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Title: Adaptation and divergence in experimental populations of the bacterium escherichia coli : the roles of environment, phylogeny and chance
Creator: Travisano, Michael
Date: 1993
Collection: Electronic Theses & Dissertations

Title: An analysis of fitness in long-term asexual evolution experiments
Creator: Wiser, Michael J.
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Evolution is the central unifying concept of modern biology. Yet it can be hard to study in natural system, as it unfolds across generations. Experimental evolution allows us to ask questions about the process of evolution itself: How repeatable is the evolutionary process? How predictable is it? How general are the results? To address these questions, my collaborators and I carried out experiments both within the Long-Term Evolution Experiment (LTEE) in the bacteria Escherichia coli, and the...
Show moreEvolution is the central unifying concept of modern biology. Yet it can be hard to study in natural system, as it unfolds across generations. Experimental evolution allows us to ask questions about the process of evolution itself: How repeatable is the evolutionary process? How predictable is it? How general are the results? To address these questions, my collaborators and I carried out experiments both within the Long-Term Evolution Experiment (LTEE) in the bacteria Escherichia coli, and the digital evolution software platform Avida. In Chapter 1, I focused on methods. Previous research in the LTEE has relied on one particular way of measuring fitness, which we know becomes less precise as fitness differentials increase. I therefore decided to test whether two alternate ways of measuring fitness would improve precision, using one focal population. I found that all three methods yielded similar results in both fitness and coefficient of variation, and thus we should retain the traditional method.In Chapter 2, I turned to measuring fitness in each of the populations. Previous work had considered fitness to change as a hyperbola. A hyperbolic function is bounded, and predicts that fitness will asymptotically approach a defined upper bound; however, we knew that fitness in these populations routinely exceeded the asymptotic limit calculated from a hyperbola fit to the earlier data. I instead used to a power law, a mathematical function that does not have an upper bound. I found that this function substantially better describes fitness in this system, both among the whole set of populations, and in most of the individual populations. I also found that the power law models fit on just early subsets of the data accurately predict fitness far into the future. This implies that populations, even after 50,000 generations of evolution in consistent environment, are so far from the tops of fitness peaks that we cannot detect evidence of those peaks.In Chapter 3, I examined to how variance in fitness changes over long time scales. The among-population variance over time provides us information about the adaptive landscape on which the populations have been evolving. I found that among-population variance remains significant. Further, competitions between evolved pairs of populations reveal additional details about fitness trajectories than can be seen from competitions against the ancestor. These results demonstrate that our populations have been evolving on a complex adaptive landscape.In Chapter 4, I examined whether the patterns found in Chapter 2 apply to a very different evolutionary system, Avida. This system incorporates many similar evolutionary pressures as the LTEE, but without the details of cellular biology that underlie nearly all organic life. I find that in both the most complex and simplest environments in Avida, fitness also follows the same power law dynamics as seen in the LTEE. This implies that power law dynamics may be a general feature of evolving systems, and not dependent on the specific details of the system being studied.
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Title: Anti-late RNA and transcriptional control in T4 bacteriophage infected Escherichia coli
Creator: Frederick, Robert John, 1944-
Date: 1976
Collection: Electronic Theses & Dissertations

Title: Bioactivities of bovine anti-coliform antibodies elicited by J5 vaccinations
Creator: Chaiyotwittayakun, Anatachai
Date: 2003
Collection: Electronic Theses & Dissertations

Title: Bioavailability of tetracycline in water and soil to Escherichia coli for expression of antibiotic resistance
Creator: Zhang, Yingjie
Date: 2013
Collection: Electronic Theses & Dissertations
Description: Tetracyclines are a class of antimicrobials extensively used as human and veterinary medicine, and in livestock production since they were discovered in the 1940s. A large portion of tetracyclines administered to humans and animals are excreted and subsequently released into the environment, where they pose potential risks to ecosystem and human health. There is a growing concern that the presence of antibiotics such as tetracycline at trace levels in the environment is related to the...
