Extracellular Vesicles from 50,000 Generation Clones of the Escherichia coli Long-Term Evolution Experiment.

Extracellular vesicles (EVs) are critical elements of cell-cell communication. Here, we characterized the outer membrane vesicles (OMVs) released by specific clones of Escherichia coli isolated from the Long-Term Evolution Experiment after 50,000 generations (50K) of adaptation to glucose minimal medium. Compared with their ancestor, the evolved clones produce small OMVs but also larger ones which display variable amounts of both OmpA and LPS. Tracking ancestral, fluorescently labelled OMVs revealed that they fuse with both ancestral- and 50K-evolved cells, albeit in different proportions. We quantified that less than 2% of the cells from one 50K-evolved clone acquired the fluorescence delivered by OMVs from the ancestral strain but that one cell concomitantly fuses with several OMVs. Globally, our results showed that OMV production in E. coli is a phenotype that varies along bacterial evolution and question the contribution of OMVs-mediated interactions in bacterial adaptation.

Evolution of Bacterial Persistence to Antibiotics during a 50,000-Generation Experiment in an Antibiotic-Free Environment.

Failure of antibiotic therapies causes > 700,000 deaths yearly and involves both bacterial resistance and persistence. Persistence results in the relapse of infections by producing a tiny fraction of pathogen survivors that stay dormant during antibiotic exposure. From an evolutionary perspective, persistence is either a 'bet-hedging strategy' that helps to cope with stochastically changing environments or an unavoidable minimal rate of 'cellular errors' that lock the cells in a low activity state. Here, we analyzed the evolution of persistence over 50,000 bacterial generations in a stable environment by improving a published method that estimates the number of persister cells based on the growth of the reviving population. Our results challenged our understanding of the factors underlying persistence evolution. In one case, we observed a substantial decrease in persistence proportion, suggesting that the naturally observed persistence level is not an unavoidable minimal rate of 'cellular errors'. However, although there was no obvious environmental stochasticity, in 11 of the 12 investigated populations, the persistence level was maintained during 50,000 bacterial generations.

Insertion-sequence-mediated mutations both promote and constrain evolvability during a long-term experiment with bacteria.

Insertion sequences (IS) are ubiquitous bacterial mobile genetic elements, and the mutations they cause can be deleterious, neutral, or beneficial. The long-term dynamics of IS elements and their effects on bacteria are poorly understood, including whether they are primarily genomic parasites or important drivers of adaptation by natural selection. Here, we investigate the dynamics of IS elements and their contribution to genomic evolution and fitness during a long-term experiment with Escherichia coli. IS elements account for ~35% of the mutations that reached high frequency through 50,000 generations in those populations that retained the ancestral point-mutation rate. In mutator populations, IS-mediated mutations are only half as frequent in absolute numbers. In one population, an exceptionally high ~8-fold increase in IS150 copy number is associated with the beneficial effects of early insertion mutations; however, this expansion later slowed down owing to reduced IS150 activity. This population also achieves the lowest fitness, suggesting that some avenues for further adaptation are precluded by the IS150-mediated mutations. More generally, across all populations, we find that higher IS activity becomes detrimental to adaptation over evolutionary time. Therefore, IS-mediated mutations can both promote and constrain evolvability.

Genomic trajectories to fluoroquinolone resistance in Francisella tularensis subsp. holarctica live vaccine strain.

