The Evolutionary Potential of the TrpA gene of Escherichia coli

Transcription

1 The Evolutionary Potential of the TrpA gene of Escherichia coli Stephanie Ebnet, Biology Dr. Ralph Seelke, Department of Biological and Earth Sciences ABSTRACT For bacteria to evolve, mutations must occur which impart a selective advantage to the organism. An ongoing evolutionary question is the capability of mutation and selection to produce an advantageous change, when two or more mutations are needed. We investigated this question using Escherichia coli RS202-5 containing two inactivating mutations in the trpa tryptophan biosynthesis gene. RS202-5 was grown by serial transfer under conditions selective for evolution of TrpA +. After approximately 2000 generations, TrpA + evolvants have failed to appear. However, RS202-5 has evolved to grow faster under low tryptophan conditions. We conclude that, when evolution of a Trp + phenotype requires two independent mutations, it is not observed. Long-term selection when tryptophan is limited does result in improved growth rates. The reversion rate of single inactivating mutations was also investigated. Introduction My advisor, Dr. Ralph Seelke, is interested in a major research question: What can evolution be shown to do experimentally? This is a question that is interesting in its own right, but it is also one that has enormous practical importance. Both cancer and antibiotic resistance can be viewed as the result of evolutionary events. In the case of antibiotic resistance, some of the resistance is the result of mutation generating variants that are resistant to a particular antibiotic, and such variants being selected for growth in the presence of that antibiotic. The early history of our use of antibiotics is replete with cases where we did not understand the capabilities of evolution, resulting in evolution of antibiotic resistance, or the limitations of evolution, resulting in our failure to wisely utilize our growing collection of antibiotics. One can safely state that, had we more fully understood both the capabilities and limitations of evolution 60 years ago, the dangers that we face due to multiply resistant microbes would be much reduced today. Antibiotic resistance is a major threat to the welfare of society. Microbes can acquire antibiotic resistance through either horizontal evolution (transmission of genetic information from one microbe to another) or vertical evolution (obtaining resistance through mutation and selection; Nester, Anderson, Roberts, Pearsall, Nester, 2004). Knowledge of acquired resistance can be helpful for the development of new antibiotics. However, the knowledge of acquired resistance, particularly vertical evolution, is limited. Information about vertical evolution would allow us to answer questions such as, Is the evolution of antibiotic resistance inevitable? and Can we predict the development of resistance? Dr. Barry Hall has suggested that the answer to the first question is Not necessarily, and to the second question is Yes (Hall, 1999). Based on Hall s research findings, he has proposed that microbes have the ability to evolve new traits, a concept he has coined as evolutionary potential. The basic idea behind evolutionary potential is that preexisting genetic information can undergo mutation and selection to produce new traits. However, that potential is probably limited: a particular gene may be able to evolve one type of new function, but may not be able to evolve a different type of function. For instance, Hall s work has shown that some antibiotic resistance genes are capable of evolving to produce resistance to newer types of antibiotics, while others are unable to evolve (2004). The concept of evolutionary potential immediately raises the question: What might allow, or limit, the evolutionary potential of a particular gene? During the 1970 s and early 1980 s, there were a number of microbial systems that showed the impressive ability of microbes to evolve new functions; many of these are found in Robert Mortlock s 1984 book, Microorganisms as 1