Show moreTetracyclines are a class of antimicrobials extensively used as human and veterinary medicine, and in livestock production since they were discovered in the 1940s. A large portion of tetracyclines administered to humans and animals are excreted and subsequently released into the environment, where they pose potential risks to ecosystem and human health. There is a growing concern that the presence of antibiotics such as tetracycline at trace levels in the environment is related to the emergence and ever-increasing abundance of antibiotic resistance genes in natural and engineered microbial populations. However, basic knowledge at the molecular scale of bacterial access to tetracyclines present in environmental matrices and expression of antibiotic resistance genes remain nearly unknown. In this study, we used the E. coli MC4100/pTGM whole-cell bioreporter as an effective tool to investigate bioavailability of tetracycline in water and soil to bacteria for expression of antibiotic resistance genes. Our hypothesis was that the speciation of tetracycline dissolved in water and sorption by soil minerals controls the bioavailabilities for bacterial uptake and subsequent activation of antibiotic resistance genes. The results revealed that activation of antibiotic resistance in the E. coli bioreporter responded linearly to intracellular tetracycline concentration. The extent of tetracycline uptake by E. coli was modulated by tetracycline speciation. We have identified that zwitterionic tetracycline as the primary species favorable for bacterial uptake. Geochemical factors such as pH, salt composition and concentration influenced the fractional distributions of tetracycline species in aqueous solution and hence altered uptake by E. coli. In addition, the presence of organic ligands could also alter tetracycline speciation by releasing tetracycline from its metal complexes in aqueous solution. For tetracycline associated with Mg-smectite, desorption of tetracycline from clay to solution was the major exposure pathway for bacterial uptake and subsequent activation of antibiotic resistance in the diluted clay suspensions. In clay film cultivation, clay-sorbed tetracycline was still bioaccessible to E. coli evoking strong expression of antibiotic resistance. Direct contact of the E. coli bioreporters with clay surfaces and further formation of biofilms plausibly facilitated tetracycline transfer to bacteria. Overall, this study greatly advances the fundamental understanding of bioavailability of tetracycline in the environment to bacteria for expression of antibiotic resistance genes.
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Title: Biological activity of the lipids from Escherichia coli and Shigella dysenteriae and their characterization by means of infrared spectrophotometry
Creator: Neblett, Thomas Randolph, 1928-
Date: 1957
Collection: Electronic Theses & Dissertations

Title: Breaking biofilms : regulation of Type II secretion system in V. cholerae and the formation of the hyper-pseudopilus
Creator: Sloup, Rudolph E.
Date: 2016
Collection: Electronic Theses & Dissertations
Description: Vibrio cholerae is the causative agent of the human disease cholera, it resides in aquatic resevoirs and forms biofilms, which are closely associated communities of bacteria embedded in polysaccharides, DNA, and proteins. In V. cholerae biofilm formation is regulated by the second messenger molecule cyclic di-GMP (c-di-GMP). A genetic screen for promoters regulated by the c-di-GMP revealed a novel promoter (PepsG) in the eps operon encoding the V. cholerae Type 2 secretion system (T2SS). The...