OBJECTIVES: Fluoroquinolone (FQ)-resistant mutants were previously selected from the live vaccine strain (LVS) of Francisella tularensis (F. tularensis) subsp. holarctica. This study further characterised all genetic changes that occurred in these mutants during the evolutionary trajectory toward high-level FQ resistance, and their potential impact on F. tularensis antibiotic resistance and intracellular fitness. METHODS: The whole genomes of FQ-resistant mutants were determined and compared with those of their parental strain. All detected mutations were evaluated for their potential impact on FQ resistance and intracellular multiplication of F. tularensis. RESULTS: As compared with the parental LVS genome, 28 mutations were found in the derived FQ-resistant mutants. These mutations involved all genes encoding type II topoisomerases (i.e. gyrA, gyrB, parC, and parE). Interestingly, some of them were not previously associated with FQ resistance, warranting further characterisation. Mutations associated with FQ resistance were also found in other genes, including three encoding proteins involved in transport processes. Most of the detected mutations did not alter multiplication of the corresponding mutants in J774 cells. In contrast, all mutations at locus FTL_0439 encoding FupA/B, a membrane protein involved in iron transport, were associated with FQ resistance and fitness loss. CONCLUSION: FQ resistance in F. tularensis is complex and may involve single or combined mutations in genes encoding type II topoisomerases, transport systems and FupA/B. In vivo studies are now required to assess the potential role of these mutations in FQ treatment failures.

Bacterial genome architecture shapes global transcriptional regulation by DNA supercoiling.

DNA supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes' local orientation in their supercoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates convergent genes in several enterobacteria, while conversely, an increase in DNA supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA supercoiling as a global but fine-tuned transcriptional regulator.

Changes in Intrinsic Antibiotic Susceptibility during a Long-Term Evolution Experiment with Escherichia coli.

High-level resistance often evolves when populations of bacteria are exposed to antibiotics, by either mutations or horizontally acquired genes. There is also variation in the intrinsic resistance levels of different bacterial strains and species that is not associated with any known history of exposure. In many cases, evolved resistance is costly to the bacteria, such that resistant types have lower fitness than their progenitors in the absence of antibiotics. Some longer-term studies have shown that bacteria often evolve compensatory changes that overcome these tradeoffs, but even those studies have typically lasted only a few hundred generations. In this study, we examine changes in the susceptibilities of 12 populations of Escherichia coli to 15 antibiotics after 2,000 and 50,000 generations without exposure to any antibiotic. On average, the evolved bacteria were more susceptible to most antibiotics than was their ancestor. The bacteria at 50,000 generations tended to be even more susceptible than after 2,000 generations, although most of the change occurred during the first 2,000 generations. Despite the general trend toward increased susceptibility, we saw diverse outcomes with different antibiotics. For streptomycin, which was the only drug to which the ancestral strain was highly resistant, none of the evolved lines showed any increased susceptibility. The independently evolved lineages often exhibited correlated responses to the antibiotics, with correlations usually corresponding to their modes of action. On balance, our study shows that bacteria with low levels of intrinsic resistance often evolve to become even more susceptible to antibiotics in the absence of corresponding selection.IMPORTANCE Resistance to antibiotics often evolves when bacteria encounter antibiotics. However, bacterial strains and species without any known exposure to these drugs also vary in their intrinsic susceptibility. In many cases, evolved resistance has been shown to be costly to the bacteria, such that resistant types have reduced competitiveness relative to their sensitive progenitors in the absence of antibiotics. In this study, we examined changes in the susceptibilities of 12 populations of Escherichia coli to 15 antibiotics after 2,000 and 50,000 generations without exposure to any drug. The evolved bacteria tended to become more susceptible to most antibiotics, with most of the change occurring during the first 2,000 generations, when the bacteria were undergoing rapid adaptation to their experimental conditions. On balance, our findings indicate that bacteria with low levels of intrinsic resistance can, in the absence of relevant selection, become even more susceptible to antibiotics.

Plasticity of Promoter-Core Sequences Allows Bacteria to Compensate for the Loss of a Key Global Regulatory Gene.

