Trade-offs, trade-ups, and high mutational parallelism underlie microbial adaptation during extreme cycles of feast and famine.

Microbes are evolutionarily robust organisms capable of rapid adaptation to complex stress, which enables them to colonize 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 in repeated feast/famine conditions with 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 positively 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.

Rapid evolution of mutation rate and spectrum in response to environmental and population-genetic challenges.

Ecological and demographic factors can significantly shape the evolution of microbial populations both directly and indirectly, as when changes in the effective population size affect the efficiency of natural selection on the mutation rate. However, it remains unclear how rapidly the mutation-rate responds evolutionarily to the entanglement of ecological and population-genetic factors over time. Here, we directly assess the mutation rate and spectrum of Escherichia coli clones isolated from populations evolving in response to 1000 days of different transfer volumes and resource-replenishment intervals. The evolution of mutation rates proceeded rapidly in response to demographic and/or environmental changes, with substantial bidirectional shifts observed as early as 59 generations. These results highlight the remarkable rapidity by which mutation rates are shaped in asexual lineages in response to environmental and population-genetic forces, and are broadly consistent with the drift-barrier hypothesis for the evolution of mutation rates, while also highlighting situations in which mutator genotypes may be promoted by positive selection.

Complex Ecotype Dynamics Evolve in Response to Fluctuating Resources.

Ecotypic diversification and its associated cooperative behaviors are frequently observed in natural microbial populations whose access to resources is often sporadic. However, the extent to which fluctuations in resource availability influence the emergence of cooperative ecotypes is not fully understood. To determine how exposure to repeated resource limitation affects the establishment and long-term maintenance of ecotypes in a structured environment, we followed 32 populations of Escherichia coli evolving to either 1-day or 10-day feast/famine cycles for 900 days. Population-level analysis revealed that compared to populations evolving to 1-day cycles, 10-day populations evolved increased biofilm density, higher parallelism in mutational targets, and increased mutation rates. As previous investigations of evolution in structured environments have identified biofilm formation as the earliest observable phenotype associated with diversification of ecotypes, we revived cultures midway through the evolutionary process and conducted additional genomic, transcriptional, and phenotypic analyses of clones isolated from these evolving populations. We found not only that 10-day feast/famine cycles support multiple ecotypes but also that these ecotypes exhibit cooperative behavior. Consistent with the black queen hypothesis, or evolution of cooperation by gene loss, transcriptomic evidence suggests the evolution of bidirectional cross-feeding behaviors based on essential resources. These results provide insight into how analogous cooperative relationships may emerge in natural microbial communities. IMPORTANCE Despite regular feast and famine conditions representing an environmental pressure that is commonly encountered by microbial communities, the evolutionary outcomes of repeated cycles of feast and famine have been less studied. By experimentally evolving initially isogenic Escherichia coli populations to 10-day feast/famine cycles, we observed rapid diversification into ecotypes with evidence of bidirectional cross-feeding on costly resources and frequency-dependent fitness. Although unidirectional cross-feeding has been repeatedly observed to evolve in laboratory culture, most investigations of bidirectional cooperative behaviors in microbial populations have been conducted in engineered communities. This work demonstrates the de novo evolution of black queen relationships in a microbial population originating from a single ancestor, providing a model for investigation of the eco-evolutionary processes leading to mutualistic cooperation.

Evolutionary Dynamics of Asexual Hypermutators Adapting to a Novel Environment.

How microbes adapt to a novel environment is a central question in evolutionary biology. Although 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 strong genetic linkage. Despite these linkage effects, the evolved population exhibits parallelism in fixed mutations, including those potentially related to biofilm formation, transcription regulation, and mutation-rate evolution. Together, these results are generally inconsistent with a hypothesized positive relationship between the mutation rate and the adaptive speed of evolution, and provide insight into how clonal adaptation occurs in novel environments.

Low base-substitution mutation rate and predominance of insertion-deletion events in the acidophilic bacterium Acidobacterium capsulatum.