Show moreVibrio cholerae is the causative agent of the human disease cholera, it resides in aquatic resevoirs and forms biofilms, which are closely associated communities of bacteria embedded in polysaccharides, DNA, and proteins. In V. cholerae biofilm formation is regulated by the second messenger molecule cyclic di-GMP (c-di-GMP). A genetic screen for promoters regulated by the c-di-GMP revealed a novel promoter (PepsG) in the eps operon encoding the V. cholerae Type 2 secretion system (T2SS). The T2SS, which exports proteins from the periplasm to the extracellular space, is phylogenetically related to Type 4 pili. The major pseudopilin is encoded by epsG which forms a short piston like structure necessary for secretion. I hypothesized that differential regulation of the eps operon extends the pseudopilin forming a structure called a hyper-pseudopilus outside the cell where it promotes biofilm development. In Chapter 2, I determined that the promoter upstream of the operon (PepsC1) is induced four fold by c-di-GMP and this induction is mediated by the c-di-GMP binding transcription factor VpsR directly. High levels of c-di-GMP were found to decrease the activity of extra cellular proteases secreted by the T2SS, however this effect was not a direct result of regulation of the T2SS as determined by mutation of the VpsR binding site in PepsC1. I was unable to establish a phenotype for the transcriptional control of the eps operon. This work establishes T2S as a new phenotype which is transcriptionally controlled by c-di-GMP and the biofilm associated transcription factor VpsR. In Chapter 3, I show that overexpression of epsG in a continuous flow cell system increased V. cholerae biofilms while a ΔepsG strain showed no biofilm formation. However, there was no change in activity of T2S dependent serine proteases while epsG was over expressed indicating increased biofilms is not likely due to increased secretion. Polyclonal antibody stained EpsG was also detectable on the surface of WT cells and long pseudopili were visualized with over expression of epsG. This evidence suggests the T2SS forms a hyper-pseudopilus important for biofilm formation. In Chapter 4, I present my work identifying novel anti-biofilm compounds. In 2011 Escherichia coli O104:H4 caused the deadliest E. coli outbreak in modern times resulting in 54 deaths and the highest rate of hemolytic uremic syndrome ever recorded. Subsequently, we showed a correlation between biofilm gene expression and virulence factor expression. I sought to identify small molecule compounds effective at inhibiting O104:H4 biofilms. I discovered at a concentration of 0.01% the nonionic surfactants polysorbate 80 (PS80) and polysorbate 20 (PS20) were found to inhibit biofilm formation by 90% and 91% respectively. These compounds were able to disperse preformed biofilms. Treatment of mice infected with E. coli O104:H4 resulted in high bacterial loads and inflammation. While addition of PS80 in the drinking water of the mice did not reduce bacterial loads, it completely abolished inflammation symptoms. PS80 is an FDA approved compound, well studied and effective at low nanomolar concentrations that reduces symptoms of infection in mice. which establishes it as an excellent candidate for further study as an anti-infective agent with anti-biofilm capabilities
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Title: Cell division in cis-platinum(II)diamminodichloride-induced filaments of Escherichia coli : dependence upon completion of deoxyribonucleic acid repair and a protease activity
Creator: Markham, Bruce Edward
Date: 1980
Collection: Electronic Theses & Dissertations

Title: Characterization of OmpC and OmpF porins from Escherichia coli K-12
Creator: Rocque, Warren J.
Date: 1990
Collection: Electronic Theses & Dissertations

Title: Cloning of the polysaccharide depolymerase gene of bacteriophage pea1 (h) and its expression in Erwinia amylovora
Creator: Hartung, John Stephen
Date: 1985
Collection: Electronic Theses & Dissertations

Title: Coevolution of bacterial-phage interactions
Creator: Meyer, Justin R.
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Bacteria and their viruses, phage, are the most abundant and genetically diverse group of organisms on earth. Given their prevalence, it is no wonder that recent studies have found their interactions important for ecosystem function, as well as the health of humans. Unfortunately, because of technical challenges with studying microbes, some of the most basic questions on their interactions, such as who infects whom, and how their relationships evolved in the first place, remain unanswered....
