Biology subjects

Behringer, M. G.

Publications and source records attributed to Behringer, M. G..

17 recordsLinked to original sources

bioRxiv2026

Environment-dependent epistasis shapes adaptation to fluctuating resources.

Microbial populations frequently experience fluctuations in resource availability, yet how these fluctuations reshape genetic interactions during adaptation remains unclear. Here, we identify the rapid evolution of latent antimicrobial susceptibility in Escherichia coli populations experimentally evolved under repeated feast-famine cycles. Longitudinal population genomics revealed recurrent mutations in the transport genes acrB and ompF, and reconstruction of these mutations demonstrated environment-dependent fitness effects characterized by positive epistasis after 1 day of culture and reciprocal sign epistasis after 10 days. Together, our results support a two-step adaptive trajectory in which mutations affecting efflux are followed by changes in porin permeability, generating genotypes that are jointly beneficial across feast and famine. More broadly, our findings demonstrate how fluctuating environments reshape adaptive genetic interactions, producing historically contingent evolutionary trajectories and latent phenotypes. Article SummaryHow fluctuating environments shape genetic interactions and evolutionary trajectories remains a central question in evolutionary genetics. We identified latent antimicrobial susceptibility in Escherichia coli populations evolved under repeated feast-famine cycles and reconstructed evolved mutations to resolve the genotype-to-phenotype map underlying this trait. The responsible mutations arose in key transport genes, exhibited environmental-dependent epistasis, and increased fitness across both feast and famine conditions. Our findings provide experimental evidence that fluctuating environments can select for interacting mutations that optimize fitness across a dynamic adaptive seascape.

evolutionary biology↗

bioRxiv2026

Rational engineering of facultative anaerobiosis enables commensal survival in the oxygenated gut

Life originated in the absence of oxygen. Despite its substantial energetic advantages, many modern microbes remain obligate anaerobes, confined to anoxic niches such as the mammalian gut. Why these organisms cannot tolerate oxygen has remained unresolved for more than two centuries. Here, using integrated multi-omics analyses, we identify a network of interlocking vulnerabilities in central metabolism, biosynthetic pathways, and redox homeostasis that together impose an aerobic growth barrier in the obligate anaerobic commensal Bacteroides thetaiotaomicron. Rational repair of these vulnerabilities restores metabolic integrity and progressively enhances oxygen tolerance, yielding engineered strains capable of robust growth at 10% O2 and markedly improved resilience in the oxygenated, inflamed gut. These findings define a molecular basis for obligate anaerobiosis and establish a framework for engineering commensal bacteria to function in oxidative environments, expanding their ecological range and therapeutic potential.

microbiology↗

bioRxiv2026

Bayesian analysis of longitudinal RB-TnSeq resolves the fitness seascape in fluctuating environments

Temporally structured environments are ubiquitous in nature, but time-dependent fitness effects are difficult to measure and thus understudied. To resolve temporal fitness structure at genome scale, we developed a Bayesian multilevel framework for longitudinal randomly barcoded transposon sequencing (RB-TnSeq) that stabilizes noisy mutant abundance trajectories into time-resolved selection-rate estimates with interpretable uncertainty. Applying this approach to a feast-famine starvation regime in Escherichia coli, we uncovered distinct fitness trajectories underpinned by shared molecular strategies across growth-curve phase, consistent with shifting constraints and antagonistic pleiotropy. Fitness during initial growth strongly constrained cumulative success, such that later advantages under stress could not rescue mutants that were initially deleterious. We then compressed these dynamics with a one-dimensional Fishers geometric "seascape" model that orders mutants along a latent axis aligning with generalist-specialist and growth-survival trade-offs, providing a compact quantitative description of genome-wide constraints in a fluctuating environment. Finally, our longitudinal estimates and inferred seascape are predictive of both the identity and timing of mutational targets in an extended evolution experiment under similar repeated feast-famine conditions, linking short-term competitive fitness effects, with their related trade-offs and constraints, to long-term adaptive outcomes.

evolutionary biology↗

bioRxiv2025

Historical Contingency Limits Adaptive Diversification in a Spatially Structured Environment

