The transfer of host chromosomal genes occurs in a fixed order from the point of origin, the site at which F is integrated into the host chromosome. The strain is then a "merodiploid" for the resulting duplicated sequence; subsequently, homologous regions of the chromosomes can undergo recombination, conferring on the resultant cell, a different genotype. It would take about 100 minutes to copy and transfer the 4000 genes from the Hfr to the F-. If ‘mating’ is disrupted during that transfer, only the genes closest to the first F factor block of genes will be donated. The gene farthest away on the circular DNA seldom gets transferred. This system was used to provide the first genetic map of E. coli.

Some strains of E. coli have genes that confer resistance to antibiotics. Some of these genes reside in chromosomal DNA; the majority are on plasmids (called "R plasmids" for "resistance"). Examples are strains with chromosomal genes for tetracycline resistance (tetr ) or for streptomycin (strr). A bacterium with a tetr gene, for example, would grow on media with tetracycline in it, which would otherwise be lethal for the bacterium.

One of the first demonstrations that DNA is the genetic material was the isolation of DNA from one strain of Diplococcus pneumonia (aka: pneumococcus) and its use to cause heritable changes in recipient strains of pneumococcus. This phenomenon is known as transformation and involves the uptake of naked DNA with subsequent recombination of homologous regions of the donor and receipient chromosomes. (N.B. This is referred to as "genomic" transformation, to distinguish it from transformation brought about by the uptake and expression of plasmid DNA. The latter does not require recombination; it will be dealt with in a later section.)

Not all bacteria demonstrate this phenomenon (or at least under the laboratory conditions so far used to study this phenomenon). The two other bacteria in which this has been studied are Bacillus subtilis and Hemophilus influenzae. In all three cases, it was found that cultures of cells are "competent" to undergo transformation only under special growth conditions, suggesting that preparing cells to take up DNA and undergo recombination is triggered by diffusable factors in the medium by mechanisms that are still not at all clear.

Apart from the intrinsic interest in the process of transformation, it served as a very useful research tool in the early days of molecular biology since it provided the only way to correlate physical and chemical alterations of DNA with observable effects on expression of genetic information. You are encouraged to read the material at the end of the syllabus taken from Malacinski and Freifelder on conjugation, transduction, and transformation. At the end of this section are descriptions and protocols for transduction and tranformation experimentation should you wish to work with this during the course.

B. EXPERIMENTAL PROTOCOL FOR CONJUGATION

In this exercise, the antibiotics tetracycline and streptomycin will be used to select bacteria with DNA for resistance to both. (Tetracycline is used to treat penicillin resistant strains of gonorrhea and streptomycin is used to treat acne and boils. Indiscriminate use of antibiotics will select for strains resistant to the antibiotics.) The Hfr strain (Hfr Cavelli or HfrC) used in this investigation has a transposon , called Tn10, inserted into the chromosomal DNA (at position 1'). This transposon has the gene for tetracycline resistance. The genotype is therefore: Hfr tetrstrs; phenotypically, it will grow in medium containing tetracycline but not containing streptomycin. The F- strain (AB1157) has no insert, so it has no tetracycline resistance, but is sensitive to the drug. However, it has a gene for streptomycin resistance at another locus, corresponding to the recessive strs allele on HfrC. The genotype is therefore: F- tetsstrr; phenotypically, it will grow in medium containing streptomycin but not containing tetracycline.

During conjugation, some recipient cells will receive DNA containing the tetrgene; subsequent recombination will create, among other possibilities, cells carrying both the tetr allele and the strr allele. These recombinants can be selected for by plating the cell mixture on medium containing both antibiotics.

MATERIALS

Overnight cultures of Hfr strain and F- (AB1157) strains

L broth

Sterile flasks

Plates: L, L+strep, L+tet, L+strep+tet

sterile tubes, pipets, tips.

marker

burner and loop

PROCEDURE

1. Subculture overnight Hfr and F- cultures by diluting them 1:25 in fresh L broth; grow both at 37oC with shaking to 3-8 x 108 cells/ml (OD550 ≈ 0.3-0.5; ca. 3 hrs)

2. When the cultures have attained the desired cell density:

a. Mix 0.2ml Hfr + 0.2ml F- in large sterile test tube;

---let tube sit 90min, 37oC;

---add 2ml L broth and grow in 37oC shaking water bath 1-2 hr;

---then plate 0.1ml of a 102, 103, and 104 dilutions of this culture on LB+strep+tet plates, and plate 0.1 ml of 105 and 106 dilutions on L agar plates, L+tet plates, and L+strep plates (these are the controls).

