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1. Predicting parallelism and quantifying divergence in experimental evolution

   https://doi.org/10.1101/2020.05.13.070953  

   Shoemaker WR, Lennon JT (January 2021, bioRxiv)

   The degree that the environment determines what genes contribute towards adaptation is a fundamental question in microbial evolution. Microbial populations are often experimentally passaged in different environments and sequenced in order to identify candidates for adaptation in a particular environment. However, there remains the need to develop an appropriate statistical framework to identify genes that acquired more mutations in one environment over the other (i.e., divergent evolution). Here we demonstrate how the evolutionary outcomes among replicate populations in the same environment, known as parallel evolution, can be leveraged to construct an intuitive statistical test for identifying the genes that contribute towards divergent evolution. To accomplish this task, we examined publicly available evolve-and-resequence experiment datasets and found that the distribution of mutation counts among genes can be predicted using an ensemble of independent Poisson random variables. Building on this result, we propose that the degree of divergent evolution at a given gene between populations from two different environments can be modeled as the difference between two Poisson random variables, known as the Skellam distribution. We then propose and apply a statistical test to identify specific genes that contribute towards divergent evolution. IMPORTANCE: There is currently no existing framework that can be leveraged to identify genes that contribute towards divergent evolution in microbial evolution experiments. To correct for this absence, we investigated the distribution of mutation counts among genes in order to identify an appropriate null model. Our observations suggest that divergent evolution within a given gene can be modeled as the difference in the total number of mutations observed between two environments. This quantity is described by a probability distribution known as the Skellam distribution, providing an appropriate statistical test for researchers seeking to identify the set of genes that contribute towards divergent evolution in evolution experiments. 

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2. Historical Contingency Causes Divergence in Adaptive Expression of the lac Operon

   https://doi.org/10.1093/molbev/msab077  

   Karkare, Kedar; Lai, Huei-Yi; Azevedo, Ricardo B.R.; Cooper, Tim F. (March 2021, Molecular Biology and Evolution)

   Wittkopp, Patricia (Ed.)

   Abstract Populations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that cause constitutive expression of the lac operon. These mutations occur at a high rate and provide a significant benefit. Despite this, eight of 24 populations evolved for 8,000 generations in environments containing lactose contained no detectable repressor mutations. We report here on the basis of this observation. We find that, given relevant mutation rates, repressor mutations are expected to have fixed in all evolved populations if they had maintained the same fitness effect they confer when introduced to the ancestor. In fact, reconstruction experiments demonstrate that repressor mutations have become neutral or deleterious in those populations in which they were not detectable. Populations not fixing repressor mutations nevertheless reached the same fitness as those that did fix them, indicating that they followed an alternative evolutionary path that made redundant the potential benefit of the repressor mutation, but involved unique mutations of equivalent benefit. We identify a mutation occurring in the promoter region of the uspB gene as a candidate for influencing the selective choice between these paths. Our results detail an example of historical contingency leading to divergent evolutionary outcomes. 

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3. Divergent Evolution of Mutation Rates and Biases in the Long-Term Evolution Experiment with Escherichia coli

   https://doi.org/10.1093/gbe/evaa178  

   Maddamsetti, Rohan; Grant, Nkrumah A (September 2020, Genome Biology and Evolution)

   Zhang, George (Ed.)

   Abstract All organisms encode enzymes that replicate, maintain, pack, recombine, and repair their genetic material. For this reason, mutation rates and biases also evolve by mutation, variation, and natural selection. By examining metagenomic time series of the Lenski long-term evolution experiment (LTEE) with Escherichia coli (Good BH, McDonald MJ, Barrick JE, Lenski RE, Desai MM. 2017. The dynamics of molecular evolution over 60,000 generations. Nature 551(7678):45–50.), we find that local mutation rate variation has evolved during the LTEE. Each LTEE population has evolved idiosyncratic differences in their rates of point mutations, indels, and mobile element insertions, due to the fixation of various hypermutator and antimutator alleles. One LTEE population, called Ara+3, shows a strong, symmetric wave pattern in its density of point mutations, radiating from the origin of replication. This pattern is largely missing from the other LTEE populations, most of which evolved missense, indel, or structural mutations in topA, fis, and dusB—loci that all affect DNA topology. The distribution of mutations in those genes over time suggests epistasis and historical contingency in the evolution of DNA topology, which may have in turn affected local mutation rates. Overall, the replicate populations of the LTEE have largely diverged in their mutation rates and biases, even though they have adapted to identical abiotic conditions. 

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4. Parallel Evolution in Predatory Bdellovibrio sp. NC01 during Long-Term Coculture with a Single Prey Strain

   https://doi.org/10.1128/aem.01776-22  

   Mulvey, Kathryn; Brosnan, Katherine; Galvin, Mackenzie; Mohr, Sydney; Muldowney, Lauren; Oser, Molly; Williams, Laura E. (January 2023, Applied and Environmental Microbiology)

   Villanueva, Laura (Ed.)