Transcription regulatory networks (TRNs) are of central importance for both short-term phenotypic adaptation in response to environmental fluctuations and long-term evolutionary adaptation, with global regulatory genes often being targets of natural selection in laboratory experiments. Here, we combined evolution experiments, whole-genome resequencing, and molecular genetics to investigate the driving forces, genetic constraints, and molecular mechanisms that dictate how bacteria can cope with a drastic perturbation of their TRNs. The crp gene, encoding a major global regulator in Escherichia coli, was deleted in four different genetic backgrounds, all derived from the Long-Term Evolution Experiment (LTEE) but with different TRN architectures. We confirmed that crp deletion had a more deleterious effect on growth rate in the LTEE-adapted genotypes; and we showed that the ptsG gene, which encodes the major glucose-PTS transporter, gained CRP (cyclic AMP receptor protein) dependence over time in the LTEE. We then further evolved the four crp-deleted genotypes in glucose minimal medium, and we found that they all quickly recovered from their growth defects by increasing glucose uptake. We showed that this recovery was specific to the selective environment and consistently relied on mutations in the cis-regulatory region of ptsG, regardless of the initial genotype. These mutations affected the interplay of transcription factors acting at the promoters, changed the intrinsic properties of the existing promoters, or produced new transcription initiation sites. Therefore, the plasticity of even a single promoter region can compensate by three different mechanisms for the loss of a key regulatory hub in the E. coli TRN.

Mutator genomes decay, despite sustained fitness gains, in a long-term experiment with bacteria.

Understanding the extreme variation among bacterial genomes remains an unsolved challenge in evolutionary biology, despite long-standing debate about the relative importance of natural selection, mutation, and random drift. A potentially important confounding factor is the variation in mutation rates between lineages and over evolutionary history, which has been documented in several species. Mutation accumulation experiments have shown that hypermutability can erode genomes over short timescales. These results, however, were obtained under conditions of extremely weak selection, casting doubt on their general relevance. Here, we circumvent this limitation by analyzing genomes from mutator populations that arose during a long-term experiment with Escherichia coli, in which populations have been adaptively evolving for >50,000 generations. We develop an analytical framework to quantify the relative contributions of mutation and selection in shaping genomic characteristics, and we validate it using genomes evolved under regimes of high mutation rates with weak selection (mutation accumulation experiments) and low mutation rates with strong selection (natural isolates). Our results show that, despite sustained adaptive evolution in the long-term experiment, the signature of selection is much weaker than that of mutational biases in mutator genomes. This finding suggests that relatively brief periods of hypermutability can play an outsized role in shaping extant bacterial genomes. Overall, these results highlight the importance of genomic draft, in which strong linkage limits the ability of selection to purge deleterious mutations. These insights are also relevant to other biological systems evolving under strong linkage and high mutation rates, including viruses and cancer cells.

Genetic Basis of Exploiting Ecological Opportunity During the Long-Term Diversification of a Bacterial Population.

Adaptive diversification is an essential evolutionary process, one that produces phenotypic innovations including the colonization of available ecological niches. Bacteria can diverge in sympatry when ecological opportunities allow, but the underlying genetic mechanisms are often unknown. Perhaps, the longest-lasting adaptive diversification seen in the laboratory occurred during the long-term evolution experiment, in which 12 populations of Escherichia coli have been evolving independently for more than 65,000 generations from a common ancestor. In one population, two lineages, S and L, emerged at ~6500 generations and have dynamically coexisted ever since by negative frequency-dependent interactions mediated, in part, by acetate secretion by L. Mutations in spoT, arcA, and gntR promoted the emergence of the S lineage, although they reproduced only partially its phenotypic traits. Here, we characterize the evolved mechanism of acetate consumption by the S lineage that enabled invasion and coexistence with the L lineage. We identified an additional mutation in acs that, together with the arcA mutation, drove an early restructuring of the transcriptional control of central metabolism in S, leading to improved acetate consumption. Pervasive epistatic interactions within the S genome contributed to the exploitation of this new ecological opportunity. The emergence and maintenance of this long-term polymorphism is a complex multi-step process.

Beware batch culture: Seasonality and niche construction predicted to favor bacterial adaptive diversification.

