leu + ) is mated with and F - (Leu - ) and recombinants are selected by plating the cells on a medium that lacks leucine. The F - parent cannot grow on this medium, but the Hfr parent and the recombinants can. We must then arrange for some mechanism to prevent the growth of the Hfr to be able to easily recognize the recombinants. The inhibition of the Hfr may be accomplished by a variety of techniques such as: Utilizing an Hfr that has a metabolic requirement and omit that essential nutrient from the selection medium. Lyse the donor (Hfr) cells with a virulent male specific phage. Include antibiotics to which males are susceptible in the medium. Incubate the selection medium at a temperature that is antagonistic to growth of the donor (Hfr) cells. Naturally all these methods require that the counterselection affect only the donor and that the recipient be resistant (to the antibiotic or phage), prototrophic (for the particular metabolic lesion), or temperature insensitive. In the following experiment, you will receive an Hfr which is prototrophic for all genes (i.e., Hfr is wild type) and an F - that is auxotrophic for proline, lactose, tryptophan and histidine. (Note: This is the same E. coli strain used in the transduction exercise). In addition, the male is sensitive to, and the female is resistant to streptomycin. So our cross would be: F - (pro - lac - trp - his - str R ) x (pro + lac + trp + his + str S ). We shall include streptomycin in our selection medium to counterselect against the Hfr, and the omission of an essential nutrient will select against the female. In theory, only the recombinants should grow. III. LABORATORY SUPPLIES F - culture (NR57.10), 2 ml Hfr culture (NR57.8), 1 ml Sterile dropper Sterile empty test tube (largest size) Sterile empty test tubes (small size) 2 1 tube/group 1 tube/group 2/group 10/group 7/group
1.0, 2.0 and 10 ml pipets as needed Nutrient agar plate 6/group MS + Strep plate 1/group MacConkey + Strep plate 1/group Pro - + Strep plate 4/group Trp - + Strep plate 4/group His - + Strep plate 7/group MS broth 100 ml/group MS + Strep broth 50 ml/group Glass rod spreader and alcohol beaker 1/group 37 C water bath 2/lab Vortexer 4/lab IV. PROCEDURE Note: This experiment will be performed by students at a table forming a group. Try to keep all cultures and plates at 37 C as much as possible and use sterile techniques in all your work. Observation of Recombinants 1. Obtain the Hfr and F - cultures, one MS + Strep (Mineral salts + streptomycin) and one MAC + streptomycin plate. Label plates with your name and medium type. 2. With a sterile dropper, place a small drop of F - culture on the left and middle of each plate (See figure). Do not move the plates. Use a new sterile dropper to place a small drop of Hfr culture on the right and middle of each plate. Make sure that the F - and Hfr drops at the center of the plate mix together. The center of the plate is the place where conjugation between F - and Hfr cells should take place. All other drops serve as controls for natural reversions of F - cells to prototrophy and Hfr to antibiotic resistance. 3. Return the cultures to the 37 C water bath. Allow the drops to dry before transferring the plates to 37 C for two days. 3
Finding the Order of Genes 1. Obtain a small sterile test tube and mix 0.9 ml of F - with 0.1 ml of Hfr. This is your conjugation mixture. Note the exact time (i.e. time zero), mix gently by tapping the tube and incubate in the water bath at 37 C. This tube should be kept at 37 C at all times except when taking samples. You should be very quick in taking your samples not to decrease the temperature of the mixture below 37 C. 2. Quickly take 0.1 ml of the conjugation mixture and add to 0.9 ml of MS + Strep broth. Vortex the tube for 90 seconds at top speed and then transfer 0.1 of this solution to 9.9 ml of MS + Strep and vortex at medium speed for 30 seconds. Place 0.1 ml of this dilution on each of Pro -, Trp - and His - plates. Spread with alcohol flamed glass rod. Label plates with zero time and type of plate as well as your initials. 3. At exactly 20 min into the experiment, repeat the above step. 4. At exactly 40 and 60 min into the experiment, take 0.1 ml of the conjugation mixture and add 0.9 ml of MS + Strep. Vortex 90 seconds at top speed. Take 0.1 ml of this mixture and add 0.9 ml of MS + Strep. Vortex for another 30 seconds. Place 0.1 ml of the last dilution on the 3 different plate types, spread and label as before. 5. Incubate the plates at 37 C for 2 days. Your T.A. will remove the plates to a refrigerator. Enumeration of Parental and Recombinant Cells 1. Use your knowledge of dilution series to prepare 10-6, 10-7 and 10-8 dilutions of the Hfr and F - cultures separately by using MS broth. 2. Plate 0.1 ml of each dilution on a nutrient agar plate and spread the inoculum with a sterile glass rod. Work from the lowest dilution upward so that you only use a single pipet. Spreading should be done very rapidly not to give the inoculum any chance of soaking in. Let the inocula dry completely after spreading before inverting the plates. 3. Label 3 His - plates for enumeration of recombinants as 10-1, 10-2 and 10-3. About 60 minutes into the experiment, transfer 0.1 ml of the mixture from the conjugation tube used for finding the order of genes onto the 10-1 His - plate and use a glass rod to spread it. Dilute the mixture 10-1 and 10-2 times with MS + Strep and plate onto the remaining His - plates. When the inocula have dried, label plates, invert them and incubate at 37 C for 2 days. Use of any section of this Lab Manual without the written consent of, Dept. of Biology, University of Pennsylvania is strictly prohibited. 4
Results of the Bacterial Conjugation Lab Exercise NAME DATE GROUP NAME NAME(S) OF PARTNERS Draw the results of "Observation of Recombinants" and label well. Time elapsed 0 min 20 min 40 min 60 min No. of colonies on Pro - plates No. of colonies on Trp - plates No. of colonies on His - plates What is the gene order and why? No. of colonies Plate 10-6 10-7 10-8 dilutions Number of cells per ml of original F - Hfr No. of colonies 10-1 10-2 10-3 Plate Number of cells per dilution ml of original Recombinants 5
From the data you have obtained above and the fact that you used 0.9 ml of F - and 0.1 ml of Hfr in your conjugation mixture, determine the number of F - and Hfr cells that can pair up. From the data on the number of F -, Hfr and conjugants, derive the efficiency of conjugation in your experiment (i.e., the % of the conjugants per parental pair). Why do we dilute the parental mixture less and less as the conjugation time increases? Why do we enumerate conjugants at 60 minutes and not later? 6