Show moreBacteria and their viruses, phage, are the most abundant and genetically diverse group of organisms on earth. Given their prevalence, it is no wonder that recent studies have found their interactions important for ecosystem function, as well as the health of humans. Unfortunately, because of technical challenges with studying microbes, some of the most basic questions on their interactions, such as who infects whom, and how their relationships evolved in the first place, remain unanswered. Here I report six studies on bacterial-phage interactions, each focused on understanding their pattern and the underlying biophysical, ecological, and evolutionary processes that shape them. To do this, I tested a number of hypotheses using laboratory experiments and analyses of natural microbial diversity. First, I tested whether Esherichia coli cultured without phage would counter-intuitively evolve new interactions with phage. Typically bacterial traits responsible for phage resistance have pleiotropic consequences on growth, therefore as a side-effect of adapting to an abiotic environment, bacteria may also evolve to become more or less vulnerable to their parasites. After 45,000 generations of laboratory culturing without phage, E. coli gained resistance to lambda phage, gained sensitivity to a mutant T6 phage, and remained resistant to wild type T6. Each response was explained by understanding the pleiotropic costs or benefits of mutations that confer resistance. Because of pleiotropy, interactions may even evolve in the absence of one player.For the rest of my studies I examined how interactions evolve when host and parasite co-occur. First, I found that when E. coli and phage lambda are cocultured, E. coli evolves resistance by reducing the number of phage genotypes that can infect it, whereas, lambda evolves to increase the number of bacterial genotypes it can infect. This antagonism produces a interaction matrix with a nested form where less derived host-ranges fall one within another. To determine whether this nested pattern is an artifact of the labratory environment, or if the pattern is general to natural communities, I performed a metaanalysis on already published phage-bacterial interaction matrices. The majority of networks were significantly nested (28 of 38). Lastly, I examined the molecular basis of E. coli resistance to lambda and found that resistance often evolves through mutations in E. coli's lamB, the gene for the phage receptor. Also, the strength of resistance is correlated with how the mutation perturbs the orientation specific features of the protein structure, primarily loop four which extends out of the cell membrane. For the final two chapters, I studied whether lambda could evolve to target a novel receptor and the evolutionary consequences of such an innovation. Under particular laboratory conditions, E. coli evolves resistance by down-regulating LamB, which sets the stage for lambda to evolve the necessary mutations to exploit a new protein receptor. When allowed to coevolve under this condition, lambda evolved to exploit another outer-membrane protein, OmpF. This new function is the result of a particular combination of four mutations in J, the gene for the protein ligand lambda uses to bind to its host. Once lambda evolves this novel interaction, an evolutionary arms-race begins that drives rapid diversification of the bacteria and phage. Overall, my studies show that coevolution between bacteria and phage, whether it be in the lab or in nature, produces nested interactions matrices. Secondly, antagonistic coevolution is a creative process able to generate new genotypes of host and parasite and promote the evolution of novel function. Lastly, costs for resistance have many important effects, from determining whether resistance will evolve or be lost, to the generation of diversity.
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Title: Conversion of the mitochondrial gene for mammalian cytochrome oxidase subunit II to a universal equivalent and expression in E. coli, in vitro, and in Xenopus oocytes
Creator: Cao, Jianli
Date: 1990
Collection: Electronic Theses & Dissertations

Title: Deconstructing the correlated nature of ancient and emergent traits : an evolutionary investigation of metabolism, morphology, and mortality
Creator: Grant, Nkrumah Alions
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Phenotypic correlations are products of genetic and environmental interactions, yet the nature of these correlations is obscured by the multitude of genes organisms possess. My dissertation work focused on using 12 populations of Escherichia coli from Richard Lenski's long-term evolution experiment (LTEE) to understand how genetic correlations facilitate or impede an organism's evolution. In chapter 1, I describe how ancient correlations between aerobic and anaerobic metabolism have...
