A Broad Continuum of E. coli Traits in Nature Associated with the Trade-off Between Self-preservation and Nutritional Competence.

A trade-off between reproduction and survival is a characteristic of many organisms. In bacteria, growth is constrained when cellular resources are channelled towards environmental stress protection. At the core of this trade-off in Escherichia coli is RpoS, a sigma factor that diverts transcriptional resources towards general stress resistance. The constancy of RpoS levels in natural isolates is unknown. A uniform RpoS content in E. coli would impart a narrow range of resistance properties to the species, whereas a diverse set of RpoS levels in nature should result in a diverse range of stress susceptibilities. We explore the diversity of trade-off settings and phenotypes by measuring the level of RpoS protein in strains of E. coli cohabiting in a natural environment. Strains from a stream polluted with domestic waste were investigated in monthly samples. Analyses included E. coli phylogroup classification, RpoS protein level, RpoS-dependent stress phenotypes and the sequencing of rpoS mutations. The most striking finding was the continuum of RpoS levels, with a 100-fold range of RpoS amounts consistently found in individuals in the stream. Approximately 1.8% of the sampled strains carried null or non-synonymous mutations in rpoS. The natural isolates also exhibited a broad (>100-fold) range of stress resistance responses. Our results are consistent with the view that a multiplicity of survival-multiplication trade-off settings is a feature of the species E. coli. The phenotypic diversity resulting from the trade-off permits bet-hedging and the adaptation of E. coli strains to a very broad range of environments.

Irregularities in genetic variation and mutation rates with environmental stresses.

The appearance of new mutations is determined by the equilibrium between DNA error formation and repair. In bacteria like Escherichia coli, stresses are thought shift this balance towards increased mutagenesis. Recent findings, however, suggest a very uneven relationship between stress and mutations. Only a subset of stressful environments increase the net rate of mutation and different forms of nutritional stress (such as oxygen, carbon or phosphorus limitations) result in markedly different mutation rates after similar reductions in growth rate. Moreover, different stresses result in altered mutational spectra, with some increasing transposition and others increasing indel formation. Single-base substitution rates are lower with some stresses than in unstressed bacteria. Indeed, changes to the mix of mutations with stress are more widespread than a marked increase in net mutation rate. Much remains to be learned on how environments have unique mutational signatures and why some stresses are more mutagenic than others. Even beyond stress-induced genetic variation, the fundamental unresolved question in the stress-mutation relationship is the adaptive value of different types of mutations and mutation rates; is transposition, for example, more advantageous under anaerobic conditions? It remains to be investigated whether stress-specific genetic variation impacts on evolvability differentially in distinct environments.

Escherichia coli mutation rates and spectra with combinations of environmental limitations.

Micro-organisms often face multiple stresses in natural habitats. Individual stresses are well known to influence mutation rates and the spectra of mutational types, but the extent to which multiple stresses affect the genetic variation in populations is unknown. Here we investigate pair-wise combinations of nutritional stresses in Escherichia coli to determine their effect on mutation rates and mutational types. Environmental interactions modified both the rate and spectrum of mutations in double-limited environments, but the effects were not additive or synergistic relative to single stresses. Generally, bacteria in the mixed environments behaved as if one of the two single-stress stimuli was more dominant and the genetic variation seen with every dual limitation was intermediate between known patterns with individual stresses. The composition of mutational types with double stresses was also intermediate between individual stress patterns. At least with mutations, the single stressor results available are reasonable indicators of stress-induced genetic variation in multifaceted natural habitats. With the influence of 11 conditions available on mutational patterns, we can now also see the clustering of mutational types as a function of these environments.

The impact of growth rate and environmental factors on mutation rates and spectra in Escherichia coli.

Genetic variation in bacterial populations is remarkably sensitive to environmental influences, including simple, nutritional differences. Not only the rate but also the kind of mutational changes is biased by the nutritional state of bacteria. Here we investigate the mutational consequences of a universal variable for free-living bacteria, namely the growth rate. By controlling growth in chemostats, the rate and mix of mutations was investigated for populations of Escherichia coli subject to different specific growth rates. Both aerobic and anaerobic cultures were compared with see if growth rate is a factor in the commonest respiratory conditions for E. coli. We find mutation rates are raised markedly with decreasing growth rate. Base pair substitutions and 1-bp insertions and deletions increase with reduced growth rate, but less so in anaerobic cultures. Insertion sequence movements are particularly sensitive to growth rate, with IS2 being optimal at intermediate growth rates whereas IS1 and IS150 movements are highest at the slowest tested growth rate. A comprehensive comparison of growth rate effects, as well as six other environmental factors, provides the most complete picture yet of the range of mutational signatures in bacterial genetic variation.