   ABSTRACT Experimental evolution provides a powerful tool for examining how Bdellovibrio evolves in response to unique selective pressures associated with its predatory lifestyle. We tested how Bdellovibrio sp. NC01 adapts to long-term coculture with Pseudomonas sp. NC02, which is less susceptible to predation compared to other Gram-negative bacteria. Analyzing six replicate Bdellovibrio populations across six time points spanning 40 passages and 2,880 h of coculture, we detected 30 to 40 new mutations in each population that exceeded a frequency of 5%. Nonsynonymous substitutions were the most abundant type of new mutation, followed by small indels and synonymous substitutions. After completing the final passage, we detected 20 high-frequency (>75%) mutations across all six evolved Bdellovibrio populations. Eighteen of these alter protein sequences, and most increased in frequency rapidly. Four genes acquired a high-frequency mutation in two or more evolved Bdellovibrio populations, reflecting parallel evolution and positive selection. The genes encode a sodium/phosphate cotransporter family protein (Bd2221), a metallophosphoesterase (Bd0054), a TonB family protein (Bd0396), and a hypothetical protein (Bd1601). Tested prey range and predation efficiency phenotypes did not differ significantly between evolved Bdellovibrio populations and the ancestor; however, all six evolved Bdellovibrio populations demonstrated enhanced starvation survival compared to the ancestor. These results suggest that, instead of evolving improved killing of Pseudomonas sp. NC02, Bdellovibrio evolved to better withstand nutrient limitation in the presence of this prey strain. The mutations identified here point to genes and functions that may be important for Bdellovibrio adaptation to the different selective pressures of long-term coculture with Pseudomonas . IMPORTANCE Bdellovibrio attack and kill Gram-negative bacteria, including drug-resistant pathogens of animals and plants. This lifestyle is unusual among bacteria, and it imposes unique selective pressures on Bdellovibrio . Determining how Bdellovibrio evolve in response to these pressures is valuable for understanding the mechanisms that govern predation. We applied experimental evolution to test how Bdellovibrio sp. NC01 evolved in response to long-term coculture with a single Pseudomonas strain, which NC01 can kill, but with low efficiency. Our experimental design imposed different selective pressures on the predatory bacteria and tracked the evolutionary trajectories of replicate Bdellovibrio populations. Using genome sequencing, we identified Bdellovibrio genes that acquired high-frequency mutations in two or more populations. Using phenotype assays, we determined that evolved Bdellovibrio populations did not improve their ability to kill Pseudomonas , but rather are better able to survive starvation. Overall, our results point to functions that may be important for Bdellovibrio adaptation. 

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5. Parallel Evolution towards Increased Motility in Long-Term Cultures of Escherichia coli, Even Though Motility was Not Required for Long-Term Survival

   https://doi.org/10.1128/spectrum.02330-21  

   Henderson, Autumn L.; Moreno, Angie; Kram, Karin E. (June 2022, Microbiology Spectrum)

   Gao, Beile (Ed.)

   ABSTRACT Escherichia coli can survive for long periods in batch culture in the laboratory, where they experience a stressful and heterogeneous environment. During this incubation, E. coli acquires mutations that are selected in response to this environment, ultimately leading to evolved populations that are better adapted to these complex conditions, which can lead to a better understanding of evolutionary mechanisms. Mutations in regulatory genes often play a role in adapting to heterogeneous environments. To identify such mutations, we examined transcriptional differences during log phase growth in unaged cells compared to those that had been aged for 10 days and regrown. We identified expression changes in genes involved in motility and chemotaxis after adaptation to long-term cultures. We hypothesized that aged populations would also have phenotypic changes in motility and that motility may play a role in survival and adaptation to long-term cultures. While aged populations did show an increase in motility, this increase was not essential for survival in long-term cultures. We identified mutations in the regulatory gene sspA and other genes that may contribute to the observed differences in motility. Taken together, these data provide an overall picture of the role of mutations in regulatory genes for adaptation while underscoring that all changes that occur during evolution in stressful environments are not necessarily adaptive. IMPORTANCE Understanding how bacteria adapt in long-term cultures aids in both better treatment options for bacterial infections and gives insight into the mechanisms involved in bacterial evolution. In the past, it has been difficult to study these organisms in their natural environments. By using experimental evolution in heterogeneous and stressful laboratory conditions, we can more closely mimic natural environments and examine evolutionary mechanisms. One way to observe these mechanisms is to look at transcriptomic and genomic data from cells adapted to these complex conditions. Here, we found that although aged cells increase motility, this increase is not essential for survival in these conditions. These data emphasize that not all changes that occur due to evolutionary processes are adaptive, but these observations could still lead to hypotheses about the causative mutations. The information gained here allow us to make inferences about general mechanisms underlying phenotypic changes due to evolution. 

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