Understanding how genotype-by-environment (GxE) interactions influence evolutionary trajectories and contribute to historical contingency is key to predicting evolution. In spatially structured, heterogeneous environments, populations often diversify into ecotypes, resulting in niche specialization. However, the ability to specialize depends not only on ecological opportunity but also on whether genetic variation permits access to novel niches, as genetic disruptions may inhibit adaptation unless alternative trajectories or compensatory mutations are subsequently accessible. Previously, we demonstrated that Escherichia coli populations rapidly diversify into two co-existing ecotypes in a nutrient-rich, spatially structured environment. The adaptation of both ecotypes results in benefits only perceived in the spatially structured culture tube environment. Here, diversification is initiated by first-step mutations associated with the overexpression of genes encoding the type 1 fimbria, the major attachment pilus involved in biofilm development, enabling range expansion and allowing E. coli to occupy the surface-air interface of the culture tube. To investigate how first-step mutations shape evolutionary trajectories, we experimentally evolved wild-type and fimbrial-deficient ({Delta}fimA) E. coli for 91 days in both structured (tube) and unstructured (flask) environments. While a fimA deletion initially confers a fitness benefit by avoiding the cost of insufficient biofilm formation, it ultimately prevents range expansion in structured environments and is not compensated by expression of cryptic fimbriae by the end of experimental evolution. As a result, {Delta}fimA populations show constrained adaptation in tubes compared to wild-type. Alternatively, both genotypes perform similarly in flasks, where biofilm formation is not advantageous and whole population sequencing reveals that flask-evolved populations similar early mutational trajectories. Our results highlight the ruggedness of the adaptive landscape in structured environments and show how an initially beneficial mutation can trap a lineage on a local fitness peak, underscoring the importance of GxE interactions and early mutational events in shaping the predictability and contingency of evolutionary outcomes.

evolutionary biology↗

bioRxiv2025

An Anaerobic Pathogen Rewires Host Metabolism to Fuel Oxidative Growth in the Inflamed Gut

To colonize their host and cause disease, enteric pathogens must deploy their virulence factors to establish distinct nutrient niches. How obligate anaerobic pathogens construct nutrient niches in the densely populated large intestine remains poorly understood. Enterotoxigenic Bacteroides fragilis (ETBF) is considered an obligate anaerobic bacterium and has been implicated in inflammation-associated diseases, including colitis and colorectal cancer. Here we show that ETBF uses its virulence factor, Bacteroides fragilis toxin, to reprogram colonic epithelial cell metabolism to colonize the inflamed gut. Bacteroides fragilis toxin activates colonic epithelial signaling and hijacks the host bile acid recycling pathway, inducing a metabolic shift in the epithelium from oxidative phosphorylation to glycolysis. This shift increases local concentrations of lactate and oxygen, nutrients that support an oxidative metabolism in ETBF. These findings reveal an unexpected strategy by which a pathogenic organism, previously considered to be an obligate anaerobic bacterium, generates and exploits an oxidative niche in the inflamed gut.

microbiology↗

bioRxiv2025

Starvation drives co-existence in cross-feeding bacterial populations

Cross-feeding is a universal feature of natural microbial communities that extends even into subpopulation structures, generating cooperative ecotypes. However, we still lack an understanding of the ecological parameters that support the ecotype co-existence necessary for co-evolution between partners within populations. Here, we show that repeated moderate starvation - conditions that mimic the fluctuations experienced by microbes in nature - can drive frequency-dependent co-existence for hundreds of generations, providing a new mechanism for the origin of cooperation and the maintenance of population-level diversity in natural microbial communities.

microbiology↗

bioRxiv2025

Elevated rates and biased spectra of mutations in anaerobically cultured lactic acid bacteria

