Genetically identical cells frequently display substantial heterogeneity in gene expression, cellular morphology and physiology. It has been suggested that by rapidly generating a subpopulation with novel phenotypic traits, phenotypic heterogeneity (or plasticity) accelerates the rate of adaptive evolution in populations facing extreme environmental challenges. This issue is important as cell-to-cell phenotypic heterogeneity may initiate key steps in microbial evolution of drug resistance and cancer progression. Here, we study how stochastic transitions between cellular states influence evolutionary adaptation to a stressful environment in yeast Saccharomyces cerevisiae. We developed inducible synthetic gene circuits that generate varying degrees of expression stochasticity of an antifungal resistance gene. We initiated laboratory evolutionary experiments with genotypes carrying different versions of the genetic circuit by exposing the corresponding populations to gradually increasing antifungal stress. Phenotypic heterogeneity altered the evolutionary dynamics by transforming the adaptive landscape that relates genotype to fitness. Specifically, it enhanced the adaptive value of beneficial mutations through synergism between cell-to-cell variability and genetic variation. Our work demonstrates that phenotypic heterogeneity is an evolving trait when populations face a chronic selection pressure. It shapes evolutionary trajectories at the genomic level and facilitates evolutionary rescue from a deteriorating environmental stress. Phenotypic heterogeneity of genetically identical cells can generate nonheritable variation in a population. Is this heterogeneity favorable for microbes? In a changing environment, the answer is a definite yes. While scholars have argued that stochastically generated variation precedes genetic changes and thereby facilitate the evolution of complex traits, this idea has remained disputed, not least because of the shortage of experimental studies. We address this long-standing and controversial issue by integrating synthetic biology, laboratory experimental evolution, and genomic analyses. We explicitly tested the mechanisms whereby phenotypic heterogeneity may promote evolvability. Our work demonstrates that phenotypic heterogeneity facilitates evolutionary rescue from deteriorating environmental stress by generating individuals with exceptionally high fitness. Remarkably, elevated phenotypic heterogeneity evolves as a direct response to stress and thereby it promotes evolution of rare combinations of mutations. These results indicate that phenotypic heterogeneity might have an important role in the evolution of key innovations.

Despite ongoing antibiotic development, evolution of resistance may render candidate antibiotics ineffective. Here we studied in vitro emergence of resistance to 13 antibiotics introduced after 2017 or currently in development, compared with in-use antibiotics. Laboratory evolution showed that clinically relevant resistance arises within 60 days of antibiotic exposure in Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, priority Gram-negative ESKAPE pathogens. Resistance mutations are already present in natural populations of pathogens, indicating that resistance in nature can emerge through selection of pre-existing bacterial variants. Functional metagenomics showed that mobile resistance genes to antibiotic candidates are prevalent in clinical bacterial isolates, soil and human gut microbiomes. Overall, antibiotic candidates show similar susceptibility to resistance development as antibiotics currently in use, and the corresponding resistance mechanisms overlap. However, certain combinations of antibiotics and bacterial strains were less prone to developing resistance, revealing potential narrow-spectrum antibacterial therapies that could remain effective. Finally, we develop criteria to guide efforts in developing effective antibiotic candidates.

Antibiotic resistance typically induces a fitness cost that shapes the fate of antibiotic-resistant bacterial populations. However, the cost of resistance can be mitigated by compensatory mutations elsewhere in the genome, and therefore the loss of resistance may proceed too slowly to be of practical importance. We present our study on the efficacy and phenotypic impact of compensatory evolution in Escherichia coli strains carrying multiple resistance mutations. We have demonstrated that drug-resistance frequently declines within 480 generations during exposure to an antibiotic-free environment. The extent of resistance loss was found to be generally antibiotic-specific, driven by mutations that reduce both resistance level and fitness costs of antibiotic-resistance mutations. We conclude that phenotypic reversion to the antibiotic-sensitive state can be mediated by the acquisition of additional mutations, while maintaining the original resistance mutations. Our study indicates that restricting antimicrobial usage could be a useful policy, but for certain antibiotics only.

Proteins are necessary for cellular growth. Concurrently, however, protein production has high energetic demands associated with transcription and translation. Here, we propose that activity of molecular chaperones shape protein burden, that is the fitness costs associated with expression of unneeded proteins. To test this hypothesis, we performed a genome-wide genetic interaction screen in baker's yeast. Impairment of transcription, translation, and protein folding rendered cells hypersensitive to protein burden. Specifically, deletion of specific regulators of the Hsp70-associated chaperone network increased protein burden. In agreement with expectation, temperature stress, increased mistranslation and a chemical misfolding agent all substantially enhanced protein burden. Finally, unneeded protein perturbed interactions between key components of the Hsp70-Hsp90 network involved in folding of native proteins. We conclude that specific chaperones contribute to protein burden. Our work indicates that by minimizing the damaging impact of gratuitous protein overproduction, chaperones enable tolerance to massive changes in genomic expression.

Antibiotics that target multiple cellular functions are anticipated to be less prone to bacterial resistance. Here we hypothesize that while dual targeting is crucial, it is not sufficient in preventing resistance. Only those antibiotics that simultaneously target membrane integrity and block another cellular pathway display reduced resistance development. To test the hypothesis, we focus on three antibiotic candidates, POL7306, Tridecaptin M152-P3 and SCH79797, all of which fulfill the above criteria. Here we show that resistance evolution against these antibiotics is limited in ESKAPE pathogens, including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, while dual-target topoisomerase antibiotics are prone to resistance. We discover several mechanisms restricting resistance. First, de novo mutations result in only a limited elevation in resistance, including those affecting the molecular targets and efflux pumps. Second, resistance is inaccessible through gene amplification. Third, functional metagenomics reveal that mobile resistance genes are rare in human gut, soil and clinical microbiomes. Finally, we detect rapid eradication of bacterial populations upon toxic exposure to membrane targeting antibiotics. We conclude that resistance mechanisms commonly found in natural bacterial pathogens provide only limited protection to these antibiotics. Our work provides guidelines for the future development of antibiotics.

Deleterious mutations are generally considered to be irrelevant for morphological evolution. However, they could be compensated by conditionally beneficial mutations, thereby providing access to new adaptive paths. Here we use high-dimensional phenotyping of laboratory-evolved budding yeast lineages to demonstrate that new cellular morphologies emerge exceptionally rapidly as a by-product of gene loss and subsequent compensatory evolution. Unexpectedly, the capacities for invasive growth, multicellular aggregation and biofilm formation also spontaneously evolve in response to gene loss. These multicellular phenotypes can be achieved by diverse mutational routes and without reactivating the canonical regulatory pathways. These ecologically and clinically relevant traits originate as pleiotropic side effects of compensatory evolution and have no obvious utility in the laboratory environment. The extent of morphological diversity in the evolved lineages is comparable to that of natural yeast isolates with diverse genetic backgrounds and lifestyles. Finally, we show that both the initial gene loss and subsequent compensatory mutations contribute to new morphologies, with their synergistic effects underlying specific morphological changes. We conclude that compensatory evolution is a previously unrecognized source of morphological diversity and phenotypic novelties.