Show morePhenotypic correlations are products of genetic and environmental interactions, yet the nature of these correlations is obscured by the multitude of genes organisms possess. My dissertation work focused on using 12 populations of Escherichia coli from Richard Lenski's long-term evolution experiment (LTEE) to understand how genetic correlations facilitate or impede an organism's evolution. In chapter 1, I describe how ancient correlations between aerobic and anaerobic metabolism have maintained - and even improved - the capacity of E. coli to grow in an anoxic environment despite 50,000 generations of relaxed selection for anaerobic growth. I present genomic evidence illustrating substantially more mutations have accumulated in anaerobic-specific genes and show parallel evolution at two genetic loci whose protein products regulate the aerobic-to-anaerobic metabolic switch. My findings reject the "if you don't use it, you lose it" notion underpinning relaxed selection and show modules with deep evolutionary roots can overlap more, hence making them harder to break. In chapter 2, I revisit previous work in the LTEE showing that the fitness increases measured for the 12 populations positively correlated with an increase in cell size. This finding was contrary to theory predicting smaller cells should have evolved. Sixty thousand generations have surpassed since that initial study, and new fitness data collected for the 12 populations show fitness has continued to increase over this period. Here, I asked whether cell size also continued to increase. To this end, I measured the size of cells for each of the 12 populations spanning 50,000 generations of evolution using a particle counter, microscopy, and machine learning. I show cell size has continued to increase and that it remains positively correlated with fitness. I also present several other observations including heterogeneity in cell shape and size, parallel mutations in cell-shape determining genes, and elevated cell death in the single LTEE population that evolved a novel metabolism - namely the ability to grow aerobically on citrate. This last observation formed the basis of my chapter 3 research where my collaborators and I fully examine the cell death finding and the associated genotypic and phenotypic consequences of the citrate metabolic innovation.
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Title: Design and evaluation of immunogenic Escherichia coli heat-stable enterotoxin (STa) and characterization of the immune response in laboratory animals
Creator: Aref, Nasr-Eldin Mohamed M.
Date: 2008
Collection: Electronic Theses & Dissertations

Title: Development of a nanoparticle DNA based biosensor for the Shiga-like toxin 1 detection using co-polymerization hybridization readout amplification
Creator: Anderson, Michael Jeffrey
Date: 2012
Collection: Electronic Theses & Dissertations
Description: Approximately 3.4 million deaths occur each year due to lack of clean water. The Shigatoxigenic group of Escherichia coli (STEC) bacteria causes approximately 2.2 million of these deaths and nearly a billion cases of illness. Current approved testing methods for Escherichia coli require a 12-24 hours growth of the sample in order to test for an indicator species. This indicator species only suggests possible E. coli O157:H7 presence, but does not confirm it. Direct culture testing cannot...
Show moreApproximately 3.4 million deaths occur each year due to lack of clean water. The Shigatoxigenic group of Escherichia coli (STEC) bacteria causes approximately 2.2 million of these deaths and nearly a billion cases of illness. Current approved testing methods for Escherichia coli require a 12-24 hours growth of the sample in order to test for an indicator species. This indicator species only suggests possible E. coli O157:H7 presence, but does not confirm it. Direct culture testing cannot identify the pathogenic STEC bacteria either. Confirmation of toxicity requires molecular methods of identification leading to specialized equipment needs and higher testing costs. Typical molecular methods use polymerase chain reaction (PCR) to amplify a very specific target DNA sequence, indicating the presence of a specific microorganism. PCR requires a thermocycler and a clean laboratory environment. Recent work has been successfully explored using nanomaterial based systems to identify the same genes used in a PCR reaction in a simpler detection system. The nanomaterials are used for both signal amplification and as the detection molecule. In this dissertation work a biosensor utilizing nanoparticles has been developed for the detection of Shiga-like toxin 1 gene that is present in Escherichia coli O157:H7. The system has been successfully constructed using a novel carbohydrate coated gold nanoparticle for delivery of the self assembling co-polymerization DNA oligonucleotide reporters. A set of self-assembled single-stranded DNA (ssDNA) molecules has been developed to amplify a target sequence without the use of enzymes. The full detection system incorporates the gold nanoparticle and a magnetic microparticle into a DNA recognition step. The gold nanoparticle is used for both co-polymerization detection and electrochemical detection. Input material (genomic DNA) is extracted using a modified commercial extraction method to produce PCR quality DNA from whole bacterial cells. When extraction and recognition elements are combined, a limit of detection for E. coli O157:H7 of 105 cells/mL using co-polymerization and 101 cells/mL with electrochemical detection has been achieved. Sample preparation from spiked culture samples until final detection takes a total of 7 hours. Electrochemical detection provides only a presence or absence indicator, where tethered co-polymerization is able to provide quantitative values of the input bacterial concentration. So a combination of co-polymerization amplification and electrochemical detection can provide the potential for more sensitive and quantitative measurement without the need for enzymes as in PCR applications.