2 Model Systems for Studying Evolution. However, that same work and that of others showed that along with the successes were a number of failures. In reviewing some of the successful and unsuccessful examples of evolution, it appears that the evolutionary potential of a gene to evolve a new function may follow some simple, almost self-evident rules: 1) the gene subject to evolution must be transcriptionally active, or within one or two mutations of becoming so; and 2) the gene product in question must have at least some residual ability to perform the new function, or be within one or two mutations of becoming so. These rules stem from simple probability considerations: the probability of multiple independent events all occurring is the product of their individual probabilities; when three or more independent events are required for evolution to occur, the probabilities become quite small. Suppose, for example, that an advantageous mutation has a one in a million probability of occurring. Out of a population of a million individuals, on average one of them will gain this mutation and thus evolve. However, if evolution (i.e., gaining an advantageous trait) requires two independent events, both with a probability of one in a million, then only one in a trillion individuals can be expected to have both of these events occurring. Hall (1991) has stated this problem as follows: A very general problem in evolution: how is an advantageous phenotype selected when it requires multiple mutations, none of which are advantageous until all are present?...[this presents] a barrier that would appear to be difficult when two independent random mutations are required to improve fitness and insuperable when more than two are required (emphasis added). The model system Dr. Seelke has utilized to investigate evolutionary potential is the tryptophan (trp) operon of Escherichia coli. The trp operon contains five structural genes trpa through trpe. The organization of the genes and overall biochemical reaction of the operon is depicted below. Yanofsky and Crawford have identified eight critical regions of the tryptophan synthase α subunit (1972). Dr. Seelke has constructed single and double trpa mutants by mutating at two of those locations, position 49 and 60. If a microbe is trpa due to one mutation, it should readily evolve (i.e., revert) at an observable frequency. If a microbe is trpa due to two mutations, and both must revert for it to become Trp+, then evolution should, in essence, be stopped. The number of organisms that must be tested will be well into the hundreds of trillions before one could expect to find such a reversion event. I became part of this ongoing long-term evolution study when approximately 1800 generations of the double mutants were reached using a serial transfer technique. One of my objectives was to aid in the continuation of the serial transfers of the double mutant. Also, my 2

3 goal was to determine the growth rate of the double mutant at generation 0, 500, 1000, and When the long-term evolution experiment reached 2,000 generations, I sought to analyze the DNA sequence changes that might have occurred during that period of evolution. My final goal was to determine the mutational frequency of mutants with one mutation at 49 and one mutation at 60. Methods Bacterial strains, Plasmids, and Phage Strains The Escherichia coli strain used throughout this study was FTP 3917 [ (tonbtrpab)17 glyv55]. Both the trpa and trpb genes are deleted from the chromosome. The FTP 3917 served as the host for the each of the mutated pwsi plasmids (Schneider, Nichols, & Yanofsky, 1981). Plasmid pws1 contains a chloramphenicol resistance gene as well as both the trpa and trpb genes. Thus, an FTP3917 strain harboring pws1 is Trp +. All changes shown below are in the trpa gene. Plasmid Base Amino Acid Mutation Change Change Location prs201-2 gag -> gtg glu -> val 49 prs202-1 gat -> aat asp -> asn 60 prs202-5 gag -> gtg gat -> aat glu -> val asp -> asn 49 & 60 The mutations were constructed using site directed mutagenesis (Seelke, 2005). Each of the RS plasmids is like pws1 in that it carries the gene for chloramphenical (CAM) resistance. However, because of the mutation(s) in trpa, it is Trp -. Because of the active trpb gene, the mutant plasmids still allow the cell grow when indole is supplied instead of tryptophan. For convenience, FTP3917 strains containing prs201 or prs202 variants are designated RS201-2, RS202-2, and RS Media and Growth Conditions Minimal Davis (MD-MET-CAM) plates consisted of 1.5% agar containing Minimal Davis salts (Difco), 0.2% glucose, 20µg/ml DL methionine, and 20 µg/ml chloramphenicol (CAM). Each liter of medium also contained 10 mls of a 100X stock solution of NH 4 Cl (10g/100 ml) MgSO 4 ;7H 2 O (2g/100 ml) MnCl 2 (0.02 g/100ml), CaCl 2 (0.1g/100 ml) and FeSO 4 (0.05g/100ml) (per 500 mls). L-Broth is as described (Maniatis, Fritsch, & Sambrook, 1982) and supplemented with chloramphenicol (20 µg/ml) as required. If necessary, it was supplemented with tryptophan (1-20 µg/ml) or indole (10 µg/ml). Nutrient agar (NA-YE-CAM) plates consisted of Nutrient Agar (Difco), supplemented with 5g yeast extract per liter and 20µg/ml CAM. Serial Transfers Every 24 hours, a 20 μl sample of an overnight culture of A1 & A2 was inoculated into 2 mls of MD-MET-CAM broth supplemented with 1 μg/ml tryptophan. The sample was incubated at 30º C. This is a modified version of the serial transfer technique developed by Lenski (Lenski & Travisano, 1994). Each day resulted in 6.64 generations of evolution. Growth Rates Growth rates were measured by following the increase in A 425. A 0.5 ml sample of FTP RS202-5 generation 0, 500, 1000, and 1500 were inoculated into 250 ml flasks containing 50 mls MD-MET-CAM broth supplemented with 1 μg/ml tryptophan. The samples were shaken at 37 º C and the absorbance was measured every 30 minutes. Preparation for DNA Sequencing At 2000 generations, the plasmid from RS202-5 was isolated using a Qiagen midi-prep kit. The DNA sequencing will be conducted by a commercial sequencing company. Rate of Reversion One would assume that the rate of reversion for a mutant would be easily obtained by simply dividing the number of revertants in a culture (in this case obtained by plating 3