Analyses of spontaneous mutation have shown that total genome-wide mutation rates are quantitatively similar for most prokaryotic organisms. However, this view is mainly based on organisms that grow best around neutral pH values (6.0-8.0). In particular, the whole-genome mutation rate has not been determined for an acidophilic organism. Here, we have determined the genome-wide rate of spontaneous mutation in the acidophilic Acidobacterium capsulatum using a direct and unbiased method: a mutation-accumulation experiment followed by whole-genome sequencing. Evaluation of 69 mutation accumulation lines of A. capsulatum after an average of ~2900 cell divisions yielded a base-substitution mutation rate of 1.22 × 10-10 per site per generation or 4 × 10-4 per genome per generation, which is significantly lower than the consensus value (2.5-4.6 × 10-3) of mesothermophilic (~15-40°C) and neutrophilic (pH 6-8) prokaryotic organisms. However, the insertion-deletion rate (0.43 × 10-10 per site per generation) is high relative to the base-substitution mutation rate. Organisms with a similar effective population size and a similar expected effect of genetic drift should have similar mutation rates. Because selection operates on the total mutation rate, it is suggested that the relatively high insertion-deletion rate may be balanced by a low base-substitution rate in A. capsulatum, with selection operating on the total mutation rate.

Specificity of the DNA Mismatch Repair System (MMR) and Mutagenesis Bias in Bacteria.

The mutation rate of an organism is influenced by the interaction of evolutionary forces such as natural selection and genetic drift. However, the mutation spectrum (i.e., the frequency distribution of different types of mutations) can be heavily influenced by DNA repair. Using mutation-accumulation lines of the extremophile bacterium Deinococcus radiodurans ΔmutS1 and the model soil bacterium Pseudomonas fluorescens wild-type and MMR- (Methyl-dependent Mismatch Repair-deficient) strains, we report the mutational features of these two important bacteria. We find that P. fluorescens has one of the highest MMR repair efficiencies among tested bacteria. We also discover that MMR of D. radiodurans preferentially repairs deletions, contrary to all other bacteria examined. We then, for the first time, quantify genome-wide efficiency and specificity of MMR in repairing different genomic regions and mutation types, by evaluating the P. fluorescens and D. radiodurans mutation data sets, along with previously reported ones of Bacillus subtilis subsp. subtilis, Escherichia coli, Vibrio cholerae, and V. fischeri. MMR in all six bacteria shares two general features: 1) repair efficiency is influenced by the neighboring base composition for both transitions and transversions, not limited to transversions as previously reported; and 2) MMR only recognizes indels <4 bp in length. This study demonstrates the power of mutation accumulation lines in quantifying DNA repair and mutagenesis patterns.

Escherichia coli cultures maintain stable subpopulation structure during long-term evolution.

How genetic variation is generated and maintained remains a central question in evolutionary biology. When presented with a complex environment, microbes can take advantage of genetic variation to exploit new niches. Here we present a massively parallel experiment where WT and repair-deficient (∆mutL) Escherichia coli populations have evolved over 3 y in a spatially heterogeneous and nutritionally complex environment. Metagenomic sequencing revealed that these initially isogenic populations evolved and maintained stable subpopulation structure in just 10 mL of medium for up to 10,000 generations, consisting of up to five major haplotypes with many minor haplotypes. We characterized the genomic, transcriptomic, exometabolomic, and phenotypic differences between clonal isolates, revealing subpopulation structure driven primarily by spatial segregation followed by differential utilization of nutrients. In addition to genes regulating the import and catabolism of nutrients, major polymorphisms of note included insertion elements transposing into fimE (regulator of the type I fimbriae) and upstream of hns (global regulator of environmental-change and stress-response genes), both known to regulate biofilm formation. Interestingly, these genes have also been identified as critical to colonization in uropathogenic E. coli infections. Our findings illustrate the complexity that can arise and persist even in small cultures, raising the possibility that infections may often be promoted by an evolving and complex pathogen population.

An Unbiased Genome-Wide View of the Mutation Rate and Spectrum of the Endosymbiotic Bacterium Teredinibacter turnerae.