Metabolic cross-feeding interactions between microbial strains are common in nature, and emerge during evolution experiments in the laboratory, even in homogeneous environments providing a single carbon source. In sympatry, when the environment is well-mixed, the reasons why emerging cross-feeding interactions may sometimes become stable and lead to monophyletic genotypic clusters occupying specific niches, named ecotypes, remain unclear. As an alternative to evolution experiments in the laboratory, we developed Evo2Sim, a multi-scale model of in silico experimental evolution, equipped with the whole tool case of experimental setups, competition assays, phylogenetic analysis, and, most importantly, allowing for evolvable ecological interactions. Digital organisms with an evolvable genome structure encoding an evolvable metabolic network evolved for tens of thousands of generations in environments mimicking the dynamics of real controlled environments, including chemostat or batch culture providing a single limiting resource. We show here that the evolution of stable cross-feeding interactions requires seasonal batch conditions. In this case, adaptive diversification events result in two stably co-existing ecotypes, with one feeding on the primary resource and the other on by-products. We show that the regularity of serial transfers is essential for the maintenance of the polymorphism, as it allows for at least two stable seasons and thus two temporal niches. A first season is externally generated by the transfer into fresh medium, while a second one is internally generated by niche construction as the provided nutrient is replaced by secreted by-products derived from bacterial growth. In chemostat conditions, even if cross-feeding interactions emerge, they are not stable on the long-term because fitter mutants eventually invade the whole population. We also show that the long-term evolution of the two stable ecotypes leads to character displacement, at the level of the metabolic network but also of the genome structure. This difference of genome structure between both ecotypes impacts the stability of the cross-feeding interaction, when the population is propagated in chemostat conditions. This study shows the crucial role played by seasonality in temporal niche partitioning and in promoting cross-feeding subgroups into stable ecotypes, a premise to sympatric speciation.

Evolution of Ubiquinone Biosynthesis: Multiple Proteobacterial Enzymes with Various Regioselectivities To Catalyze Three Contiguous Aromatic Hydroxylation Reactions.

The ubiquitous ATP synthase uses an electrochemical gradient to synthesize cellular energy in the form of ATP. The production of this electrochemical gradient relies on liposoluble proton carriers like ubiquinone (UQ), which is used in the respiratory chains of eukaryotes and proteobacteria. The biosynthesis of UQ requires three hydroxylation reactions on contiguous positions of an aromatic ring. In Escherichia coli, each of three UQ flavin monooxygenases (FMOs), called UbiF, UbiH, and UbiI, modifies a single position of the aromatic ring. This pattern of three hydroxylation reactions/three proteins has been accepted as a paradigm in UQ biology. Using a phylogenetic analysis, we found that UbiF, UbiH, and UbiI are detected only in a small fraction of proteobacteria, and we identified two new types of UQ FMOs: UbiM, which is distributed in members of the alpha, beta, and gamma classes of proteobacteria, and UbiL, which is restricted to members of the alphaproteobacteria. Remarkably, the ubiL and ubiM genes were found in genomes with fewer than three UQ hydroxylase-encoding genes. We demonstrated, using biochemical approaches, that UbiL from Rhodospirillum rubrum and UbiM from Neisseria meningitidis hydroxylate, respectively, two and three positions of the aromatic ring during UQ biosynthesis. We conclude that bacteria have evolved a large repertoire of hydroxylase combinations for UQ biosynthesis, including pathways with either three specialist enzymes or pathways with one or two generalist enzymes of broader regioselectivity. The emergence of the latter is potentially related to genome reduction events. IMPORTANCE UQ, a key molecule for cellular bioenergetics that is conserved from proteobacteria to humans, appeared in an ancestral proteobacterium more than 2 billion years ago. UQ biosynthesis has been studied only in a few model organisms, and thus, the diversity of UQ biosynthesis pathways is largely unknown. In the work reported here, we conducted a phylogenomic analysis of hydroxylases involved in UQ biosynthesis. Our results support the existence of at least two UQ hydroxylases in the proteobacterial ancestor, and yet, we show that their number varies from one to four in extant proteobacterial species. Our biochemical experiments demonstrated that bacteria containing only one or two UQ hydroxylases have developed generalist enzymes that are able to catalyze several steps of UQ biosynthesis. Our study documents a rare case where evolution favored the broadening of an enzyme's regioselectivity, which resulted in gene loss in several proteobacterial species with small genomes.