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Title: Development of a resistance based biosensor utilizing conducting microfibers for microbial pathogen detection
Creator: McGraw, Shannon Katie
Date: 2013
Collection: Electronic Theses & Dissertations
Description: Escherichia coli O157:H7 (E. coli O157:H7) is one of the U.S. military's top pathogens of interest for the development of rapid diagnostic systems. The enteric pathogen can cause severe gastroenteritis and is spread through the consumption of contaminated food and water. This is of concern to the U.S. military and warfighter because an outbreak of diarrheal disease in the field has the ability to rapidly render a large number of warfighters ineffective in...
Show moreEscherichia coli O157:H7 (E. coli O157:H7) is one of the U.S. military's top pathogens of interest for the development of rapid diagnostic systems. The enteric pathogen can cause severe gastroenteritis and is spread through the consumption of contaminated food and water. This is of concern to the U.S. military and warfighter because an outbreak of diarrheal disease in the field has the ability to rapidly render a large number of warfighters ineffective in performing their duties. Current field &ldquoportable&rdquo detection technologies can be cumbersome and require generous quantities of chemicals to operate. In addition, the current FDA gold standard for identification of this pathogen from food matrices takes up to 3 days to generate a confirmed positive result. The objective of this dissertation research was to develop a rapid, novel electrochemical biosensor based on the use of polypropylene microfiber membranes coated with a conductive polypyrrole and antibody functionalized for the biological capture and detection of E. coli O157:H7. In this dissertation research, an electrotextile composed of conductive polymer coated microfibers containing functional attachment sites for biorecognition elements was developed. The electrotextiles were optically and electrically assessed based on the polymerization chemicals and reaction time to determine how these factors affected the resistance of the fibers. Based on these experiments, a mathematical model was developed, optimized, and validated. Various methods of antibody immobilization and surface blocking on the fibers were also assessed. Using glutaraldehyde, pathogen specific antibodies were covalently attached to the conductive microfiber electrotextiles which were then blocked using a 5% bovine serum albumin solution. The functionalized membranes were exposed to E. coli O157:H7 cells, washed in Butterfield's phosphate buffer and added to a phosphate buffer electrolyte solution. When a voltage was applied to the system, the presence of the captured pathogen on the fiber surface resulted in an increase in resistance at the electrotextile electrode surface, indicating a positive result. It was found that the conductivity of the components of the system, other than the electrotextile fibers, was not statistically significant. Proof&ndashof&ndashconcept experiments were conducted and it was determined that the electrotextile electrode was able to differentiate between positive and negative samples using the pathogen E. coli O157:H7 cells as the target over a concentration range of 100 &ndash 109 colony forming units per milliliter (CFU/mL). The reproducibility of the sensor results was tested and it was found that the trends in the biosensor results were reproducible. By testing the significance of the biosensor response it was determined that the biosensor can successfully function as a yes / no screening system. The results show that the biosensor has an experimental lower limit of detection of 3.23 x 100 CFU/mL for the detection of E. coli O157:H7 in pure culture.
