Trade-offs predicted by metabolic network structure give rise to evolutionary specialization and phenotypic diversification.

Mitigating trade-offs between different resource-utilization functions is key to an organism's ecological and evolutionary success. These trade-offs often reflect metabolic constraints with a complex molecular underpinning; therefore, their consequences for evolutionary processes have remained elusive. Here, we investigate how metabolic architecture induces resource utilization constraints and how these constraints, in turn, elicit evolutionary specialization and diversification. Guided by the metabolic network structure of the bacterium Lactococcus cremoris, we selected two carbon sources (fructose and galactose) with predicted co-utilization constraints. By evolving L. cremoris on either fructose, galactose or a mix of both sugars, we imposed selection favoring divergent metabolic specializations or co-utilization of both resources, respectively. Phenotypic characterization revealed the evolution of either fructose or galactose specialists in the single-sugar treatments. In the mixed sugar regime, we observed adaptive diversification: both specialists coexisted, and no generalist evolved. Divergence from the ancestral phenotype occurred at key pathway junctions in the central carbon metabolism. Fructose specialists evolved mutations in the fbp and pfk genes that appear to balance anabolic and catabolic carbon fluxes. Galactose specialists evolved increased expression of pgmA (the primary metabolic bottleneck of galactose metabolism) and silencing of ptnABCD (the main glucose transporter) and ldh (regulator/enzyme of downstream carbon metabolism). Overall, our study shows how metabolic network architecture and historical contingency serve to predict targets of selection and inform the functional interpretation of evolved mutations. The elucidation of the relationship between molecular constraints and phenotypic trade-offs contributes to an integrative understanding of evolutionary specialization and diversification.

Wide lag time distributions break a trade-off between reproduction and survival in bacteria.

Many microorganisms face a fundamental trade-off between reproduction and survival: Rapid growth boosts population size but makes microorganisms sensitive to external stressors. Here, we show that starved bacteria encountering new resources can break this trade-off by evolving phenotypic heterogeneity in lag time. We quantify the distribution of single-cell lag times of populations of starved Escherichia coli and show that population growth after starvation is primarily determined by the cells with shortest lag due to the exponential nature of bacterial population dynamics. As a consequence, cells with long lag times have no substantial effect on population growth resumption. However, we observe that these cells provide tolerance to stressors such as antibiotics. This allows an isogenic population to break the trade-off between reproduction and survival. We support this argument with an evolutionary model which shows that bacteria evolve wide lag time distributions when both rapid growth resumption and survival under stressful conditions are under selection. Our results can explain the prevalence of antibiotic tolerance by lag and demonstrate that the benefits of phenotypic heterogeneity in fluctuating environments are particularly high when minorities with extreme phenotypes dominate population dynamics.

Imperfect drug penetration leads to spatial monotherapy and rapid evolution of multidrug resistance.

Infections with rapidly evolving pathogens are often treated using combinations of drugs with different mechanisms of action. One of the major goal of combination therapy is to reduce the risk of drug resistance emerging during a patient's treatment. Although this strategy generally has significant benefits over monotherapy, it may also select for multidrug-resistant strains, particularly during long-term treatment for chronic infections. Infections with these strains present an important clinical and public health problem. Complicating this issue, for many antimicrobial treatment regimes, individual drugs have imperfect penetration throughout the body, so there may be regions where only one drug reaches an effective concentration. Here we propose that mismatched drug coverage can greatly speed up the evolution of multidrug resistance by allowing mutations to accumulate in a stepwise fashion. We develop a mathematical model of within-host pathogen evolution under spatially heterogeneous drug coverage and demonstrate that even very small single-drug compartments lead to dramatically higher resistance risk. We find that it is often better to use drug combinations with matched penetration profiles, although there may be a trade-off between preventing eventual treatment failure due to resistance in this way and temporarily reducing pathogen levels systemically. Our results show that drugs with the most extensive distribution are likely to be the most vulnerable to resistance. We conclude that optimal combination treatments should be designed to prevent this spatial effective monotherapy. These results are widely applicable to diverse microbial infections including viruses, bacteria, and parasites.

Quorum sensing as a mechanism to harness the wisdom of the crowds.