2. Incubate the plates overnight at 37oC or for 1-2 days at room temperature.

OBJECTIVES

1. To become aware of the spread of antibiotic resistant bacteria in our environment.

2. To study the relative ease for exchange of antibiotic resistant genes among bacteria.

INTRODUCTION

The spread of antibiotic resistance among pathogenic bacteria has become a significant public health problem with the past few decades. This was observed clinically and epidemiologically by physicians and public health officials not too long after antibiotics became part of the arsenal used to treat bacterial infections; and researchers were able to isolate plasmids that contained multiple antibiotic resistant genes. As the use of antibiotics moved from the treatment of human disease to the treatment of diseases in animals, particularly farm animals, and to their prophylactic use among livestock, public health officials began to suspect that runoff from farms may contribute to release of antibiotic resistant bacteria in many streams and lakes. This protocol is designed to test the hypothesis that antibiotic resistant microorganisms can be isolated from local streams and lakes.

In May, 1994, a survey was conducted of the following locations: Lake Mendota at the Memorial Union, Lake Mendota in Middleton, Lake Wingra, Monona Bay, Lake Monona, and the stream next to the School of Veterinary Medicine on the UW campus. All samples were evaluated for the presence of ampicillin-resistant microorganisms. Even though coliform bacteria, an indicator of fecal contamination, were found at several sites, ampicilllin resistant organisms were found at only two sites: Lake Mendota in Middleton and the stream next to the SVM. This exercise is a follow-up on this initial study. In addition, this exercise will also test the hypothesis that antibiotic resistant genes can be transferred by conjugation from one species to another. To study this, a multi-drug resistant Salmonella anatum strain will be mated with an E. coli strain.

Genes encoding resistance to antimicrobial agents, or antibiotics, are often present on plasmids. Plasmids are autonomous, self-replicating genetic elements that usually are not essential for bacterial survival. Many plasmids are able to move from one bacterium to another by a process called conjugation, and are called mobile, self-transmissible, or conjugative plasmids. These plasmids carry a number of genes, designated tra genes, which encode proteins needed for the transfer or conjugation process. Some of the tra genes encode the proteins for the sex pilus. Because of the large number of genes required for conjugation, conjugative or self-transmissible plasmids are usually large, at least 25kbp in size. Some plasmids cannot transfer themselves but can be transferred by conjugation because they are mobilized by self-transmissible plasmids. Such plasmids are called mobilizable plasmids and are much smaller because only one or two genes (i.e. mob genes) are required to take advantage of the transfer machinery provided by a self-transmissible plasmid. Self-transmissible or mobilizable plasmids often acquire multiple antibiotic resistant genes, frequently by insertion of transposons carrying these genes into the plasmid. Bacterial containing such plasmids can then transmit these antibiotic resistance genes to other bacteria. This process happens readily in nature and it is in this way that antibiotic resistance is spread from a resistant organism to a sensitive one. In this exercise, you will determine the number of antibiotic resistant microorganism in the creek next to the SVM, based on sample taken at several locations along the creek.

RATIONALE

A frequent source of contamination of many lakes and streams is fecal material, either from humans or animals. For this experiment, you will also assess the level of fecal contamination by determining the number of coliform bacteria present in the water sample. The word, coliform, means having E.coli-like characteristics (e.g. a small gram-negative rod that can ferment the sugar, lactose, with production of acid and gas). E.coli and other members of the family, Enterobacteriaceae, can be detected easily using MacConkey agar plates: organisms that ferment lactose form pink colonies on these plates, while lactose non-fermenters are colorless. Organisms that ferment lactose include: E.coli, Klebsiella, Enterobacter (Salmonella is a non-lactose fermenting member of the family Enterobacteriaceae)

MacConkey agar is a selective and differential medium used for the isolation of gram-negative bacilli. MacConkey plates are selective in that the growth of gram-positive organisms is inhibited, because of the inclusion of crystal violet and bile salts in the medium. MacConkey are differential because they can distinguish between organisms that ferment lactose and those that do not ferment lactose. When lactose is fermented, acidic endproducts are produced; the dye, neutral red, is included in the MacConkey agar as a pH indicator to detect this acid production by lactose fermenters. Thus, lactose fermenters form pink colonies, while lactose non-fermenter colonies are colorless or beige. The typical colony morphology on MacConkey agar is as follows:

Day 1:

Sterile container with 6-8 ft. string attached (for collecting water samples)

Sterile test tube containing 9 ml buffered saline (for dilution of sample)

Plate spreader, alcohol and burner

1 ml pipet

plates: 2 each of MacConkey, LB, LB + amp, LB+tet, LBB+kan.