4 Growth Rate, Generations/hr The Evolutionary Potential of the TrpA gene of Escherichia coli large numbers of bacteria on a plate lacking tryptophan) by the total number of bacteria in the culture (obtained by dilution and plating onto a plate that supports growth of all bacteria). However, this number is complicated by the fact that the revertants could all be the result of individual reversion events, or they may all be the offspring of a single event. To obtain a more accurate estimate, the Luria fluctuation test (Stanier, Ingraham, Wheelis, & Painter, 1986) is used. In this test, known numbers of microbes are allowed to grow up in multiple tubes from small initial inocula. Each tube is then plated on medium lacking tryptophan. The number of tubes not having revertants, along with the total number of bacteria in each tube, allows us to estimate the reversion frequency. In our use of this test, approximately 100 cells of RS201-2 and RS was inoculated into 0.1 or 0.11 mls of L-Broth + CAM. A 10 ul sample was removed from the two 0.11 ml tubes, diluted and plated onto NA-YE-CAM plates to determine the total number of bacteria present. The tubes which contained 0.1 mls of the sample, were concentrated and resuspended in 100 ul buffered DI H 2 O. Each sample was inoculated onto MD-MET-CAM plates and incubated for two days at 37 o C. The plates were then examined for the presence of revertant colonies able to grow in the absence of tryptophan. Results Long term evolution As part of this study, we followed the evolution of RS202-5 cultures A1 and A2 by serial transfer for 2,000 generations. My involvement in this study began at about 1800 generations. These cultures are growing in a limited amount (1 µg/ml) of tryptophan, providing strong selective pressure for evolution of the ability to synthesize tryptophan. At this time of writing, 2071 generations of growth under selective conditions have occurred without either culture evolving to become Trp+. In total, over 1.5 trillion cells have been tested for evolution of a Trp+ phenotype, and over 2,000 generations of selection for evolution of a Trp+ phenotype have occurred, without such an evolutionary event (reversion) being observed (Myers, Ebnet, & Radulovic, 2006). Growth Rates of RS202-5 with evolution As shown in Figure 2, the growth rate of RS202-5 improved dramatically with evolution in the low tryptophan medium. By 500 generations, the growth rate had more than doubled, from 0.53 to 1.15 generations/hour. However, no further evolution occurred, since the growth rate at 1000 and 1500 generations was essentially the same as at 500 generations Fig. 2: Increased Fitness of RS202-5 in Low-Trp Medium Generations of Evolution 4