Bacteria release and sense small molecules called autoinducers in a process known as quorum sensing. The prevailing interpretation of quorum sensing is that by sensing autoinducer concentrations, bacteria estimate population density to regulate the expression of functions that are only beneficial when carried out by a sufficiently large number of cells. However, a major challenge to this interpretation is that the concentration of autoinducers strongly depends on the environment, often rendering autoinducer-based estimates of cell density unreliable. Here we propose an alternative interpretation of quorum sensing, where bacteria, by releasing and sensing autoinducers, harness social interactions to sense the environment as a collective. Using a computational model we show that this functionality can explain the evolution of quorum sensing and arises from individuals improving their estimation accuracy by pooling many imperfect estimates - analogous to the 'wisdom of the crowds' in decision theory. Importantly, our model reconciles the observed dependence of quorum sensing on both population density and the environment and explains why several quorum sensing systems regulate the production of private goods.

How bacteria navigate varying environments.

Collective sensing and phenotypic diversification aid response to environmental fluctuations.

Microfluidics for Single-Cell Study of Antibiotic Tolerance and Persistence Induced by Nutrient Limitation.

Nutrient limitation is one of the most common triggers of antibiotic tolerance and persistence. Here, we present two microfluidic setups to study how spatial and temporal variation in nutrient availability lead to increased survival of bacteria to antibiotics. The first setup is designed to mimic the growth dynamics of bacteria in spatially structured populations (e.g., biofilms) and can be used to study how spatial gradients in nutrient availability, created by the collective metabolic activity of a population, increase antibiotic tolerance. The second setup captures the dynamics of feast-and-famine cycles that bacteria recurrently encounter in nature, and can be used to study how phenotypic heterogeneity in growth resumption after starvation increases survival of clonal bacterial populations. In both setups, the growth rates and metabolic activity of bacteria can be measured at the single-cell level. This is useful to build a mechanistic understanding of how spatiotemporal variation in nutrient availability triggers bacteria to enter phenotypic states that increase their tolerance to antibiotics.

Quorum sensing integrates environmental cues, cell density and cell history to control bacterial competence.

Streptococcus pneumoniae becomes competent for genetic transformation when exposed to an autoinducer peptide known as competence-stimulating peptide (CSP). This peptide was originally described as a quorum-sensing signal, enabling individual cells to regulate competence in response to population density. However, recent studies suggest that CSP may instead serve as a probe for sensing environmental cues, such as antibiotic stress or environmental diffusion. Here, we show that competence induction can be simultaneously influenced by cell density, external pH, antibiotic-induced stress, and cell history. Our experimental data is explained by a mathematical model where the environment and cell history modify the rate at which cells produce or sense CSP. Taken together, model and experiments indicate that autoinducer concentration can function as an indicator of cell density across environmental conditions, while also incorporating information on environmental factors or cell history, allowing cells to integrate cues such as antibiotic stress into their quorum-sensing response. This unifying perspective may apply to other debated quorum-sensing systems.Peptide CSP regulates natural competence in pneumococci and has been proposed as a quorum-sensing signal or a probe for sensing environmental cues. Here, the authors show that CSP levels can indeed act as an indicator of cell density and also incorporate information on environmental factors or cell history.

Speed of adaptation and genomic footprints of host-parasite coevolution under arms race and trench warfare dynamics.

Coevolution between hosts and their parasites is expected to follow a range of possible dynamics, the two extreme cases being called trench warfare (or Red Queen) and arms races. Long-term stable polymorphism at the host and parasite coevolving loci is characteristic of trench warfare, and is expected to promote molecular signatures of balancing selection, while the recurrent allele fixation in arms races should generate selective sweeps. We compare these two scenarios using a finite size haploid gene-for-gene model that includes both mutation and genetic drift. We first show that trench warfare do not necessarily display larger numbers of coevolutionary cycles per unit of time than arms races. We subsequently perform coalescent simulations under these dynamics to generate sequences at both host and parasite loci. Genomic footprints of recurrent selective sweeps are often found, whereas trench warfare yield signatures of balancing selection only in parasite sequences, and only in a limited parameter space. Our results suggest that deterministic models of coevolution with infinite population sizes do not predict reliably the observed genomic signatures, and it may be best to study parasite rather than host populations to find genomic signatures of coevolution, such as selective sweeps or balancing selection.