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.

Reuse of medical face masks in domestic and community settings without sacrificing safety: Ecological and economical lessons from the Covid-19 pandemic.

The need for personal protective equipment increased exponentially in response to the Covid-19 pandemic. To cope with the mask shortage during springtime 2020, a French consortium was created to find ways to reuse medical and respiratory masks in healthcare departments. The consortium addressed the complex context of the balance between cleaning medical masks in a way that maintains their safety and functionality for reuse, with the environmental advantage to manage medical disposable waste despite the current mask designation as single-use by the regulatory frameworks. We report a Workflow that provides a quantitative basis to determine the safety and efficacy of a medical mask that is decontaminated for reuse. The type IIR polypropylene medical masks can be washed up to 10 times, washed 5 times and autoclaved 5 times, or washed then sterilized with radiations or ethylene oxide, without any degradation of their filtration or breathability properties. There is loss of the anti-projection properties. The Workflow rendered the medical masks to comply to the AFNOR S76-001 standard as "type 1 non-sanitory usage masks". This qualification gives a legal status to the Workflow-treated masks and allows recommendation for the reuse of washed medical masks by the general population, with the significant public health advantage of providing better protection than cloth-tissue masks. Additionally, such a legal status provides a basis to perform a clinical trial to test the masks in real conditions, with full compliance with EN 14683 norm, for collective reuse. The rational reuse of medical mask and their end-of-life management is critical, particularly in pandemic periods when decisive turns can be taken. The reuse of masks in the general population, in industries, or in hospitals (but not for surgery) has significant advantages for the management of waste without degrading the safety of individuals wearing reused masks.

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.

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.

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.

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.

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.

Insertion sequence-driven evolution of Escherichia coli in chemostats.

Insertion sequence (IS) elements are present in almost all bacterial genomes and are mobile enough to provide genomic tools to differentiate closely related isolates. They can be used to estimate genetic diversity and identify fitness-enhancing mutations during evolution experiments. Here, we determined the genomic distribution of eight IS elements in 120 genomes sampled from Escherichia coli populations that evolved in glucose- and phosphate-limited chemostats by comparison to the ancestral pattern. No significant differential transposition of the various IS types was detected across the environments. The phylogenies revealed substantial diversity amongst clones sampled from each chemostat, consistent with the phenotypic diversity within populations. Two IS-related changes were common to independent chemostats, suggesting parallel evolution. One of them corresponded to insertions of IS1 elements within rpoS encoding the master regulator of stress conditions. The other parallel event was an IS5-dependent deletion including mutY involved in DNA repair, thereby providing the molecular mechanism of generation of mutator clones in these evolving populations. These deletions occurred in different co-existing genotypes within single populations and were of various sizes. Moreover, differential locations of IS elements combined with their transpositional activity provided evolved clones with different phenotypic landscapes. Therefore, IS elements strongly influenced the evolutionary processes in continuous E. coli cultures by providing a way to modify both the global regulatory network and the mutation rates of evolving cells.

Identification of three tomato flower and fruit MADS-box proteins with a putative histone deacetylase binding domain.

MADS-box transcription factors play crucial roles in organ and cell differentiation in organisms ranging from yeast to humans. Most of the work on plant MADS-box proteins focused on their roles in floral development whereas less information is available on their function in fruit maturation. We cloned three distinct tomato cDNAs using a RT-PCR approach, encoding LeMADS1, LeMADS5 and LeMADS6 factors and whose mRNAs mostly accumulate in tomato flowers and fruits. Phylogeny analysis indicates that LeMADS1, 5 and 6 belong to the MEF2-like family. When transiently expressed in tobacco leaves or in human cells, LeMADS1, 5 and 6 are targeted to the cell nucleus. As the endogenous target genes of these putative transcription factors are unknown, the transcriptional activity of these proteins was characterized in a heterologous system and we showed that, when fused to a Gal4-DNA-binding domain, they repress the transcription of heterologous reporter genes. Since histone deacetylases control MEF2 transcriptional activity and since a putative histone deacetylase binding site was present in LeMADS1, 5 and 6, we tested the potential interaction between these factors and HDAC5 deacetylase. Surprisingly, in this heterologous system, LeMADS1, 5 and 6 interacted with HDAC5 N-terminal region. Our data suggest that, like mammalian MEF2A, plant MADS-box transcriptional activity might be regulated by enzymes controlling chromatin acetylation.

Genomic identification of a novel mutation in hfq that provides multiple benefits in evolving glucose-limited populations of Escherichia coli.

Beneficial mutations in diversifying glucose-limited Escherichia coli populations are mostly unidentified. The genome of an evolved isolate with multiple differences from that of the ancestor was fully assembled. Remarkably, a single mutation in hfq was responsible for the multiple benefits under glucose limitation through changes in at least five regulation targets.

Parallel genetic and phenotypic evolution of DNA superhelicity in experimental populations of Escherichia coli.