Mutations contribute to genetic variation in all living systems. Thus, precise estimates of mutation rates and spectra across a diversity of organisms are required for a full comprehension of evolution. Here, a mutation-accumulation (MA) assay was carried out on the endosymbiotic bacterium Teredinibacter turnerae. After ∼3,025 generations, base-pair substitutions (BPSs) and insertion-deletion (indel) events were characterized by whole-genome sequencing analysis of 47 independent MA lines, yielding a BPS rate of 1.14 × 10-9 per site per generation and indel rate of 1.55 × 10-10 events per site per generation, which are among the highest within free-living and facultative intracellular bacteria. As in other endosymbionts, a significant bias of BPSs toward A/T and an excess of deletion mutations over insertion mutations are observed for these MA lines. However, even with a deletion bias, the genome remains relatively large (∼5.2 Mb) for an endosymbiotic bacterium. The estimate of the effective population size (Ne) in T. turnerae is quite high and comparable to free-living bacteria (∼4.5 × 107), suggesting that the heavy bottlenecking associated with many endosymbiotic relationships is not prevalent during the life of this endosymbiont. The efficiency of selection scales with increasing Ne and such strong selection may have been operating against the deletion bias, preventing genome erosion. The observed mutation rate in this endosymbiont is of the same order of magnitude of those with similar Ne, consistent with the idea that population size is a primary determinant of mutation-rate evolution within endosymbionts, and that not all endosymbionts have low Ne.

Evolutionary determinants of genome-wide nucleotide composition.

One of the long-standing mysteries of evolutionary genomics is the source of the wide phylogenetic diversity in genome nucleotide composition (G + C versus A + T), which must be a consequence of interspecific differences in mutation bias, the efficiency of selection for different nucleotides or a combination of the two. We demonstrate that although genomic G + C composition is strongly driven by mutation bias, it is also substantially modified by direct selection and/or as a by-product of biased gene conversion. Moreover, G + C composition at fourfold redundant sites is consistently elevated above the neutral expectation-more so than for any other class of sites.

The Rate and Spectrum of Spontaneous Mutations in Mycobacterium smegmatis, a Bacterium Naturally Devoid of the Postreplicative Mismatch Repair Pathway.

Mycobacterium smegmatis is a bacterium that is naturally devoid of known postreplicative DNA mismatch repair (MMR) homologs, mutS and mutL, providing an opportunity to investigate how the mutation rate and spectrum has evolved in the absence of a highly conserved primary repair pathway. Mutation accumulation experiments of M. smegmatis yielded a base-substitution mutation rate of 5.27 × 10(-10) per site per generation, or 0.0036 per genome per generation, which is surprisingly similar to the mutation rate in MMR-functional unicellular organisms. Transitions were found more frequently than transversions, with the A:T→G:C transition rate significantly higher than the G:C→A:T transition rate, opposite to what is observed in most studied bacteria. We also found that the transition-mutation rate of M. smegmatis is significantly lower than that of other naturally MMR-devoid or MMR-knockout organisms. Two possible candidates that could be responsible for maintaining high DNA fidelity in this MMR-deficient organism are the ancestral-like DNA polymerase DnaE1, which contains a highly efficient DNA proofreading histidinol phosphatase (PHP) domain, and/or the existence of a uracil-DNA glycosylase B (UdgB) homolog that might protect the GC-rich M. smegmatis genome against DNA damage arising from oxidation or deamination. Our results suggest that M. smegmatis has a noncanonical Dam (DNA adenine methylase) methylation system, with target motifs differing from those previously reported. The mutation features of M. smegmatis provide further evidence that genomes harbor alternative routes for improving replication fidelity, even in the absence of major repair pathways.

Antibiotic treatment enhances the genome-wide mutation rate of target cells.