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Title: Electronic sensor array incorporating artificial neural network algorithms for rapid identification and quantification of Escherichia coli and Salmonella enterica serovar Typhimurium and their volatile metabolites
Creator: Ubonrat Siripatrawan
Date: 2002
Collection: Electronic Theses & Dissertations

Title: Engineering applications of microbial chemotaxis
Creator: Widman, Mark Thomas
Date: 1997
Collection: Electronic Theses & Dissertations

Title: Enteric colibacillosis in gnotobiotic swine : fluorescence and electron microscopic studies
Creator: Drees, David T., 1933-
Date: 1969
Collection: Electronic Theses & Dissertations

Title: Experimental evolution and ecological consequences : new niches and changing stoichiometry
Creator: Turner, Caroline B.
Date: 2015
Collection: Electronic Theses & Dissertations
Description: Evolutionary change can alter the ecological conditions in which organisms live and continue to evolve. My dissertation research used experimental evolution to study two aspects of evolutionary change with ecological consequences: the generation of new ecological niches and evolution of the elemental composition of biomass. I worked with the long-term evolution experiment (LTEE), which is an ongoing experiment in which E. coli have evolved under laboratory conditions for more than 60,000...
Show moreEvolutionary change can alter the ecological conditions in which organisms live and continue to evolve. My dissertation research used experimental evolution to study two aspects of evolutionary change with ecological consequences: the generation of new ecological niches and evolution of the elemental composition of biomass. I worked with the long-term evolution experiment (LTEE), which is an ongoing experiment in which E. coli have evolved under laboratory conditions for more than 60,000 generations. The LTEE began with extremely simple ecological conditions. Twelve populations were founded from a single bacterial genotype and growth was limited by glucose availability. In Chapter 1, I focused on a population within the LTEE in which some of the bacteria evolved the ability to consume a novel resource, citrate. Citrate was present in the growth media throughout the experiment, but E. coli is normally unable to consume it under aerobic conditions. The citrate consumers (Cit+) coexisted with a clade of bacteria which were unable to consume citrate (Cit-). Specialization on glucose, the standard carbon source in the LTEE, was insufficient to explain the frequency-dependent coexistence of Cit- with Cit+. Instead Cit– evolved to cross-feed on molecules released by Cit+. The evolutionary innovation of citrate consumption led to a more complex ecosystem in which two co-existing ecotypes made use of five different carbon sources.After 10,000 generations of coexistence, Cit- went extinct from the population (Chapter 2). I conducted replay experiments, re-evolving for 500 generations 20 replicate populations from prior to extinction. Cit- was retained in all populations, indicating that the extinction was not deterministic. Furthermore, when I added small numbers of Cit- to the population after extinction, Cit- was able to reinvade. It therefore appears that the Cit- extinction was not due to exclusion by Cit+, but rather to unknown laboratory variation.Chapter 3 shifts focus to studying evolutionary changes in stoichiometry, the ratio of different elements within organisms’ biomass. Variation in stoichiometry between organisms has important ecological consequences, but the evolutionary origin of that variation had not previously been studied experimentally. Growth in the LTEE is carbon limited and nitrogen and phosphorus are abundant. Additionally, daily transfer to fresh media selects for increased growth rate, which other research has suggested correlates to higher phosphorus content. Consistent with our predictions based on this environment, clones isolated after 50,000 generations of evolution had significantly higher nitrogen and phosphorus content than ancestral clones. There was no change in the proportion of carbon in biomass, but the total amount of carbon retained in biomass increased, indicating that the bacteria also evolved higher carbon use efficiency.To test whether the increases in nitrogen and phosphorus observed in the LTEE were a result of carbon limitation or were side effects of other selective factors in the experiment, I evolved clones from the LTEE for 1000 generations under nitrogen rather than carbon limitation (Chapter 4). The stoichiometry of the bacteria did change over the course of 1000 generations, indicating that evolution of stoichiometry can occur over relatively short time frames. Unexpectedly however, the evolved bacteria had higher nitrogen and phosphorus content. It appears that the bacteria were initially poor at incorporating nitrogen into biomass, but evolved improved nitrogen uptake.
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