DNA supercoiling is the master function that interconnects chromosome structure and global gene transcription. This function has recently been shown to be under strong selection in Escherichia coli. During the evolution of 12 initially identical populations propagated in a defined environment for 20,000 generations, parallel increases in DNA supercoiling were observed in ten populations. The genetic changes associated with the increased supercoiling were examined in one population, and beneficial mutations in the genes topA (encoding topoisomerase I) and fis (encoding a histone-like protein) were identified. To elucidate the molecular basis and impact of these changes, we quantified the level of genetic, phenotypic, and molecular parallelism linked to DNA supercoiling in all 12 evolving populations. First, sequence determination of DNA topology-related loci revealed strong genetic parallelism, with mutations concentrated in three genes (topA, fis, and dusB), although the populations had different alleles at each locus. Statistical analyses of these polymorphisms implied the action of positive selection and, moreover, suggested that fis and dusB, which belong to the same operon, have related functions. Indeed, we demonstrated that DusB regulates the expression of fis by both experimental and phylogenetic analyses. Second, molecular analyses of five mutations in fis and dusB affecting the transcription, translation, and protein activity of Fis also revealed strong parallelism in the resulting phenotypic effects. Third, artificially increasing DNA supercoiling in one of the two populations that lacked DNA topology changes led to a significant fitness increase. The high levels of molecular and genetic parallelism, targeting a small subset of the many genes involved in DNA supercoiling, indicate that changes in DNA superhelicity have been important in the evolution of these populations. Surprisingly, however, most of the evolved alleles we tested had either no detectable or slightly deleterious effects on fitness, despite these signatures of positive selection.

Plant lipid-associated fibrillin proteins condition jasmonate production under photosynthetic stress.

The role of a subfamily of lipid globule-associated proteins, referred to as plant fibrillins (FIB1a, -1b, -2), was determined using a RNA interference (RNAi) strategy. We show that Arabidopsis plants with reduced levels of these plastid structural proteins are impaired in long-term acclimation to environmental constraint, namely photooxidative stress imposed by high light combined with cold. As a result, their photosynthetic apparatus is inefficiently protected. This leads to the prevalence of an abnormal granal and stromal membrane arrangement, as well as higher photosystem II photoinhibition under stress. The visible phenotype of FIB1-2 RNAi lines also includes retarded shoot growth and a deficit in anthocyanin accumulation under stress. All examined phenotypic effects of lower FIB levels are abolished by jasmonate (JA) treatment. An atypical expression pattern of several JA-induced genes was observed in RNAi plants. A JA-deficient mutant was found to share similar stress phenotypic characteristics with FIB RNAi plants. We conclude a new physiological role for JA, namely acclimation of chloroplasts, and that light/cold stress-related JA biosynthesis is conditioned by the accumulation of plastoglobule-associated FIB1-2 proteins. Consistent correlative data suggest that this FIB effect is mediated by plastoglobule (and triacylglycerol) accumulation as the potential site for initiating the chloroplast stress-related JA biosynthesis.

Fibrillin influence on plastid ultrastructure and pigment content in tomato fruit.

The protein termed fibrillin is involved in the formation of lipoprotein structures, such as plastoglobules and fibrils in certain chromoplast types, which have been implicated in the over-production of pigments due to a sink effect. In order to examine its effect in differentiating chromoplasts of a non-fibrillar type, the pepper fibrillin gene was expressed in tomato fruit. Both the transcript and protein were found to accumulate during tomato fruit ripening from an early mature green stage. However, formation of carotenoid deposition structures in tomato chromoplasts, such as fibrils, was not observed. Nevertheless, a two-fold increase in carotenoid content and associated carotenoid derived flavour volatiles (6-methyl-5-hepten-2-one, geranylacetone, beta-ionone and beta-cyclocitral) was observed. An unexpected phenotypic observation in the transgenic fruit was the delayed loss of thylakoids in differentiating chromoplasts, leading to the transient formation of plastids exhibiting a typical chromoplastic zone adjacent to a protected chloroplastic zone with preserved thylakoids. An in vitro assay has been developed to monitor fibrillin activity on thylakoids: data were obtained suggesting a membrane protection role for fibrillin, more specifically against moderate uncoupling effects.

Isolation and study of a ubiquitously expressed tomato pectin methylesterase regulatory region

Pectin methylesterase (PME) is an enzyme located in the plant cell wall of higher plants whose physiological role is largely unknown. We had isolated a PME gene from a tomato genomic library, including 2.59 kb of 5üL flanking region and the coding region. Both coding and promoter region were sequenced and computer analyzed. Tobacco transgenic plants were created harboring constructs in which 2.596 Kb, 1.306 Kb and 0.267 Kb sizes of the promoter were driving the expression of âÀ-Glucuronidase gene (GUS). GUS activity was studied by histochemical and fluorometric assays. Two introns of 106 and 1039 bp were found in the coding region and phylogenetic analysis placed this PME gene closer to genes from Citrus sinensis and Arabidopsis thaliana than tomato fruit-specific PME genes. In the promoter, it was found direct repeats, perfect inverted repeats and light responsive elements. GUS histochemical analysis showed activity in all plant tissues with the exception of pollen. The reduction in the promoter size induced a reduction in GUS activity in root, stem and leaf. Furthermore, root and leaf showed the highest and lowest activity, respectively. We had isolated a tomato PME gene with novel characteristics as compared with other known PME genes from tomato.