The fitness costs and benefits of antibiotic resistance in drug-free microenvironments encountered in the human body.

The relationship between bacterial drug resistance and growth fitness is a contentious topic, but some antibiotic resistance mutations clearly have a fitness cost in the laboratory. Whether these costs translate into deleterious effects in natural habitats is less certain however. Previously, fitness effects of resistance mutations were mostly characterized in nutrient-rich, fast-growth conditions, which bacteria rarely encounter in natural habitats. Carbon, phosphate, iron or oxygen limitations are conditions met by bacterial pathogens in various compartments of the human body. Here, we measured the fitness of four different rpoB mutations commonly found in rifampicin-resistant bacterial isolates. The fitness properties and the emergence of these and other alleles were studied in Escherichia coli populations growing under nutrient excess and in four different nutrient-limited states. Consistent with previous findings, all four mutations exhibited deleterious fitness effects under nutrient-rich conditions. In stark contrast, we found positive or neutral fitness effects under nutrient-limited conditions. Two particular rpoB alleles had a remarkable fitness increase under phosphate limitation and these alleles arose to high frequencies specifically under phosphate limitation. These findings suggest that it is not meaningful to draw general conclusions on fitness costs without considering bacterial microenvironments in humans and other animals.

A shifting mutational landscape in 6 nutritional states: Stress-induced mutagenesis as a series of distinct stress input-mutation output relationships.

Environmental stresses increase genetic variation in bacteria, plants, and human cancer cells. The linkage between various environments and mutational outcomes has not been systematically investigated, however. Here, we established the influence of nutritional stresses commonly found in the biosphere (carbon, phosphate, nitrogen, oxygen, or iron limitation) on both the rate and spectrum of mutations in Escherichia coli. We found that each limitation was associated with a remarkably distinct mutational profile. Overall mutation rates were not always elevated, and nitrogen, iron, and oxygen limitation resulted in major spectral changes but no net increase in rate. Our results thus suggest that stress-induced mutagenesis is a diverse series of stress input-mutation output linkages that is distinct in every condition. Environment-specific spectra resulted in the differential emergence of traits needing particular mutations in these settings. Mutations requiring transpositions were highest under iron and oxygen limitation, whereas base-pair substitutions and indels were highest under phosphate limitation. The unexpected diversity of input-output effects explains some important phenomena in the mutational biases of evolving genomes. The prevalence of bacterial insertion sequence transpositions in the mammalian gut or in anaerobically stored cultures is due to environmentally determined mutation availability. Likewise, the much-discussed genomic bias towards transition base substitutions in evolving genomes can now be explained as an environment-specific output. Altogether, our conclusion is that environments influence genetic variation as well as selection.

Natural Escherichia coli isolates rapidly acquire genetic changes upon laboratory domestication.

The adaptation of environmental bacteria to laboratory conditions was analysed through the exploration of genomic changes in four strains of Escherichia coli freshly isolated from their natural habitats and belonging to different taxonomic clusters. Up to 25 mutations were present in all cultures of natural isolates within 10 days of transfer in rich media or with a single growth cycle involving an extended stationary phase. Among numerous individual mutations, two genes were affected in parallel in distinct backgrounds. Mutations in rpoS (encoding sigma factor RpoS), altering a multiplication-survival trade-off in E. coli, were present in isolates derived from all four different ancestors. More surprisingly, two different natural isolates acquired mutations in mutL, affecting DNA mismatch repair, and a third also involved higher mutation rates. The elevated mutation rates in these isolates indicate the danger of increased genetic instability arising from laboratory domestication. Neither rpoS nor mutator mutations were detected in the already-acclimatized MG1655 laboratory strain; only one or no new mutations were present in the laboratory strain under the same culture conditions. Our results indicate rapid adaptation to the laboratory environment. Ancestor-specific responses also arise in the laboratory and mutational events are also sensitive to culture conditions such as extended stationary phase. To maintain natural isolates in a stable state, our data suggest that the transition of strains to the laboratory should minimize culture cycles and extended stationary phase.

The fitness costs and trade-off shapes associated with the exclusion of nine antibiotics by OmpF porin channels.