Day 2:

Inoculation loop

Test tube containing 1 m LB broth (creek organism)

3 tubes containing

1 ml E. coli Rif^r

1 ml E.coli Rif^r + 1 ml Salmonella donor

1 ml E.coli Rif^r + 1 lm creek donor

Day 3:

2 x 1 ml pipets

plate spreader

plates: LB + amp, LB + tet, LB + rif (x2), MAC + rifLB + amp + rif, LB + tet + rif, MAC + amp + rif, MAC + tet + rif,

1. Let T = number of teams + 5. Pour the following plates

2xT MAC

2xT MAC + 100 m g/ml RIf

1xT MAC + 100 m g/ml RIf + 50 m g/ml AMP

1xT MAC + 100 m g/ml RIf + 50 m g/ml AMP + 25 m g/ml TET

2xT LB

3xT LB + 50 m g/ml AMP

3xT LB + 25 m g/ml TET

2xT LB + 50 m g/ml KAN

2xT LB + 100 m g/ml RIF

1xT LB + 100 m g/ml RIF + 50 m g/ml AMP

1xT LB + 100 m g/ml RIF + 25 m g/ml TET

Cover RIF ant TET plates; dry overnihgt and store at 4^oC.

2. From plates, inoculate overnight cultures of E. coli and Salmonella strains the day prior to the experiment. Grow enough to have, per team:

3 x 1ml per tube E.coli recipient

1 x 1 ml per tube Salmonella donor

For the creek organism, each team needs 1 tube containing 1 ml LB broth.

Procedure

1. Count the number of colonies on each of the plates. For the MacConkey plates, record the number of lactose fermenting (pink/red) colonies and the number of lactose non-fermenting colonies. Record in the attached table.

CONJUGATION

2. Each team will perform two conjugations: one with an antibiotic resistant organism from the creek and the other with a multi-drug resistant, Salmonella anatum.

3. We will arbitrarily designate the creek organism as F+ since we suspect that it contains a conjugative plasmid. This is the donor strain which will be donating antibiotic resistance genes to a sensitive E.coli strain. The E.coli recipient does not contain any plasmids and therefore is designated as F-. However, it is resistant to rifampicin. In these matings we are using resistance to rifampicin as a way of selecting for and distinguishing the recipient from the rifampicin sensitive donor strain.

4. With a loop, carefully scrape off one colony from either the ampicillin or tetracycline plate and inoculate a tube of L broth and a tube of L broth containing the rifampicin resistant E.coli recipient.

5. Each team will also be given a culture of Salmonella anatum. This organism is sensitive to rifampicin. Since it contains conjugative plasmids, it is designated, F+. You will also be given a 1 ml culture of the rifampicin resistant F- E.coli recipient. Inoculate the E.coli culture with a loopful of the Salmonella culture.

6. Incubate both tubes from step 4 and the mating mixture from step 5 overnight at 37oC.

1. Pipet 0.1ml of the mating mixture from the creek onto each of the following plates and spread out evenly:

4. Streak the creek organism onto LB agar containing 100µg/ml rifampicin. This plate is a control to demonstrate that this organism is sensitive to rifampicin and therefore cannot grow on any plates containing 100µg/ml rifampicin.

5. Streak the Salmonella anatum culture onto MacConkey agar containing 100µg/ml rifampicin. This plate is a control to demonstrate that this organism is sensitive to rifampicin and therefore cannot grow on any plates containing 100µg/ml rifampicin.

6. Incubate all plates overnight at 37oC.

1. Count the number of colonies on each plate and record your results using the attached table.

COMMENTS AND QUESTIONS

1. The following controls were done to check the experimental results against the working hypothesis:

2. Why was MacConkey agar used for the S.anatum/E.coli mating? It was used to differentiate between the two bacterial species: S.anatum forms yellowish colonies while E.coli forms dark pink colonies. Do you know why?

3. Did the creek organism transfer drug resistance to the E.coli strain? Explain why you came to this conclusion.

4. Did the S.anatum transfer drug resistance to the E.coli strain? Consider the implications of this, both scientifically and from a public health standpoint. (The first time this phenomenon surfaced in the popular press was the discovery of penicillin-resistant strains of Neisseria gonococcus isolated from soldiers in or returning from Vietnam.)