Tempo and mode of genome evolution in a 50,000-generation experiment.

Adaptation by natural selection depends on the rates, effects and interactions of many mutations, making it difficult to determine what proportion of mutations in an evolving lineage are beneficial. Here we analysed 264 complete genomes from 12 Escherichia coli populations to characterize their dynamics over 50,000 generations. The populations that retained the ancestral mutation rate support a model in which most fixed mutations are beneficial, the fraction of beneficial mutations declines as fitness rises, and neutral mutations accumulate at a constant rate. We also compared these populations to mutation-accumulation lines evolved under a bottlenecking regime that minimizes selection. Nonsynonymous mutations, intergenic mutations, insertions and deletions are overrepresented in the long-term populations, further supporting the inference that most mutations that reached high frequency were favoured by selection. These results illuminate the shifting balance of forces that govern genome evolution in populations adapting to a new environment.

Metabolic modelling in a dynamic evolutionary framework predicts adaptive diversification of bacteria in a long-term evolution experiment.

BACKGROUND: Predicting adaptive trajectories is a major goal of evolutionary biology and useful for practical applications. Systems biology has enabled the development of genome-scale metabolic models. However, analysing these models via flux balance analysis (FBA) cannot predict many evolutionary outcomes including adaptive diversification, whereby an ancestral lineage diverges to fill multiple niches. Here we combine in silico evolution with FBA and apply this modelling framework, evoFBA, to a long-term evolution experiment with Escherichia coli. RESULTS: Simulations predicted the adaptive diversification that occurred in one experimental population and generated hypotheses about the mechanisms that promoted coexistence of the diverged lineages. We experimentally tested and, on balance, verified these mechanisms, showing that diversification involved niche construction and character displacement through differential nutrient uptake and altered metabolic regulation. CONCLUSION: The evoFBA framework represents a promising new way to model biochemical evolution, one that can generate testable predictions about evolutionary and ecosystem-level outcomes.

Contrasting effects of historical contingency on phenotypic and genomic trajectories during a two-step evolution experiment with bacteria.

BACKGROUND: The impact of historical contingency, i.e. the past evolutionary history of a population, on further adaptation is mostly unknown at both the phenotypic and genomic levels. We addressed this question using a two-step evolution experiment. First, replicate populations of Escherichia coli were propagated in four different environmental conditions for 1000 generations. Then, all replicate populations were transferred and propagated for further 1000 generations to a single new environment. RESULTS: Using this two-step experimental evolution strategy, we investigated, at both the phenotypic and genomic levels, whether and how adaptation in the initial historical environments impacted evolutionary trajectories in a new environment. We showed that both the growth rate and fitness of the evolved populations obtained after the second step of evolution were contingent upon past evolutionary history. In contrast however, the genes that were modified during the second step of evolution were independent from the previous history of the populations. CONCLUSIONS: Our work suggests that historical contingency affects phenotypic adaptation to a new environment. This was however not reflected at the genomic level implying complex relationships between environmental factors and the genotype-to-phenotype map.

Hidden Selection of Bacterial Resistance to Fluoroquinolones In Vivo: The Case of Legionella pneumophila and Humans.