The trade-off relationship between antibiotic exclusion and nutrient access across the Gram-negative outer membrane is determined by structural constraints in porin channels. The precise nutritional cost of exclusion is unknown for different antibiotics, as are the shapes of the nutrition-susceptibility trade-off. Using a library of 10 engineered isogenic Escherichia coli strains with structural modifications of OmpF porin expressed at a constant level, susceptibilities were measured for nine antibiotics and the nutritional fitness costs estimated by competitions in chemostats. Different antibiotics exhibited a remarkably varied range of geometries in the nutrition-susceptibility trade-off, including convex, concave and sigmoidal trade-off shapes. The trade-off patterns predict the possibility of adaptations in contributing to antibiotic resistance; exclusion of amoxicillin or trimethoprim in ompF mutants can occur with little loss of fitness whereas kanamycin and streptomycin exclusion has a high cost. Some individual OmpF changes even allow positive correlations (trade-ups), resulting in increased fitness and decreased susceptibility specifically to cephalexin or ciprofloxacin. The surprising plasticity of the nutrition-exclusion relationship means that there are no generalisable rules that apply to decreasing susceptibility for all antibiotics. The protein changes are exquisitely specific in determining nutritional fitness and adaptive outcomes in a structural constraint trade-off.

Metastable coexistence of multiple genotypes in a constant environment with a single resource through fixed settings of a multiplication-survival trade-off.

The biological complexity of trade-offs has been a major obstacle in understanding bacterial diversity and coexistence. Here we reduce the biological complexity by using isogenic Escherichia coli strains differing only in a multiplication-survival trade-off regulated by RpoS. The contribution of trade-off characteristics to fitness in different environments was determined. We then designed an environment with intermediate-stress levels that elicits an equivalent fitness. We found metastable coexistence of three strains in steady-state chemostats until mutations changed the relative fitness of competing strains. Our results help explain the rich intra- and inter-species diversity of bacteria through alternative settings of relatively few trade-offs.

Trade-off Mechanisms Shaping the Diversity of Bacteria.

Strain-to-strain variations in bacterial biofilm formation, metabolism, motility, virulence, evolvability, DNA repair and resistance (to phage, antibiotics, or environmental stresses) each contribute to bacterial diversity. Microbiologists should be aware that all of these traits are subject to constraints imposed by trade-offs, so adaptations improving one trait may be at the cost of another. A deeper appreciation of trade-offs is thus crucial for assessing the mechanistic limits on important bacterial characteristics. Studies of the negative correlations between various traits have revealed three molecular mechanisms, namely, trade-offs involving resource allocation, design constraint, and information processing. This review further discusses why these trade-off mechanisms are important in the establishment of models capable of predicting bacterial competition, coexistence, and sources of diversity.

How Porin Heterogeneity and Trade-Offs Affect the Antibiotic Susceptibility of Gram-Negative Bacteria.

Variations in porin proteins are common in Gram-negative pathogens. Altered or absent porins reduce access of polar antibiotics across the outer membrane and can thus contribute to antibiotic resistance. Reduced permeability has a cost however, in lowering access to nutrients. This trade-off between permeability and nutritional competence is the source of considerable natural variation in porin gate-keeping. Mutational changes in this trade-off are frequently selected, so susceptibility to detergents and antibiotics is polymorphic in environmental isolates as well as pathogens. Understanding the mechanism, costs and heterogeneity of antibiotic exclusion by porins will be crucial in combating Gram negative infections.

Simple phenotypic sweeps hide complex genetic changes in populations.

Changes in allele frequencies and the fixation of beneficial mutations are central to evolution. The precise relationship between mutational and phenotypic sweeps is poorly described however, especially when multiple alleles are involved. Here, we investigate these relationships in a bacterial population over 60 days in a glucose-limited chemostat in a large population. High coverage metagenomic analysis revealed a disconnection between smooth phenotypic sweeps and the complexity of genetic changes in the population. Phenotypic adaptation was due to convergent evolution and involved soft sweeps by 7-26 highly represented alleles of several genes in different combinations. Allele combinations spread from undetectably low baselines, indicating that minor subpopulations provide the basis of most innovations. A hard sweep was also observed, involving a single combination of rpoS, mglD, malE, sdhC, and malT mutations sweeping to greater than 95% of the population. Other mutant genes persisted but at lower abundance, including hfq, consistent with its demonstrated frequency-dependent fitness under glucose limitation. Other persistent, newly identified low-frequency mutations were in the aceF, galF, ribD and asm genes, in noncoding regulatory regions, three large indels and a tandem duplication; these were less affected by fluctuations involving more dominant mutations indicating separate evolutionary paths. Our results indicate a dynamic subpopulation structure with a minimum of 42 detectable mutations maintained over 60 days. We also conclude that the massive population-level mutation supply in combination with clonal interference leads to the soft sweeps observed, but not to the exclusion of an occasional hard sweep.