5 Preparation of DNA for sequencing Plasmid prs202-5 from cultures A1 and A2 was isolated using a Qiagen midi-prep. Estimated from gel electrophoresis indicate that 3-5 μg of purified plasmid was obtained, and is in a form suitable for DNA sequencing. Mutation frequency of RS201-2 This mutant, with one mutation at amino acid position 49, was tested twice for reversion frequency. Frequencies of 6 X 10-9 and 1.6 X 10-8 per cell generation were obtained. The main source of variation in these two results was due to the fact that the first test had twice the number of cells/ml as the second test, but similar numbers of plates lacking revertants. These results are quite consistent with published rates of reversion in E. coli (Klug & Cummings, 2002). Mutation Frequency of RS202-1 This mutant has a mutation at position 60. However, it may also have a frameshift mutation at position 257, near the end of the trpa gene. When ~ 10 8 RS202-1 cells are plated on a medium lacking tryptophan, thousands of small colonies appear to grow. These proved to be partially Trp +, i.e., able to grow slowly in the absence of tryptophan. No estimate of the reversion frequency was obtained because of the unusual results. Thus, RS202-1 may have a very high rate of reversion. However, because of uncertainty about the DNA sequence of this mutant, no conclusions can be reached at this time. Discussion Evolution of RS202-5 This work, and previous work, confirms that the requirement for two independent mutations presents a formidable barrier to evolution. Meyers, et al. (2006) showed that 1.5 trillion RS202-5 cells and 1600 generations were unable to evolve the Trp+ phenotype. This work extends that to over 2000 generations. This does not mean that no evolution occurred. As shown in figure 2, RS202-5 indeed evolved to become much better at growing in a medium in which tryptophan was the limiting nutrient. We do not know the details of this evolution. However, it is reasonable to assume that RS202-5 increased its ability to transport tryptophan, becoming more efficient at extracting the little tryptophan that was available. Reversion frequency of RS201-2 and RS202-1 RS201-2, with a mutation at position 49, reverts at a frequency between 6 and 16 X 10-9 per cell generation; that is, one in million cells in a culture would have this mutation. This is not an unexpected reversion frequency, considering the fact that the gene is on a plasmid, and thus has approximately 15 copies per cell that are able to evolve. We were not able to determine the reversion frequency for RS This is a mutant that was intended to have mutations at both positions 49 and 60, but DNA sequencing only showed a mutation at position 60 (Seelke, personal communication). It has an unusual tendency to produce partial revertants at a very high frequency. This plasmid may be the subject of future investigations, but more work needs to be done to determine the exact reversion frequency at position 60. In general, however, one would expect that trpa mutants of this type should revert at a similar frequency to those at position 49. If so, the expected reversion frequency for RS202-5 (which has mutations at both 49 and 60) should be 3.9 X Thus, the fact that, in 1-2 X cells and 2000 generations no evolution of this gene is observed is not unexpected. Future Goals Dr. Seelke plans to continue the serial transfers of RS202-5 in order to determine if and when reversion would take place. Also, the DNA sequence of prs202-5 generation 2000 will be compared to the DNA sequence of prs202-5 generation 0 to determine if evolution has already occurred, but is not observable. Dr. Seelke would also like to determine the impact of exaggerated mutation rates on evolution. This will be achieved by the construction of a mutator strain (MutH) and by chemical mutagenesis utilizing ethyl methansulfonate (EMS). The cause for the increased fitness of RS202-5 will also be examined. Finally, a new plasmid RS202-1 well be re-constructed and it will be used to determine the reversion frequency of mutations at position 60 in the trpa gene. 5

6 References Hall, B. G. (1991). Adaptive evolution that requires multiple spontaneous mutations: Mutations involving base substitution. Proceedings of the National Academy of Sciences of the United States of America, 88, Hall, B. G. (1999). Toward an understanding of evolutionary potential. FEMS Microbiology Letters, 178, 1-6. Hall, B. G. (2004). Predicting the evolution of antibiotic resistance genes. Nature Reviews Microbiology, 2, Klug, W., & Cummings, M. (2002). Essentials of Genetics (4 th ed.). Upper Saddle River, NJ: Prentice Hall. Lenski, R. E., & M. Travisano. (1994). Dynamics of adaptation and diversification: A 10,000- generation experiment with bacterial populations. The Proceedings of the National Academy of Sciences, 91, Maniatis, T., Fritsch, E. F., & Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual. Cold Springs Harbor, New York:Cold Springs Harbor Laboratory. Mortlock, R. P. (1984). Microorganisms as Model Systems for Studying Evolution. New York:Plenum Press. Myers, L., Ebnet, S., & Radulovic, M. (2006, May 5). Evolutionary Potential of the TrpA Gene of Escherichia Coli. Presented at the 8 th Annual UW-Superior Undergraduate Research Symposium, University of Wisconsin-Superior. Nester, E. W., Anderson, D. G., Roberts, C. E., Pearsall, N. N., & Nester, M. T. (2003). Microbiology: A human perspective (4 ed.). New York:McGraw-Hill. Schneider, W., Nichols, B., & Yanofsky, C. (1981). Procedure for production of hybrid genes and proteins and its use in assessing significance of amino acid differences in homologus tryptophan synthetase α polypeptides. The Proceedings of the National Academy of Sciences, 78, Seelke, R. (2005, June 5-9). A set of mutants for examining the evolutionary potential of the trpa gene. Presented at the 105 th Annual Meeting, Abstract R-032, American Society for Microbiology, Atlanta, GA. Stanier, R., Ingraham, J., Wheelis, M., & Painter, P. (1986). The Microbial World (5 ed.) (pp ). Englewood Cliffs, NJ: Prentice-Hall. Yanofsky, C., & Crawford. (1972). Tryptophan Synthetase. In P.D. Boyer (Eds.), The Enzymes (3 ed) (pp. 1-31). Orlando, FL:Academic Press. 6