The rate, spectrum, and biases of mutations represent a fundamental force shaping biological evolution. Convention often attributes oxidative DNA damage as a major driver of spontaneous mutations. Yet, despite the contribution of oxygen to mutagenesis and the ecological, industrial, and biomedical importance of anaerobic organisms, relatively little is known about the mutation rates and spectra of anaerobic species. Here, we present the rates and spectra of spontaneous mutations assessed anaerobically over 1000 generations for three fermentative lactic acid bacteria species with varying levels of aerotolerance: Lactobacillus acidophilus, Lactobacillus crispatus, and Lactococcus lactis. Our findings reveal highly elevated mutation rates compared to the average rates observed in aerobically respiring bacteria with mutations strongly biased towards transitions, emphasizing the prevalence of spontaneous deamination in these anaerobic species and highlighting the inherent fragility of purines even under conditions that minimize oxidative stress. Beyond these overarching patterns, we identify several novel mutation dynamics: positional mutation bias around the origin of replication in Lb. acidophilus, a significant disparity between observed and equilibrium GC content in Lc. lactis, and repeated independent deletions of spacer sequences from within the CRISPR locus in Lb. crispatus providing mechanistic insights into the evolution of bacterial adaptive immunity. Overall, our study provides new insights into the mutational landscape of anaerobes, revealing how non-oxygenic factors shape mutation rates and influence genome evolution.

genetics↗

bioRxiv2024

Bacterial cross-feeding can promote gene retention by lowering gene expression costs

Cross-feeding is prevalent in microbial communities. Through time, cross-feeding is thought to enrich for loss-of-function mutations, thereby creating or reinforcing dependencies between community members. However, few studies have compared how cross-feeding affects the evolutionary trajectory of partners compared to monoculture conditions. Here we compared mutations that were differentially enriched in bacterial monocultures versus cocultures pairing phototrophic Rhodopseudomonas palustris with fermentative Escherichia coli in an obligate cross-feeding relationship based on the exchange of nitrogen and carbon for 650-800 generations. Opposite trends for the number of differentially enriched mutations were observed for each species; R. palustris accumulated more unique mutations in monoculture whereas E. coli accumulated more unique mutations in coculture. Contrary to expectations, the emergence of additional dependencies was more apparent for both species in monoculture, even though additional layers of cross-feeding involving iron and adenine were present in coculture. We reasoned that iron and adenine cross-feeding occurred at levels sufficient to repress gene expression in the recipient, thereby promoting gene retention by lowering gene cost. Thus, the influence of cross-feeding on evolutionary trajectories can vary with organisms and conditions, and there are situations where cross-feeding can limit, rather than promote, emergent dependencies. IMPORTANCEBacteria commonly engage in cross-feeding where nutrients are transferred between neighbors. Cross-feeding is thought to alleviate energy expenditures for genes whose role can be met by cross-fed nutrients, leading to eventual gene loss. However, few examples have been documented, especially in comparison to monocultures where bacteria are grown without a cross-feeding partner. Here we grew cocultures pairing phototrophic Rhodopseudomonas palustris with fermentative Escherichia coli alongside corresponding monocultures for 650-800 generations. While coculture conditions required obligate exchange of nitrogen and carbon, additional cross-feeding of adenine and iron likely occurred. Contrary to expectations, iron and uncharacterized dependencies emerged in monocultures but not in cocultures. Low expression for iron scavenging and adenine synthesis genes in cocultures suggested that cross-feeding repressed gene expression, thereby lowering gene cost. Thus, whereas there are likely cases where cross-feeding makes costly genes dispensable, there are also cases where cross-feeding lowers gene cost, thereby promoting gene retention.

microbiology↗

bioRxiv2024

Evolution of pH-sensitive transcription termination during adaptation to repeated long-term starvation.

Fluctuating environments that consist of regular cycles of co-occurring stress are a common challenge faced by cellular populations. For a population to thrive in constantly changing conditions, an ability to coordinate a rapid cellular response is essential. Here, we identify a mutation conferring an arginine-to-histidine (Arg to His) substitution in the transcription terminator Rho. The rho R109H mutation frequently arose in E. coli populations experimentally evolved under repeated long-term starvation conditions, during which feast and famine result in drastic environmental pH fluctuations. Metagenomic sequencing revealed that populations containing the rho mutation also possess putative loss-of-function mutations in ydcI, which encodes a recently characterized transcription factor associated with pH homeostasis. Genetic reconstructions of these mutations show that the rho allele confers a plastic alkaline-induced reduction of Rho function that, when found in tandem with a {Delta}ydcI allele, leads to intracellular alkalinization and genetic assimilation of Rho mutant function. We further identify Arg to His substitutions at analogous sites in rho alleles from species originating from fluctuating alkaline environments. Our results suggest that Arg to His substitutions in global regulators of gene expression can serve to rapidly coordinate complex responses through pH sensing and shed light on how cellular populations across the tree of life use environmental cues to coordinate rapid responses to complex, fluctuating environments.