Mutational heterogeneity: a key ingredient of bet-hedging and evolutionary divergence?: The broad spectrum of mutations and their flexible frequency in populations provides a source of risk avoidance and alternative evolutionary strategies.

Here, we propose that the heterogeneity of mutational types in populations underpins alternative pathways of evolutionary adaptation. Point mutations, deletions, insertions, transpositions and duplications cause different biological effects and provide distinct adaptive possibilities. Experimental evidence for this notion comes from the mutational origins of adaptive radiations in large, clonal bacterial populations. Independent sympatric lineages with different phenotypes arise from distinct genetic events including gene duplication, different insertion sequence movements and several independent point mutations. The breadth of the mutational spectrum in the ancestral population should be viewed as a form of bet-hedging, reducing the risk of evolutionary dead ends and complementing the phenotypic and epigenetic heterogeneities that improve the survival capabilities of a population. Different mutational events arise from distinct cellular processes and are subject to separate environmental impacts, so the availability of any particular type of mutation may constrain or promote adaptive pathways in populations.

Mutational signatures indicative of environmental stress in bacteria.

Evolutionary innovations are dependent on mutations. Mutation rates are increased by adverse conditions in the laboratory, but there is no evidence that stressful environments that do not directly impact on DNA leave a mutational imprint on extant genomes. Mutational spectra in the laboratory are normally determined with unstressed cells but are unavailable with stressed bacteria. To by-pass problems with viability, selection effects, and growth rate differences due to stressful environments, in this study we used a set of genetically engineered strains to identify the mutational spectrum associated with nutritional stress. The strain set members each had a fixed level of the master regulator protein, RpoS, which controls the general stress response of Escherichia coli. By assessing mutations in cycA gene from 485 cycloserine resistant mutants collected from as many independent cultures with three distinct perceived stress (RpoS) levels, we were able establish a dose-dependent relationship between stress and mutational spectra. The altered mutational patterns included base pair substitutions, single base pair indels, longer indels, and transpositions of different insertion sequences. The mutational spectrum of low-RpoS cells closely matches the genome-wide spectrum previously generated in laboratory environments, while the spectra of high RpoS, high perceived stress cells more closely matches spectra found in comparisons of extant genomes. Our results offer an explanation of the uneven mutational profiles such as the transition-transversion biases observed in extant genomes and provide a framework for assessing the contribution of stress-induced mutagenesis to evolutionary transitions and the mutational emergence of antibiotic resistance and disease states.

Stress-induced mutation rates show a sigmoidal and saturable increase due to the RpoS sigma factor in Escherichia coli.

Stress-induced mutagenesis was investigated in the absence of selection for growth fitness by using synthetic biology to control perceived environmental stress in Escherichia coli. We find that controlled intracellular RpoS dosage is central to a sigmoidal, saturable three- to fourfold increase in mutation rates and associated changes in DNA repair proteins.

A metabolic trade-off between phosphate and glucose utilization in Escherichia coli.

Getting the most out of available nutrients is a key challenge that all organisms face. Little is known about how they optimize and balance the simultaneous utilization of multiple elemental resources. We investigated the effects of long-term phosphate limitation on carbon metabolism of the model organism Escherichia coli using chemostat cultures. We profiled metabolic changes in the growth medium over time and found evidence for an increase in fermentative metabolism despite the aerobic conditions. Using full-genome sequencing and competition experiments, we found that fitness under phosphate-limiting conditions was reproducibly increased by a mutation preventing flux through succinate in the tricarboxylic acid cycle. In contrast, these mutations reduced competitive ability under carbon limitation, and thus reveal a conflicting metabolic benefit in the role of the TCA cycle in environments limited by inorganic phosphate and glucose.

The nature of laboratory domestication changes in freshly isolated Escherichia coli strains.

Adaptation of environmental bacteria to laboratory conditions can lead to modification of important traits, what we term domestication. Little is known about the rapidity and reproducibility of domestication changes, the uniformity of these changes within a species or how diverse these are in a single culture. Here, we analysed phenotypic changes in nutrient-rich liquid media or on agar of four Escherichia coli strains newly isolated through minimal steps from different sources. The laboratory-cultured populations showed changes in metabolism, morphotype, fitness and in some phenotypes associated with the sigma factor RpoS. Domestication events and phenotypic diversity started to emerge within 2-3 days in replicate subcultures of the same ancestor. In some strains, increased amino acid usage and higher fitness under nutrient limitation resembled those in mutants with the GASP (growth advantage in stationary phase) phenotype. The domestication changes are not uniform across a species or even within a single domesticated population. However, some parallelism in adaptation within repeat cultures was observed. Differences in the laboratory environment also determine domestication effects, which differ between liquid and solid media or with extended stationary phase. Important lessons for the handling and storage of organisms can be based on these studies.

