Genomic Epidemiology and Evolution of Escherichia coli in Wild Animals in Mexico

Escherichia coli is a common bacterial species in the gastrointestinal tracts of warm-blooded animals and humans. Pathogenicity and antimicrobial resistance in E. coli may emerge via host switching from animal reservoirs. Despite its potential clinical importance, knowledge of the population structure of commensal E. coli within wild hosts and the epidemiological links between E. coli in nonhuman hosts and E. coli in humans is still scarce. In this study, we analyzed the whole-genome sequencing data of a collection of 119 commensal E. coli strains recovered from the guts of 55 mammal and bird species in Mexico and Venezuela in the 1990s. We observed low concordance between the population structures of E. coli isolates colonizing wild animals and the phylogeny, taxonomy, and ecological and physiological attributes of the host species, with distantly related E. coli strains often colonizing the same or similar host species and distantly related host species often hosting closely related E. coli strains. We found no evidence for recent transmission of E. coli genomes from wild animals to either domesticated animals or humans. However, multiple livestockand human-related virulence factor genes were present in E. coli of wild animals, including virulence factors characteristic of Shiga toxin-producing E. coli (STEC) and atypical enteropathogenic E. coli (aEPEC), where several isolates from wild hosts harbored the locus of enterocyte effacement (LEE) pathogenicity island. Moreover, E. coli isolates from wild animal hosts often harbored known antibiotic resistance determinants, including those against ciprofloxacin, aminoglycosides, tetracyclines, and beta-lactams, with some determinants present in multiple, distantly related E. coli lineages colonizing very different host animals. We conclude that genome pools of E. coli colonizing the guts of wild animals and humans share virulence and antibiotic resistance genes, underscoring the idea that wild animals could serve as reservoirs for E. coli pathogenicity in human and livestock infections. IMPORTANCE Escherichia coli is a clinically important bacterial species implicated in humanand livestock-associated infections worldwide. The bacterium is known to reside in the guts of humans, livestock, and wild animals. Although wild animals are recognized as potential reservoirs for pathogenic E. coli strains, the knowledge of the population structure of E. coli in wild hosts is still scarce. In this study, we used fine resolution of whole-genome sequencing to provide novel insights into the evolution of E. coli genomes from a small yet diverse collection of strains recovered within a broad range of wild animal species (including mammals and birds), the coevolution of E. coli strains with their hosts, and the genetics of pathogenicity of E. coli strains in wild hosts in Mexico. Our results provide evidence for the clinical importance of wild animals as reservoirs for pathogenic strains and highlight the need to include nonhuman hosts in the surveillance programs for E. coli infections.Peer reviewe

The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells

The eukaryotic translation initiation factor 2α (eIF2α) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs. We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation. Disruption of genes encoding components of the ribosome P-stalk, uL10 and P1, selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR, mediated by the related eIF2α kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2α phosphorylation in vitro. These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation

Type II and type IV toxin–antitoxin systems show different evolutionary patterns in the global Klebsiella pneumoniae population

The Klebsiella pneumoniae species complex includes important opportunistic pathogens which have become public health priorities linked to major hospital outbreaks and the recent emergence of multidrug-resistant hypervirulent strains. Bacterial virulence and the spread of multidrug resistance have previously been linked to toxin-antitoxin (TA) systems. TA systems encode a toxin that disrupts essential cellular processes, and a cognate antitoxin which counteracts this activity. Whilst associated with the maintenance of plasmids, they also act in bacterial immunity and antibiotic tolerance. However, the evolutionary dynamics and distribution of TA systems in clinical pathogens are not well understood. Here we present a comprehensive survey and description of the diversity of TA systems in 259 clinically relevant genomes of K. pneumoniae. We show that TA systems are highly prevalent with a median of 20 loci per strain. Importantly, these toxins differ substantially in their distribution patterns and in their range of cognate antitoxins. Classification along these properties suggests different roles of TA systems and highlights the association and co-evolution of toxins and antitoxins

Segregating YKU80 and TLC1 Alleles Underlying Natural Variation in Telomere Properties in Wild Yeast

In yeast, as in humans, telomere length varies among individuals and is controlled by multiple loci. In a quest to define the extent of variation in telomere length, we screened 112 wild-type Saccharomyces sensu stricto isolates. We found extensive telomere length variation in S. paradoxus isolates. This phenotype correlated with their geographic origin: European strains were observed to have extremely short telomeres (<150 bp), whereas American isolates had telomeres approximately three times as long (>400 bp). Insertions of a URA3 gene near telomeres allowed accurate analysis of individual telomere lengths and telomere position effect (TPE). Crossing the American and European strains resulted in F1 spores with a continuum of telomere lengths consistent with what would be predicted if many quantitative trait loci (QTLs) were involved in length maintenance. Variation in TPE is similarly quantitative but only weakly correlated with telomere length. Genotyping F1 segregants indicated several QTLs associated with telomere length and silencing variation. These QTLs include likely candidate genes but also map to regions where there are no known genes involved in telomeric properties. We detected transgressive segregation for both phenotypes. We validated by reciprocal hemizygosity that YKU80 and TLC1 are telomere-length QTLs in the two S. paradoxus subpopulations. Furthermore, we propose that sequence divergence within the Ku heterodimer generates negative epistasis within one of the allelic combinations (American-YKU70 and European-YKU80) resulting in very short telomeres

SLING: a tool to search for linked genes in bacterial datasets.