Although it is well known that microbial populations can respond adaptively to challenges from antibiotics, empirical difficulties in distinguishing the roles of de novo mutation and natural selection have left several issues unresolved. Here, we explore the mutational properties of Escherichia coli exposed to long-term sublethal levels of the antibiotic norfloxacin, using a mutation accumulation design combined with whole-genome sequencing of replicate lines. The genome-wide mutation rate significantly increases with norfloxacin concentration. This response is associated with enhanced expression of error-prone DNA polymerases and may also involve indirect effects of norfloxacin on DNA mismatch and oxidative-damage repair. Moreover, we find that acquisition of antibiotic resistance can be enhanced solely by accelerated mutagenesis, i.e., without direct involvement of selection. Our results suggest that antibiotics may generally enhance the mutation rates of target cells, thereby accelerating the rate of adaptation not only to the antibiotic itself but to additional challenges faced by invasive pathogens.

Asymmetric Context-Dependent Mutation Patterns Revealed through Mutation-Accumulation Experiments.

Despite the general assumption that site-specific mutation rates are independent of the local sequence context, a growing body of evidence suggests otherwise. To further examine context-dependent patterns of mutation, we amassed 5,645 spontaneous mutations in wild- type (WT) and mismatch-repair deficient (MMR(-)) mutation-accumulation (MA) lines of the gram-positive model organism Bacillus subtilis. We then analyzed>7,500 spontaneous base-substitution mutations across B. subtilis, Escherichia coli, and Mesoplasma florum WT and MMR(-) MA lines, finding a context-dependent mutation pattern that is asymmetric around the origin of replication. Different neighboring nucleotides can alter site-specific mutation rates by as much as 75-fold, with sites neighboring G:C base pairs or dimers involving alternating pyrimidine-purine and purine-pyrimidine nucleotides having significantly elevated mutation rates. The influence of context-dependent mutation on genome architecture is strongest in M. florum, consistent with the reduced efficiency of selection in organisms with low effective population size. If not properly accounted for, the disparities arising from patterns of context-dependent mutation can significantly influence interpretations of positive and purifying selection.

Mutation rate, spectrum, topology, and context-dependency in the DNA mismatch repair-deficient Pseudomonas fluorescens ATCC948.

High levels of genetic diversity exist among natural isolates of the bacterium Pseudomonas fluorescens, and are especially elevated around the replication terminus of the genome, where strain-specific genes are found. In an effort to understand the role of genetic variation in the evolution of Pseudomonas, we analyzed 31,106 base substitutions from 45 mutation accumulation lines of P. fluorescens ATCC948, naturally deficient for mismatch repair, yielding a base-substitution mutation rate of 2.34 × 10(-8) per site per generation (SE: 0.01 × 10(-8)) and a small-insertion-deletion mutation rate of 1.65 × 10(-9) per site per generation (SE: 0.03 × 10(-9)). We find that the spectrum of mutations in prophage regions, which often contain virulence factors and antibiotic resistance, is highly similar to that in the intergenic regions of the host genome. Our results show that the mutation rate varies around the chromosome, with the lowest mutation rate found near the origin of replication. Consistent with observations from other studies, we find that site-specific mutation rates are heavily influenced by the immediately flanking nucleotides, indicating that mutations are context dependent.

Drift-barrier hypothesis and mutation-rate evolution.

Mutation dictates the tempo and mode of evolution, and like all traits, the mutation rate is subject to evolutionary modification. Here, we report refined estimates of the mutation rate for a prokaryote with an exceptionally small genome and for a unicellular eukaryote with a large genome. Combined with prior results, these estimates provide the basis for a potentially unifying explanation for the wide range in mutation rates that exists among organisms. Natural selection appears to reduce the mutation rate of a species to a level that scales negatively with both the effective population size (N(e)), which imposes a drift barrier to the evolution of molecular refinements, and the genomic content of coding DNA, which is proportional to the target size for deleterious mutations. As a consequence of an expansion in genome size, some microbial eukaryotes with large N(e) appear to have evolved mutation rates that are lower than those known to occur in prokaryotes, but multicellular eukaryotes have experienced elevations in the genome-wide deleterious mutation rate because of substantial reductions in N(e).