microbiology↗

bioRxiv2023

Trade-offs, trade-ups, and high mutational parallelism underlie microbial adaptation to extreme feast/famine

Microbes are robust organisms capable of rapidly adapting to complex stress, enabling the colonization of harsh environments. In nature, microbes are regularly challenged by starvation, which is a particularly complex stress because resource limitation often co-occurs with changes in pH, osmolarity, and toxin accumulation created by metabolic waste. Often overlooked are the additional complications introduced by eventual resource replenishment as successful microbes must withstand rapid environmental shifts before swiftly capitalizing on replenished resources to avoid invasion by competing species. To understand how microbes navigate trade-offs between growth and survival, ultimately adapting to thrive in environments with extreme fluctuations, we experimentally evolved 16 Escherichia coli populations for 900 days to repeated feast/famine cycles of 100-day starvation before resource replenishment. Using longitudinal population-genomic analysis, we found that evolution in response to extreme feast/famine is characterized by narrow adaptive trajectories with high mutational parallelism and notable mutational order. Genetic reconstructions reveal that early mutations result in trade-offs for biofilm and motility but trade-ups for growth and survival, as these mutations conferred correlated advantages during both short-term and long-term culture. Our results demonstrate how microbes can navigate the adaptive landscapes of regularly fluctuating conditions and ultimately follow mutational trajectories that confer benefits across diverse environments.

evolutionary biology↗

bioRxiv2022

Differential adenine methylation analysis reveals increased variability in 6mA in the absence of methyl-directed mismatch repair

Methylated DNA adenines (6mA) are an important epigenetic modification in bacteria that affect varied cell processes like replication, stress response, and pathogenesis. While much work has been done characterizing the influence of 6mA on specific loci, very few studies have examined the evolutionary dynamics of 6mA over long time scales. Utilizing third-generation sequencing technology, we produced a detailed analysis of 6mA methylation across the Escherichia coli K-12 substr. MG1655 genome. 6mA levels were consistently high across GATC sites; however, we identified regions where 6mA is decreased, particularly in intergenic regions, especially around the -35 promoter element, and within cryptic prophages and IS elements. We further examined 6mA in WT and methyl-directed mismatch repair-knockout (MMR-) populations after 2400 generations of experimental evolution. We find that, after evolution, MMR-populations acquire significantly more epimutations resulting in a genome-wide decrease in 6mA methylation. Here, clones from evolved MMR-populations display non-deterministic sets of epimutations, consistent with reduced selection on these modifications. Thus, we show that characterization of 6mA in bacterial populations is complementary to genetic sequencing and informative for molecular evolution.

microbiology↗

bioRxiv2021

Evolutionary dynamics of asexual hypermutators adapting to a novel environment

How microbes adapt to a novel environment is a central question in evolutionary biology. While adaptive evolution must be fueled by beneficial mutations, whether higher mutation rates facilitate the rate of adaptive evolution remains unclear. To address this question, we cultured Escherichia coli hypermutating populations, in which a defective methyl-directed mismatch repair pathway causes a 140-fold increase in single-nucleotide mutation rates. In parallel with wild-type E. coli, populations were cultured in tubes containing Luria-Bertani broth, a complex medium known to promote the evolution of subpopulation structure. After 900 days of evolution, in three transfer schemes with different population-size bottlenecks, hypermutators always exhibited similar levels of improved fitness as controls. Fluctuation tests revealed that the mutation rates of hypermutator lines converged evolutionarily on those of wild-type populations, which may have contributed to the absence of fitness differences. Further genome-sequence analysis revealed that, although hypermutator populations have higher rates of genomic evolution, this largely reflects the effects of genetic draft under strong linkage. Despite these linkage effects, the evolved populations exhibit parallelism in fixed mutations, including those potentially related to biofilm formation, transcription regulation, and mutation-rate evolution. Together, these results generally negate the presumed relationship between high mutation rates and high adaptive speed of evolution, providing insight into how clonal adaptation occurs in novel environments. Significance statementWhile mutations are critical source for the adaptation in a new environment, whether or not the elevated mutation rates can empirically lead to the elevated adaptation rates remains unclear, especially when the environment is more heterogenous. To answer this question, we evolved E. coli populations with different starting mutation rates in a complex medium for 900 days and then examined their fitness and genome profiles. In the populations that have a higher starting mutation rate, despite faster genome evolution, their fitness improvement is not significantly faster. Our results reveal that the effect of elevated mutation rates is only very limited, and the mutations accumulated in hypermutators are largely due to linkage effect.

