Structural Features of Single-Stranded Integron Cassette attC Sites and Their Role in Strand Selection

We recently showed that cassette integration and deletion in integron platforms were occurring through unconventional site-specific recombination reactions involving only the bottom strand of attC sites. The lack of sequence conservation among attC sites led us to hypothesize that sequence-independent structural recognition determinants must exist within attC sites. The structural data obtained from a synaptic complex of the Vibrio cholerae integrase with the bottom strand of an attC site has shown the importance of extra helical bases (EHB) inside the stem-loop structure formed from the bottom strand. Here, we systematically determined the contribution of three structural elements common to all known single-stranded attC site recombination substrates (the EHBs, the unpaired central spacer (UCS), and the variable terminal structure (VTS)) to strand choice and recombination. Their roles have been evaluated in vivo in the attl x attC reaction context using the suicide conjugation assay we previously developed, but also in an attC x attC reaction using a deletion assay. Conjugation was used to deliver the attC sites in single-stranded form. Our results show that strand choice is primarily directed by the first EHB, but the presence of the two other EHBs also serves to increase this strand selection. We found that the structure of the central spacer is essential to achieve high level recombination of the bottom strand, suggesting a dual role for this structure in active site exclusion and for hindering the reverse reaction after the first strand exchange. Moreover, we have shown that the VTS has apparently no role in strand selectivity

Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases

Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. The DNA targets of RGNs can be undesirable genes or polymorphisms, including antibiotic resistance and virulence determinants in carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia coli. Delivery of RGNs significantly improves survival in a Galleria mellonella infection model. We also show that RGNs enable modulation of complex bacterial populations by selective knockdown of targeted strains based on genetic signatures. RGNs constitute a class of highly discriminatory, customizable antimicrobials that enact selective pressure at the DNA level to reduce the prevalence of undesired genes, minimize off-target effects and enable programmable remodeling of microbiota.National Institutes of Health (U.S.) (New Innovator Award 1DP2OD008435)National Centers for Systems Biology (U.S.) (Grant 1P50GM098792)United States. Defense Threat Reduction Agency (HDTRA1-14-1-0007)Massachusetts Institute of Technology. Institute for Soldier Nanotechnologies (W911NF13D0001)National Institute of General Medical Sciences (U.S.) (Interdepartmental Biotechnology Training Program 5T32 GM008334)Fonds de la recherche en sante du Quebec (Master's Training Award

Population gene introgression and high genome plasticity for the zoonotic pathogen Streptococcus agalactiae

The influence that bacterial adaptation (or niche partitioning) within species has on gene spillover and transmission among bacteria populations occupying different niches is not well understood. Streptococcus agalactiae is an important bacterial pathogen that has a taxonomically diverse host range making it an excellent model system to study these processes. Here we analyze a global set of 901 genome sequences from nine diverse host species to advance our understanding of these processes. Bayesian clustering analysis delineated twelve major populations that closely aligned with niches. Comparative genomics revealed extensive gene gain/loss among populations and a large pan-genome of 9,527 genes, which remained open and was strongly partitioned among niches. As a result, the biochemical characteristics of eleven populations were highly distinctive (significantly enriched). Positive selection was detected and biochemical characteristics of the dispensable genes under selection were enriched in ten populations. Despite the strong gene partitioning, phylogenomics detected gene spillover. In particular, tetracycline resistance (which likely evolved in the human-associated population) from humans to bovine, canines, seals, and fish, demonstrating how a gene selected in one host can ultimately be transmitted into another, and biased transmission from humans to bovines was confirmed with a Bayesian migration analysis. Our findings show high bacterial genome plasticity acting in balance with selection pressure from distinct functional requirements of niches that is associated with an extensive and highly partitioned dispensable genome, likely facilitating continued and expansive adaptation

Frequency-dependent selection predicts patterns of radiations and biodiversity

Most empirical studies support a decline in speciation rates through time, although evidence for constant speciation rates also exists. Declining rates have been explained by invoking niche-filling processes, whereas constant rates have been attributed to non-adaptive processes such as sexual selection, mutation, and dispersal. Trends in speciation rate and the processes underlying it remain unclear, representing a critical information gap in understanding patterns of global diversity. Here we show that the speciation rate is driven by frequency dependent selection. We used a frequency-dependent and DNA sequence-based model of populations and genetic-distance-based speciation, in the absence of adaptation to ecological niches. We tested the frequency-dependent selection mechanism using cichlid fish and Darwin's finches, two classic model systems for which speciation rates and richness data exist. Using negative frequency dependent selection, our model both predicts the declining speciation rate found in cichlid fish and explains their species richness. For groups like the Darwin's finches, in which speciation rates are constant and diversity is lower, the speciation rate is better explained by a model without frequency-dependent selection. Our analysis shows that differences in diversity are driven by larger incipient species abundance (and consequent lower extinction rates) with frequency-dependent selection. These results demonstrate that mutations, genetic-distance-based speciation, sexual and frequency-dependent selection are sufficient not only for promoting rapid proliferation of new species, but also for maintaining the high diversity observed in natural systems

