Research Topics
1) Bacterial adaptation in response to repeated resource limitation.
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Microbes are robust organisms capable of persisting in the face of extreme environments. In nature, access to nutrients is not always consistent and populations commonly experience cycles of feast and famine. To further understand how microbes persist and eventually thrive in environments where nutrient availability is sporadic, we are performing evolution experiments varying intervals between feeding times in Escherichia coli populations. We will then characterize these lines to understand how microbes adapt to starvation and how these adaptations are constrained by the need to maintain fitness when resources are replenished.
Current questions include:
1) How does evolutionary history affects future adaptive trajectories?
(How does previous exposure to a specific interval of feast/famine affect evolution when the environment changes)
2) Is evolution predictable across a gradient of feast/famine intervals?
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3) How can mutations that arise during experimental evolution increase our understanding of basic microbial physiology?
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Evolution in response to feast/famine is specific to the duration and severity of starvation. Examples here include: A) Diversification is common during evolution under 10-day feast/famine conditions. Here, E. coli populations initiated from a single isogenic ancestor rapidly diversify into two distinct ecotypes. B) Diversification can be confirmed by sequencing clones isolated at the Day 300 timepoint. C-E) Transcriptomics reveals patterns of complementary gene expression between ecotypes as Ecotype A [red] invests less in fatty acid biosynthesis and Ecotype B invests less in the scavenging of metals. Due to the cost of fatty-acid biosynthesis and metal scavenging, these processes are ideal targets for the evolution of cooperation by gene loss as described by the Black Queen Hypothesis. (Behringer et al, 2022. mBio) F) Evolution under 100-day feast/famine conditions results in an extreme pattern of mutational order, shedding light on the rugged fitness landscape associated with adaptation to extended feast/famine cycles. (Behringer et al, 2024. Current Biology) G) Mutational parallelism at the nucleotide level is observed under 100-day feast/famine conditions with Rho R109H substitutions arising in many populations. Rho R109H confers a pH-sensing phenotype to the transcriptional terminator, which is amplified when it is found in conjunction with a loss of ydcI, a gene involved in pH homeostasis. These mutations enable E. coli to respond to the fluctuating nutrient availability associated with feast/famine cycles, as replenishment of resources triggers a rapid pH change from 9 to 7. (Worthan et al, 2024 PNAS).
2) Effects of genotype, environment, and microbial life history on genetic and epigenetic mutation rates.
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Microbes exhibit some of the lowest spontaneous mutation rates across the Tree of Life. Powered by low mutation rates and extremely large population sizes, microbes can efficiently sample new alleles and rapidly adapt to environmental changes while minimizing the burden of genetic load. However, because microbes have colonized every habitable niche on Earth, many species have evolved unique adaptations that make them difficult or impossible to culture in standard laboratory conditions and have been overlooked in studies of microbial mutation rates. Moreover, many microbes, particularly opportunistic pathogens, have life histories consisting of very different environmental conditions, which may result in mutation rate plasticity and greatly affect the molecular clock. As such, we are very interested in cataloging mutation rates in such organisms (obligate anaerobes, fastidious species) and determining the molecular and physiological features of these organisms that contribute to mutation/fidelity of DNA replication.
In addition to genetic mutations, microbes also maintain a variety of nucleotide base modifications across their genomes. This epigenetic information can be gained or lost, resulting in semi-permanent heritable change. Recent work by our lab (Stone et al, 2023, in press) has shown that the pattern of 6-methyladenine (6mA) modifications across the E. coli genome can evolve differently depending on genotype. In E. coli, the primary function of 6mA is to direct the mismatch repair (MMR) machinery and aid in the correction of DNA replication errors. In the absence of MMR, we found that the selective pressure maintaining 6mA methylation is relaxed and that genome-wide methylation erodes in a non-deterministic fashion. Future investigation by our lab will focus on estimates of 6mA epimutation rate and how the environment affects epimutation rates.
Evolution of Mutation and Epimutation. A) Lactic Acid Bacteria exhibit some of the highest mutation rates recorded, even in their preferred anaerobic conditions. In particular, G:C -> A:T mutations are elevated in Lb. acidophilus, a signature usually associated with oxidative stress, but in the absence of oxygen, may indicate other vulnerabilities of G:C nucleotides. B) Domestication may be a major factor influencing mutation rates in Lb. acidophilus, as loss of DNA repair genes is widespread in laboratory and commercial strains. As such, Lb. acidophilus, may be an ideal species to investigate the processes underlying mutation rate evolution and the drift-barrier hypothesis. (Hale et al, 2005. mBio.)