Epistatic interactions determine the mutational pathways and coexistence of lineages in clonal Escherichia coli populations.

Understanding how diversity emerges in a single niche is not fully understood. Rugged fitness landscapes and epistasis between beneficial mutations could explain coexistence among emerging lineages. To provide an experimental test of this notion, we investigated epistasis among four pleiotropic mutations in rpoS, mglD, malT, and hfq present in two coexisting lineages that repeatedly fixed in experimental populations of Escherichia coli. The mutations were transferred into the ancestral background individually or in combination of double or triple alleles. The combined competitive fitness of two or three beneficial mutations from the same lineage was consistently lower than the sum of the competitive fitness of single mutants--a clear indication of negative epistasis within lineages. We also found sign epistasis (i.e., the combined fitness of two beneficial mutations lower than the ancestor), not only from two different lineages (i.e., hfq and rpoS) but also from the same lineage (i.e., mglD and malT). The sign epistasis between loci of different lineages indeed indicated a rugged fitness landscape, providing an epistatic explanation for the coexistence of distinct rpoS and hfq lineages in evolving populations. The negative and sign epistasis between beneficial mutations within the same lineage can further explain the order of mutation acquisition.

A case of adaptation through a mutation in a tandem duplication during experimental evolution in Escherichia coli.

BACKGROUND: DNA duplications constitute important precursors for genome variation. Here we analyzed an unequal duplication harboring a beneficial mutation that may provide alternative evolutionary outcomes. RESULTS: We characterized this evolutionary event during experimental evolution for only 100 generations of an Escherichia coli strain under glucose limitation within chemostats. By combining Insertion Sequence based Restriction Length Polymorphism experiments, pulsed field gel electrophoresis and two independent genome re-sequencing experiments, we identified an evolved lineage carrying a 180 kb duplication of the 46' region of the E. coli chromosome. This evolved duplication revealed a heterozygous state, with one copy harboring a 2668 bp deletion that included part of the ogrK gene and both the yegR and yegS genes. By genetically manipulating ancestral and evolved strains, we showed that the single yegS inactivation was sufficient to confer a frequency dependent fitness increase under the chemostat selective conditions in both the ancestor and evolved genetic contexts, implying that the duplication itself was not a direct fitness contributor. Nonetheless, the heterozygous duplicated state was relatively stable in the conditions prevailing during evolution in chemostats, in striking contrast to non selective conditions in which the duplication resolved at high frequency into either its ancestral or deleted copy. CONCLUSIONS: Our results suggest that the duplication state may constitute a second order selection process providing higher evolutionary potential. Moreover, its heterozygous nature may provide differential evolutionary opportunities in alternating environments. Our results also highlighted how careful analyses of whole genome data are needed to identify such complex rearrangements.

A design-constraint trade-off underpins the diversity in ecologically important traits in species Escherichia coli.

Bacterial species are internally diverse in genomic and multi-locus gene comparisons. The ecological causes of phenotypic and genotypic diversity within species are far less well understood. Here, we focus on the competitive fitness for growth on nutrients within Escherichia coli, an internally rich species. Competition experiments in nutrient-limited chemostats revealed that members of the ECOR collection exhibited a wide continuum of competitive abilities, with some fitter and some less fit than the lab strain MG1655. We observed an inverse relationship between competitiveness and the resistance of strains to detergent and antibiotic, consistent with the notion that membrane permeability and competitive fitness are linked by a trade-off between self-preservation and nutritional competence (SPANC); high permeability has a postulated cost in antibacterial sensitivity whereas a low permeability has a cost in nutrient affinity. Isolates moved along the markedly nonlinear trade-off curve by mutational adaptation; an ECOR strain sensitive to antibacterials and a good competitor was easily converted by mutation into a mutant with higher resistance but poorer competition in the presence of low antibiotic concentrations. Conversely, a resistant ECOR strain changed into a better competitor after a short period of selection under nutrient limitation. In both directions, mutations can affect porin proteins and outer membrane permeability, as indicated by protein analysis, gene sequencing and an independent assay of outer membrane permeability. The extensive, species-wide diversity of E. coli in ecologically important traits can thus be explained as an evolutionary consequence of a SPANC trade-off driven by antagonistic pleiotropy.