Gene arrays and operons that encode functionally linked proteins form the most basic unit of transcriptional regulation in bacteria. Rules that govern the order and orientation of genes in these systems have been defined; however, these were based on a small set of genomes that may not be representative. The growing availability of large genomic datasets presents an opportunity to test these rules, to define the full range and diversity of these systems, and to understand their evolution. Here we present SLING, a tool to Search for LINked Genes by searching for a single functionally essential gene, along with its neighbours in a rule-defined proximity (https://github.com/ghoresh11/sling/wiki). Examining this subset of genes enables us to understand the basic diversity of these genetic systems in large datasets. We demonstrate the utility of SLING on a clinical collection of enteropathogenic Escherichia coli for two relevant operons: toxin antitoxin (TA) systems and RND efflux pumps. By examining the diversity of these systems, we gain insight on distinct classes of operons which present variable levels of prevalence and ability to be lost or gained. The importance of this analysis is not limited to TA systems and RND pumps, and can be expanded to understand the diversity of many other relevant gene arrays

The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells.

The eukaryotic translation initiation factor 2a (eIF2a) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs. We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation. Disruption of genes encoding components of the ribosome P-stalk, uL10 and P1, selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR, mediated by the related eIF2a kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2a phosphorylation in vitro. These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation

Predicting the mutations generated by repair of Cas9-induced double-strand breaks.

The DNA mutation produced by cellular repair of a CRISPR-Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR-Cas9 reagents. In total, we gathered data for >109 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions or longer microhomology-mediated deletions. Each gRNA has an individual cell-line-dependent bias toward particular outcomes. We uncover sequence determinants of the mutations produced and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments

The genetic architecture of low-temperature adaptation in the wine yeast Saccharomyces cerevisiae

[Background] Low-temperature growth and fermentation of wine yeast can enhance wine aroma and make them highly desirable traits for the industry. Elucidating response to cold in Saccharomyces cerevisiae is, therefore, of paramount importance to select or genetically improve new wine strains. As most enological traits of industrial importance in yeasts, adaptation to low temperature is a polygenic trait regulated by many interacting loci.[Results] In order to unravel the genetic determinants of low-temperature fermentation, we mapped quantitative trait loci (QTLs) by bulk segregant analyses in the F13 offspring of two Saccharomyces cerevisiae industrial strains with divergent performance at low temperature. We detected four genomic regions involved in the adaptation at low temperature, three of them located in the subtelomeric regions (chromosomes XIII, XV and XVI) and one in the chromosome XIV. The QTL analysis revealed that subtelomeric regions play a key role in defining individual variation, which emphasizes the importance of these regions’ adaptive nature.[Conclusions] The reciprocal hemizygosity analysis (RHA), run to validate the genes involved in low-temperature fermentation, showed that genetic variation in mitochondrial proteins, maintenance of correct asymmetry and distribution of phospholipid in the plasma membrane are key determinants of low-temperature adaptation.This work has been financially supported from the Spanish Government through MINECO and FEDER funds (AGL2013-47300-C3-3-R and PCIN-2015-143 grants) and from Generalitat Valenciana through PROMETEOII/2014/042 grant, awarded to JMG. This study has been carried out in the context of the European Project ERA-IB “YeastTempTation” EGR thanks the Spanish government for an FPI grant BES-2011-044498 and MM also thanks the Generalitat Valenciana for a VALi+d ACIF/2015/194 grant. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Population genomics of domestic and wild yeasts

The natural genetics of an organism is determined by the distribution of sequences of its genome. Here we present one- to four-fold, with some deeper, coverage of the genome sequences of over seventy isolates of the domesticated baker&#x27;s yeast, _Saccharomyces cerevisiae_, and its closest relative, the wild _S. paradoxus_, which has never been associated with human activity. These were collected from numerous geographic locations and sources (including wild, clinical, baking, wine, laboratory and food spoilage). These sequences provide an unprecedented view of the population structure, natural (and artificial) selection and genome evolution in these species. Variation in gene content, SNPs, indels, copy numbers and transposable elements provide insights into the evolution of different lineages. Phenotypic variation broadly correlates with global genome-wide phylogenetic relationships however there is no correlation with source. _S. paradoxus_ populations are well delineated along geographic boundaries while the variation among worldwide _S. cerevisiae_ isolates show less differentiation and is comparable to a single _S. paradoxus_ population. Rather than one or two domestication events leading to the extant baker&#x27;s yeasts, the population structure of _S. cerevisiae_ shows a few well defined geographically isolated lineages and many different mosaics of these lineages, supporting the notion that human influence provided the opportunity for outbreeding and production of new combinations of pre-existing variation