Among the phage that are best able to transfer genes from one bacterium to another is a virulent type of the temperate phage, P1, called P1vir . We shall use P1vir to infect the E.coli strain, HfrC. Recall that this strain contains the transposon, Tn10 which, in turn, contains a gene that confers resistance to the antibiotic, tetracycline, to the host. In these experiments, we will select for transduction events in which Tn10 has been transferred to the E. coli strain, AB1157, and has been incorporated into the recipient cell's chromosome. This results in a permanent alteration of the recipient cell genotype (by "natural genetic engineering") and, if we use the appropriate selection technique we can detect the resulting altered phenotype. As in the conjugation experiments, we shall do this by plating the transduction mixture on agar with different antibiotics: if we plate the mixture on agar containing tetracycline, transduced cells containing Tn10 will grow, but not the recipient strain, AB1157----but so will uninfected donor HfrC cells. Likewise, since the recipient AB1157 cells contain the gene for streptomycin resistance, plating the mixture on agar containing streptomycin will allow both transduced cells and uninfected recipient AB1157 cells to grow. But, if we plate on agar containing both antibiotics, only transduced cell that have received the Tn10 DNA will grow.

In addition to carrying the strr allele, the genome of AB1157 lacks functional genes for synthesizing five amino acids: threonine, leucine, histidine, arginine, and proline. Without these amino acids present in the growth medium, AB1157 cannot grow . The HfrC strain has wild type (i.e. functional) copies of these genes. Thus, by transducing AB1157 with P1vir phage previously grown on HfrC, some of the amino acid-synthesis genes from the HfrC may get incorporated in the AB1157 DNA. Such transduced cells can be detected by their growth in medium lacking one or more of these amino acids. (The use of two different antibiotic "markers" in the donor and recipient cells is crucial for this experimental design; do you see why?)

In this exercise, the antibiotics tetracycline and streptomycin will be used to select bacteria with DNA for resistance to both. (Tetracycline is used to treat penicillin-resistant strains of gonorrhea and streptomycin is used to treat acne and boils. Indiscriminate use of antibiotics will select for strains resistant to the antibiotics.) The Hfr strain (Hfr Cavelli or HfrC) used here is tetracycline resistant due to the transposon, Tn10. The genotype (with respect to the genetic markers of interest in this experiment) is: tetrstrs; phenotypically, it will grow in medium containing tetracycline, but not streptomycin. The F- strain (AB1157) has no Tn10 insert, so it is sensitive to tetracycline; however, it has a gene for streptomycin resistance at another locus, corresponding to the recessive strs allele on HfrC. The genotype is therefore: F- tetsstrr; phenotypically, it will grow in medium containing streptomycin but not containing tetracycline.

During infection of Hfr by P1vir, some of the phage heads will have packaged chunks of Hfr DNA---including those with a piece containing the tetr locus. Upon infection of AB1157 with the P1vir(Hfr) lysate, some recipient cells will be "infected" by phage containing DNA with the tetrgene; subsequent recombination will create, among other possibilities, cells carrying both the tetr allele and the strr allele. These recombinants can be selected for by plating the cell mixture on medium containing both antibiotics.

2. Materials

3. Procedure

6 Incubate plates overnight at 37oC; count the placques and enter into the box below.

FOR THOUGHT AND DISCUSSION

Phenotype of colonies Number counted .

Transformation

BACKGROUND

One of the first demonstrations that DNA is the genetic material was the isolation of DNA from one strain of cells and its use to cause heritable changes in recipient cells. This phenomenon is known as transformation. In this laboratory you have the opportunity to repeat this experiment with DNA isolated from Bacillus subtilis, a common nonpathogenic soil bacterium.

Wild type B. subtilis can grow on a simple salts medium supplemented with a carbon source such as glucose. Mutants can be isolated, however, that cannot synthesize various monomers and consequently cannot grow unless the required nutrient is supplied in the medium. You are going to work with a strain (trp-) that cannot synthesize the amino acid tryptophan and show that these cells can be transformed to wild type (trp+) by incubating them with DNA from wild type B. subtilis.

The process by which recipient cells take up donor DNA still is not understood. It is facilitated by growing the recipients under specific conditions and using them just before they enter the stationary phase of growth. (It is interesting to note that RNA is not taken up by the recipient cells.) After the donor DNA enters the cell, it recombines with the chromosome to give wild type recombinants.

This is a laboratory where you will be able to design your own experiment. One of the reasons for the great interest in transformation experiments is that they allow one to correlate effects of physical or chemical alterations in the structure of DNA with its biological activity. You may want to show that the transforming principle is indeed DNA and not RNA or protein. (The DNA that you isolate from donor trp+ cells will be far from pure.) This can be done by showing that the material which gives transformants is inactivated by DNase but not by enzymes that degrade RNA or protein (RNase and pronase.) An example of the effect of some treatment on the ability of the DNA to transform recipient cells. Some possibilities are suggested below. Be sure to include appropriate controls and to show that neither the trp- recipient cells nor the DNA alone can yield colonies on plates lacking tryptophan.