evolutionary biology↗

bioRxiv2021

Microbial population dynamics and evolutionary outcomes under extreme energy-limitation

As the most abundant and diverse form of life on Earth, microorganisms commonly inhabit energy-limited environments where cellular maintenance and growth is highly constrained. To gain insight into how microorganisms persist under such conditions, we derived demographic parameters from a diverse collection of bacteria by censusing 100 populations in a closed system for 1,000 days. All but one taxon survived prolonged resource scarcity, yielding estimated times-to-extinction ranging over four orders of magnitude from 100 - 105 years. These findings corroborate reports of long-lived bacteria that have been recovered from ancient environmental samples, while providing insight into mechanisms of persistence. Critically, we found that as death rates declined over time, lifespan was extended through the scavenging of dead cells. Although growth and reproduction were dramatically suppressed in the absence of an exogenous resource supply, bacterial populations continued to evolve. Hundreds of mutations were acquired, contributing to genome-wide signatures of negative selection as well as molecular signals of adaptation. Remarkable consistency in the ecological and evolutionary dynamics indicate that distantly related bacteria respond to energy-limitation in a similar and predictable manner, which likely contributes to the stability and robustness of microbial life.

microbiology↗

bioRxiv2020

No evidence for whole-chromosome dosage compensation or global transcriptomic expression differences in spontaneously-aneuploid mutation accumulation lines of Saccharomyces cerevisiae

Aneuploidy, the state in which an organisms genome contains one or more missing or additional chromosomes, often causes widespread genotypic and phenotypic effects. Most often, aneuploidies are deleterious; the most common examples in humans being Downs syndrome (Trisomy 21) and Turners syndrome (monosomy X). However, aneuploidy is surprisingly common in wild yeast populations. In recent years, there has been debate as to whether yeast contain an innate dosage compensation response that operates at the gene, chromosome, or the whole-genome level, or if natural isolates are robust to aneuploidy without such a mechanism. In this study, we tested for differential gene expression in 20 aneuploid and 16 euploid lines of yeast from two previous mutation accumulation experiments, where selection was minimized and therefore aneuploidies arose spontaneously. We found no evidence for whole-chromosome dosage compensation in aneuploid yeast but did find some evidence for attenuation of expression on a gene-by-gene basis. We additionally found that aneuploidy has no effect on the expression of the rest of the genome (i.e. "trans" genes), and that very few mutually exclusive aneuploid lines shared differentially expressed genes. However, we found there was a small set of genes that exhibited a shared expression response in the euploid lines, suggesting an effect of mutation accumulation on gene expression. Our findings contribute to our understanding of aneuploidy in yeast and support the hypothesis that there is no innate dosage compensation mechanism at the whole-chromosome level.

genetics↗

bioRxiv2019

Antagonism in Evolutionary Opportunities Results in Non-Monotonic Evolution Across an Environmental Gradient