BACKGROUND: Infectious diseases are the leading cause of human morbidity and mortality worldwide. One dramatic issue is the emergence of microbial resistance to antibiotics which is a major public health concern. Surprisingly however, such in vivo adaptive ability has not been reported yet for many intracellular human bacterial pathogens such as Legionella pneumophila. METHODS: We examined 82 unrelated patients with Legionnaire's disease from which 139 respiratory specimens were sampled during hospitalization and antibiotic therapy. We both developed a real time PCR assay and used deep-sequencing approaches to detect antibiotic resistance mutations in L. pneumophila and follow their selection and fate in these samples. FINDINGS: We identified the in vivo selection of fluoroquinolone resistance mutations in L. pneumophila in two infected patients treated with these antibiotics. By investigating the mutational dynamics in patients, we showed that antibiotic resistance occurred during hospitalization most likely after fluoroquinolone treatment. INTERPRETATION: In vivo selection of antibiotic resistances in L. pneumophila may be associated with treatment failures and poor prognosis. This hidden resistance must be carefully considered in the therapeutic management of legionellosis patients and in the control of the gradual loss of effectiveness of antibiotics.

RNA chaperones buffer deleterious mutations in E. coli.

Both proteins and RNAs can misfold into non-functional conformations. Protein chaperones promote native folding of nascent polypeptides and refolding of misfolded species, thereby buffering mutations that compromise protein structure and function. Here, we show that RNA chaperones can also act as mutation buffers that enhance organismal fitness. Using competition assays, we demonstrate that overexpression of select RNA chaperones, including three DEAD box RNA helicases (DBRHs) (CsdA, SrmB, RhlB) and the cold shock protein CspA, improves fitness of two independently evolved Escherichia coli mutator strains that have accumulated deleterious mutations during short- and long-term laboratory evolution. We identify strain-specific mutations that are deleterious and subject to buffering when introduced individually into the ancestral genotype. For DBRHs, we show that buffering requires helicase activity, implicating RNA structural remodelling in the buffering process. Our results suggest that RNA chaperones might play a fundamental role in RNA evolution and evolvability.

Novel synthetic bis-indolic derivatives with antistaphylococcal activity, including against MRSA and VISA strains.

OBJECTIVES: We report the synthesis, antibacterial activity and toxicity of 24 bis-indolic derivatives obtained during the development of new ways of synthesis of marine bis-indole alkaloids from the spongotine, topsentin and hamacanthin classes. METHODS: Innovative ways of synthesis and further structural optimizations led to bis-indoles presenting either the 1-(1H-indol-3'-yl)-1,2-diaminoethane unit or the 1-(1H-indol-3-yl)ethanamine unit. MIC determination was performed for reference and clinical strains of Staphylococcus aureus and CoNS species. MBC, time-kill kinetics, solubility, hydrophobicity index, plasma protein-binding and cytotoxicity assays were performed for lead compounds. Inhibition of the S. aureus NorA efflux pump was also tested for bis-indoles with no antistaphylococcal activity. RESULTS: Lead compounds were active against both S. aureus and CoNS species, with MICs between 1 and 4 mg/L. Importantly, the same MICs were found for MRSA and vancomycin-intermediate S. aureus strains. Early concentration-dependent bactericidal activity was observed for lead derivatives. Compounds with no intrinsic antibacterial activity could inhibit the S. aureus NorA efflux pump, which is involved in resistance to fluoroquinolones. At 0.5 mg/L, the most effective compound led to an 8-fold reduction of the ciprofloxacin MIC for the SA-1199B S. aureus strain, which overexpresses NorA. However, the bis-indole compounds displayed a high hydrophobicity index and high plasma protein binding, which significantly reduced antibacterial activity. CONCLUSIONS: We have synthesized and characterized novel bis-indole derivatives as promising candidates for the development of new antistaphylococcal treatments, with preserved activity against MDR S. aureus strains.

Large chromosomal rearrangements during a long-term evolution experiment with Escherichia coli.