Here are some ideas for things to test (each group of two can have up to 15 plates):

A. The size of the donor DNA. (It can be sheared to small pieces by squirting it rapidly through a small bore (26 gauge) syringe needle several times.)

B. Denaturing the DNA. (This can be done by heating the DNA to various temperatures then quick cooling it on ice.)

C. Non-homologous DNA added to the donor DNA. (Purified calf thymus DNA will be available.)

D. A time course of transformation. (How do you stop the reaction?)

E. Making the experiment quantitative and calculating the per cent of recipient cells transformed.

F. UV irradiation of the donor DNA. (Wear goggles to protect your eyes.) One of the effects of UV is to cause the formation of dimers between adjacent thymines in DNA.

You will be given a tube of trp+ B. subtilis at a concentration of approximately 5x109 cells/ml. Save out a small aliquot (a few drops) to be used later so that you can show that these cells can indeed grow in the absence of tryptophan. Add 5 drops of 2 mg/ml lysozyme, and enzyme which breaks down the cell wall, mix gently, and incubate at 37oC for 10 minutes or until the solution clears. Add 6 drops of 10% (w/v) sodium dodecyl (SDS) and mix by rotating the tube at a shallow angle. SDS is a detergent which dissolves the cell membrane and denatures proteins, including enzymes which degrade DNA. The solution should become somewhat viscous due to the long, rigid DNA molecules which are released from the cells.

DNA is insoluble in most organic solvents. This property allows you to "spool out" the DNA directly from the cell lysate. Slowly and carefully layer 4 ml 100% ethanol over the cell lysate. Do not mix the two layers. (Have your lab partner hold the tube at a 45o angle on the bench top while you allow the ethanol to run slowly down the side of the tube.) Holding the tube upright, insert the etched end of a sterile glass rod through the ethanol layer and into the lysate about 1 cm below the interface. Now stir the rod in a circular motion. As the ethanol concentration in the lysate increases at the interface, the DNA will begin to form white fibers that wind around the glass rod. Continue, lowering the glass rod slowly as the ethanol into the lower phase, stopping as soon as you have a "glop" of DNA wound around your rod or when two phases are no longer visible. At this point (it should take about 5 minutes) most of the DNA, along with contaminating RNA, will be wound around the glass rod. Transfer it to 2 ml of sterile NaCl - EDTA (0.15M NaCl, 0.001M ethylene diamine tetraacetic acid- -the EDTA protects the DNA from any DNase that might still be around by chelating Mg++ which is required for DNase to act) and shake gently. Notice that the DNA swells on the rod and becomes transparent. It will take about 10 minutes to dissolve the DNA. If you have a lot of DNA, not all of it will dissolve; that is all right.

Dilute your DNA 1:10 in sterile saline (0.15M NaCl.) For most experiments, 2.0ml of diluted DNA is plenty. Treat samples of this diluted DNA according to the experiment that you have planned, and the mix 0.1ml of each with recipient cells as described below. Be sure to include a tube containing untreated DNA as well.

1. You will be provided with 2.0ml of 10-fold concentrated recipient cells in a tiny tube on ice. Add 0.8ml of Medium II and transfer the entire 1.0 ml to a small sterile test tube with a Pasteur pipette.

2. Set up a series of small test tubes according to the experiment you have planned. Use 0.1ml of DNA where desired, 1 drop of enzyme (if your experiment calls for this), and 0.1 ml of diluted recipient cells (or Medium II in the case of controls lacking recipient cells.) Recipient cells should be added last when all other components are ready. Vortex the tubes and incubate at 37oC for 15 minutes or under the conditions that you have chosen.

3. Plate out the cells after the incubation period by pouring or pipetting the contents of each tube onto numbered plates of salts - glucose - agar medium with or without tryptophan. Dip a glass spreader into ethanol and flame it to sterilize it. (Be careful that you do not flame yourself!) Allow a few seconds for cooling. Spread the liquid across the surface of the agar while rotating the plate.

4. Incubate your plates for a day at 37oC and then count the colonies (piles of cells derived from a single cell) on them to determine your results. If there are a large number of colonies on a plate (more that a couple hundred), you may wish to estimate the number or simply write down "too many to count." Note that putting a very large number of cells on a plate will result, the next day, in a "lawn" of growth rather than individual colonies. Discard your plates after you have noted your results.

Explain your results, the controls you used, and the purpose of each. What can you say about the properties of DNA that are necessary for it to be biologically active in transformation? How could the experiment be refined to the point where you could be certain that DNA and not some trace material is the transforming principle?