Establishing reliable frameworks for predicting unknown outcomes from empirical observations is of great interest to ecologists and evolutionary biologists. Strong predictability in evolutionary responses has been previously demonstrated by the repeated observation of similar phenotypes or genotypes across multiple natural or experimental populations in analogous environments. However, the degree to which evolutionary outcomes can be predicted across environmental gradients, or in fluctuating environments, remains largely unexplored. Presumably, the phenotypic evolution in an intermediate environment could be interpolated from the evolved phenotypes observed in two extreme environments, but this assumption remains to be fully tested. Here, we report on the experimental evolution of Escherichia coli under three nutritional transfer periods: every day, every 10 days, and every 100 days, representing increasing severity in feast/famine cycles. After 900 days of experimental evolution, populations experiencing intermediate durations of starvation had evolved longer times to reach maximum growth rate, smaller colony sizes, higher biofilm formation, and higher mutation rates than populations evolving in the other environmental extremes. Because the intermediately starved populations exhibit significantly high molecular parallelism, these distinct phenotypes are likely due to non-monotonic deterministic forces instead of increased stochastic forces commonly associated with fluctuating environments. Our results demonstrate novel complexities associated with evolutionary predictability across environmental gradients and highlight the risk of using interpolation in evolutionary biology.

evolutionary biology↗

bioRxiv2019

Enhanced nutrient uptake is sufficient to drive emergent cross-feeding between bacteria

Interactive microbial communities are ubiquitous, influencing biogeochemical cycles and host health. One widespread interaction is nutrient exchange, or cross-feeding, wherein metabolites are transferred between microbes. Some cross-fed metabolites, such as vitamins, amino acids, and ammonium (NH4+), are communally valuable and impose a cost on the producer. The mechanisms that enforce cross-feeding of communally valuable metabolites are not fully understood. Previously we engineered mutualistic cross-feeding between N2-fixing Rhodopseudomonas palustris and fermentative Escherichia coli. Engineered R. palustris excreted essential nitrogen as NH4+ to E. coli while E. coli excreted essential carbon as fermentation products to R. palustris. Here, we enriched for nascent cross-feeding in cocultures with wild-type R. palustris, not known to excrete NH4+. Emergent NH4+ cross-feeding was driven by adaptation of E. coli alone. A missense mutation in E. coli NtrC, a regulator of nitrogen scavenging, resulted in constitutive activation of an NH4+ transporter. This activity likely allowed E. coli to subsist on the small amount of leaked NH4+ and better reciprocate through elevated excretion of organic acids from a larger E. coli population. Our results indicate that enhanced nutrient uptake by recipients, rather than increased excretion by producers, is an underappreciated yet possibly prevalent mechanism by which cross-feeding can emerge.

microbiology↗

bioRxiv2018

An Escherichia coli nitrogen starvation response is important for mutualistic coexistence with Rhodopseudomonas palustris

Microbial mutualistic cross-feeding interactions are ubiquitous and can drive important community functions. Engaging in cross-feeding undoubtedly affects the physiology and metabolism of individual species involved. However, the nature in which an individuals physiology is influenced by cross-feeding and the importance of those physiological changes for the mutualism have received little attention. We previously developed a genetically tractable coculture to study bacterial mutualisms. The coculture consists of fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris. In this coculture, E. coli anaerobically ferments sugars into excreted organic acids as a carbon source for R. palustris. In return, a genetically-engineered R. palustris constitutively converts N2 into NH4+, providing E. coli with essential nitrogen. Using RNA-seq and proteomics, we identified transcript and protein levels that differ in each partner when grown in coculture versus monoculture. When in coculture with R. palustris, E. coli gene-expression changes resembled a nitrogen starvation response under the control of the transcriptional regulator NtrC. By genetically disrupting E. coli NtrC, we determined that a nitrogen starvation response is important for a stable coexistence, especially at low R. palustris NH4+ excretion levels. Destabilization of the nitrogen starvation regulatory network resulted in variable growth trends and in some cases, extinction. Our results highlight that alternative physiological states can be important for survival within cooperative cross-feeding relationships.\n\nImportanceMutualistic cross-feeding between microbes within multispecies communities is widespread. Studying how mutualistic interactions influence the physiology of each species involved is important for understanding how mutualisms function and persist in both natural and applied settings. Using a bacterial mutualism consisting of Rhodopseudomonas palustris and Escherichia coli growing cooperatively through bidirectional nutrient exchange, we determined that an E. coli nitrogen starvation response is important for maintaining a stable coexistence. The lack of an E. coli nitrogen starvation response ultimately destabilized the mutualism and, in some cases, led to community collapse after serial transfers. Our findings thus inform on the potential necessity of an alternative physiological state for mutualistic coexistence with another species compared to the physiology of species grown in isolation.

microbiology↗