Principles of genetic circuit design

Cells navigate environments, communicate and build complex patterns by initiating gene expression in response to specific signals. Engineers seek to harness this capability to program cells to perform tasks or create chemicals and materials that match the complexity seen in nature. This Review describes new tools that aid the construction of genetic circuits. Circuit dynamics can be influenced by the choice of regulators and changed with expression 'tuning knobs'. We collate the failure modes encountered when assembling circuits, quantify their impact on performance and review mitigation efforts. Finally, we discuss the constraints that arise from circuits having to operate within a living cell. Collectively, better tools, well-characterized parts and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.National Institute of General Medical Sciences (U.S.) (Grant P50 GM098792)National Institute of General Medical Sciences (U.S.) (Grant R01 GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (EEC0540879)Life Technologies, Inc. (A114510)National Science Foundation (U.S.). Graduate Research FellowshipUnited States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant 4500000552

Genetic differentiation and admixture between sibling allopolyploids in the Dactylorhiza majalis complex

Allopolyploidization often happens recurrently, but the evolutionary significance of its iterative nature is not yet fully understood. Of particular interest are the gene flow dynamics and the mechanisms that allow young sibling polyploids to remain distinct while sharing the same ploidy, heritage and overlapping distribution areas. By using eight highly variable nuclear microsatellites, newly reported here, we investigate the patterns of divergence and gene flow between 386 polyploid and 42 diploid individuals, representing the sibling allopolyploids Dactylorhiza majalis s.s. and D. traunsteineri s.l. and their parents at localities across Europe. We make use in our inference of the distinct distribution ranges of the polyploids, including areas in which they are sympatric (that is, the Alps) or allopatric (for example, Pyrenees with D. majalis only and Britain with D. traunsteineri only). Our results show a phylogeographic signal, but no clear genetic differentiation between the allopolyploids, despite the visible phenotypic divergence between them. The results indicate that gene flow between sibling Dactylorhiza allopolyploids is frequent in sympatry, with potential implications for the genetic patterns across their entire distribution range. Limited interploidal introgression is also evidenced, in particular between D. incarnata and D. traunsteineri. Altogether the allopolyploid genomes appear to be porous for introgression from related diploids and polyploids. We conclude that the observed phenotypic divergence between D. majalis and D. traunsteineri is maintained by strong divergent selection on specific genomic areas with strong penetrance, but which are short enough to remain undetected by genotyping dispersed neutral markers.UE FWF; P22260UE: Y66

Genetic Incompatibility Dampens Hybrid Fertility More Than Hybrid Viability: Yeast as a Case Study

Genetic incompatibility is believed to be the major cause of postzygotic reproductive isolation. Despite huge efforts seeking for speciation-related incompatibilities in the past several decades, a general understanding of how genetic incompatibility evolves in affecting hybrid fitness is not available, primarily due to the fact that the number of known incompatibilities is small. Instead of further mapping specific incompatible genes, in this paper we aimed to know the overall effects of incompatibility on fertility and viability, the two aspects of fitness, by examining 89 gametes produced by yeast S. cerevisiae - S. paradoxus F1 hybrids. Homozygous F2 hybrids formed by autodiploidization of F1 gametes were subject to tests for growth rate and sporulation efficiency. We observed much stronger defects in sporulation than in clonal growth for every single F2 hybrid strain, indicating that genetic incompatibility affects hybrid fertility more than hybrid viability in yeast. We related this finding in part to the fast-evolving nature of meiosis-related genes, and proposed that the generally low expression levels of these genes might be a cause of the observation

Gene cassette transcription in a large integron-associated array

<p>Abstract</p> <p>Background</p> <p>The integron/gene cassette system is a diverse and effective adaptive resource for prokaryotes. Short cassette arrays, with less than 10 cassettes adjacent to an integron, provide this resource through the expression of cassette-associated genes by an integron-borne promoter. However, the advantage provided by large arrays containing hundreds of cassettes is less obvious. In this work, using the 116-cassette array of <it>Vibrio </it>sp. DAT722 as a model, we investigated the theory that the majority of genes contained within large cassette arrays are widely expressed by intra-array promoters in addition to the integron-borne promoter.</p> <p>Results</p> <p>We demonstrated that the majority of the cassette-associated genes in the subject array were expressed. We further showed that cassette expression was conditional and that the conditionality varied across the array. We finally showed that this expression was mediated by a diversity of cassette-borne promoters within the array capable of responding to environmental stressors.</p> <p>Conclusions</p> <p>Widespread expression within large gene cassette arrays could provide an adaptive advantage to the host in proportion to the size of the array. Our findings explained the existence and maintenance of large cassette arrays within many prokaryotes. Further, we suggested that repeated rearrangement of cassettes containing genes and/or promoters within large arrays could result in the assembly of operon-like groups of co-expressed cassettes within an array. These findings add to our understanding of the adaptive repertoire of the integron/gene cassette system in prokaryotes and consequently, the evolutionary impact of this system.</p

Multiple Molecular Mechanisms Cause Reproductive Isolation between Three Yeast Species

Incompatibility between nuclear and mitochondrial genomes in yeast species may represent a general mechanism of reproductive isolation during yeast evolution