C) Genome-wide 6mA methylation in E. coli K-12 substr. MG1655. The origin of replication (green), IS elements (purple) and cryptic prophages (orange) are shown for reference. (A) Percent methylation at GATC sites. Each point represents one GATC site and points are colored to reflect percent methylation from 0% (blue, inside) to 100% (red, outside). (B-C) Differentially methylated GATC sites in evolved WT (A) and MMR- (B) clones. Red bars are sites with increased methylation and blue bars are decreased methylation, and the height of the bar reflects the difference in percent methylation between the ancestor and the evolved clones. Each ring ranges from -25% to 25%. (Stone et al, 2023. mBio).
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3) Genetic and environmental drivers of diversification in experimentally evolving Escherichia coli populations.
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When provided with a rich array of nutrients and an opportunity for spatial segregation, microbes can take advantage of newly acquired genetic variation and rapidly diversify to occupy new unique niches. In a long-term evolution experiment, we have been evolving populations of E. coli in spatially complex (culture tubes) and spatially homogeneous (flasks) environments containing a nutritionally complex media (LB broth). In spatially complex environments, the first visual phenotypic change we observe is the presence of a biofilm at the surface/air interface of the culture tube. Whole-population shotgun genomic sequencing revealed that these populations contained parallel mutations, particularly affecting genes that influence biofilm formation (fimE, regulator of the type-I fimbriae (FimA); hns, global regulator of environmental-change and stress-response genes; and acrB, a subunit of the MDR family efflux pump AcrAB/TolC). Deletion of fimA renders E. coli populations unable to evolve the ability to form biofilms. Without the ability to evolve increases in biofilm formation, adaptation is hindered in the culture tube environment but not in the flask environment.
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Once diversified, the fates of the evolved ecotypes within the population are highly dependent on the feast/famine cycle. To assess this we generated a crossfeeding community consisting of a methionine overproducer (MetJ-) and a methionine-requiring strain (MetB-). We find that short feast/famine cycles results in the ecotypes settling into a stable eqilibrium while longer feast/famine cycles result in chaotic population dynamics. Future work will examine the ecological parameters that shape population dynamics and their effect on evolution.
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When cultivated in the presence of a rich, complex culture medium and in a culture vessel that provides the opportunity for spatial segregation, E. coli populations are observed to rapidly evolve subpopulation structure. A) Whole-population shotgun sequencing reveals diversification under 1- and 10-day feeast/famine cycles, but repeated sweeps in 100-day feast/famine cycles. B) When evolution under 1-day feast/famine cycles is replayed with a genetic background that is unable to form biofilms (FimA-, blue), adaptation is hindered in the culture tube environment but not the flask environment. Head to head competitions of evolved populations vs. their relative ancestor in both their "evolved" condition and their "reciprocal" condition reveals that WT populations evolve to be locally adapted (or specifically adapted to their evolved environment, left panel [red]). In contrast, FimA- populations always exhibit the greatest fitness in culture flasks regardless of their evolved environment. (Patton et al, 2025. Evolution Letters.) C) In a minimal community consisting of E. coli engineered to cross feed on methionine, we observe stable subpopulation structure under 2-day feast/famine and chaotic population dynamics under 10-day feast/famine. (McLaughlin and Beardsley et al, 2025. In review)
4) Experimental and translational investigation of interactions between pathogenic and commensal microbes in the small intestine and urogenital microbiome. ​
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It should also be noted that gene regulation by H-NS and the resulting expression of the type-I fimbriae are contributing factors to uropathogenic E. coli (UPEC) strains colonizing the bladder, resulting in urinary tract infection (UTI). As these genes are also implicated in the establishment and maintenance of subpopulation structure, it's possible that during infection the UPEC strains also evolve and diversify within the bladder - as they do in our long-term experiment - contributing to therapeutic failure in individuals with recurrent UTI. One obstacle that UPEC strains must overcome as they colonize the bladder is the presence of the native uroprotective microbiome which is comprised of a number of fastidious anaerobic microbes namely, Lactobacillus. One species, Lactobacillus crispatus, has been suggested to have an antagonistic relationship with E. coli and can exclude the growth of UPEC strains. Current work will focus on identifying strategies employed by E. coli strains to overcome Lactobacillus, and applying this knowledge to clinical E. coli isolates.