UNLABELLED: Large-scale rearrangements may be important in evolution because they can alter chromosome organization and gene expression in ways not possible through point mutations. In a long-term evolution experiment, twelve Escherichia coli populations have been propagated in a glucose-limited environment for over 25 years. We used whole-genome mapping (optical mapping) combined with genome sequencing and PCR analysis to identify the large-scale chromosomal rearrangements in clones from each population after 40,000 generations. A total of 110 rearrangement events were detected, including 82 deletions, 19 inversions, and 9 duplications, with lineages having between 5 and 20 events. In three populations, successive rearrangements impacted particular regions. In five populations, rearrangements affected over a third of the chromosome. Most rearrangements involved recombination between insertion sequence (IS) elements, illustrating their importance in mediating genome plasticity. Two lines of evidence suggest that at least some of these rearrangements conferred higher fitness. First, parallel changes were observed across the independent populations, with ~65% of the rearrangements affecting the same loci in at least two populations. For example, the ribose-utilization operon and the manB-cpsG region were deleted in 12 and 10 populations, respectively, suggesting positive selection, and this inference was previously confirmed for the former case. Second, optical maps from clones sampled over time from one population showed that most rearrangements occurred early in the experiment, when fitness was increasing most rapidly. However, some rearrangements likely occur at high frequency and may have simply hitchhiked to fixation. In any case, large-scale rearrangements clearly influenced genomic evolution in these populations. IMPORTANCE: Bacterial chromosomes are dynamic structures shaped by long histories of evolution. Among genomic changes, large-scale DNA rearrangements can have important effects on the presence, order, and expression of genes. Whole-genome sequencing that relies on short DNA reads cannot identify all large-scale rearrangements. Therefore, deciphering changes in the overall organization of genomes requires alternative methods, such as optical mapping. We analyzed the longest-running microbial evolution experiment (more than 25 years of evolution in the laboratory) by optical mapping, genome sequencing, and PCR analyses. We found multiple large genome rearrangements in all 12 independently evolving populations. In most cases, it is unclear whether these changes were beneficial themselves or, alternatively, hitchhiked to fixation with other beneficial mutations. In any case, many genome rearrangements accumulated over decades of evolution, providing these populations with genetic plasticity reminiscent of that observed in some pathogenic bacteria.

Epistasis and allele specificity in the emergence of a stable polymorphism in Escherichia coli.

Ecological opportunities promote population divergence into coexisting lineages. However, the genetic mechanisms that enable new lineages to exploit these opportunities are poorly understood except in cases of single mutations. We examined how two Escherichia coli lineages diverged from their common ancestor at the outset of a long-term coexistence. By sequencing genomes and reconstructing the genetic history of one lineage, we showed that three mutations together were sufficient to produce the frequency-dependent fitness effects that allowed this lineage to invade and stably coexist with the other. These mutations all affected regulatory genes and collectively caused substantial metabolic changes. Moreover, the particular derived alleles were critical for the initial divergence and invasion, indicating that the establishment of this polymorphism depended on specific epistatic interactions.

1-(1H-indol-3-yl)ethanamine derivatives as potent Staphylococcus aureus NorA efflux pump inhibitors.

The synthesis of 37 1-(1H-indol-3-yl)ethanamine derivatives, including 12 new compounds, was achieved through a series of simple and efficient chemical modifications. These indole derivatives displayed modest or no intrinsic anti-staphylococcal activity. By contrast, several of the compounds restored, in a concentration-dependent manner, the antibacterial activity of ciprofloxacin against Staphylococcus aureus strains that were resistant to fluoroquinolones due to overexpression of the NorA efflux pump. Structure-activity relationships studies revealed that the indolic aldonitrones halogenated at position 5 of the indole core were the most efficient inhibitors of the S. aureus NorA efflux pump. Among the compounds, (Z)-N-benzylidene-2-(tert-butoxycarbonylamino)-1-(5-iodo-1H-indol-3-yl)ethanamine oxide led to a fourfold decrease of the ciprofloxacin minimum inhibitory concentration against the SA-1199B strain when used at a concentration of 0.5 mg L(-1) . To the best of our knowledge, this activity is the highest reported to date for an indolic NorA inhibitor. In addition, a new antibacterial compound, tert-butyl (2-(3-hydroxyureido)-2-(1H-indol-3-yl)ethyl)carbamate, which is not toxic for human cells, was also found.