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Auto Tag: Recombination

September 22, 2019

Multiple convergent supergene evolution events in mating-type chromosomes.

Convergent adaptation provides unique insights into the predictability of evolution and ultimately into processes of biological diversification. Supergenes (beneficial gene linkage) are striking examples of adaptation, but little is known about their prevalence or evolution. A recent study on anther-smut fungi documented supergene formation by rearrangements linking two key mating-type loci, controlling pre- and post-mating compatibility. Here further high-quality genome assemblies reveal four additional independent cases of chromosomal rearrangements leading to regions of suppressed recombination linking these mating-type loci in closely related species. Such convergent transitions in genomic architecture of mating-type determination indicate strong selection favoring linkage of mating-type loci into cosegregating supergenes. We find independent evolutionary strata (stepwise recombination suppression) in several species, with extensive rearrangements, gene losses, and transposable element accumulation. We thus show remarkable convergence in mating-type chromosome evolution, recurrent supergene formation, and repeated evolution of similar phenotypes through different genomic changes.

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September 22, 2019

Genomes of all known members of a Plasmodium subgenus reveal paths to virulent human malaria.

Plasmodium falciparum, the most virulent agent of human malaria, shares a recent common ancestor with the gorilla parasite Plasmodium praefalciparum. Little is known about the other gorilla- and chimpanzee-infecting species in the same (Laverania) subgenus as P. falciparum, but none of them are capable of establishing repeated infection and transmission in humans. To elucidate underlying mechanisms and the evolutionary history of this subgenus, we have generated multiple genomes from all known Laverania species. The completeness of our dataset allows us to conclude that interspecific gene transfers, as well as convergent evolution, were important in the evolution of these species. Striking copy number and structural variations were observed within gene families and one, stevor, shows a host-specific sequence pattern. The complete genome sequence of the closest ancestor of P. falciparum enables us to estimate the timing of the beginning of speciation to be 40,000-60,000 years ago followed by a population bottleneck around 4,000-6,000 years ago. Our data allow us also to search in detail for the features of P. falciparum that made it the only member of the Laverania able to infect and spread in humans.

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September 22, 2019

In vitro DNA SCRaMbLE.

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The “in vitro SCRaMbLE system” uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a ß-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via the rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve.

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September 22, 2019

Precise control of SCRaMbLE in synthetic haploid and diploid yeast.

Compatibility between host cells and heterologous pathways is a challenge for constructing organisms with high productivity or gain of function. Designer yeast cells incorporating the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a platform for generating genotype diversity. Here we construct a genetic AND gate to enable precise control of the SCRaMbLE method to generate synthetic haploid and diploid yeast with desired phenotypes. The yield of carotenoids is increased to 1.5-fold by SCRaMbLEing haploid strains and we determine that the deletion of YEL013W is responsible for the increase. Based on the SCRaMbLEing in diploid strains, we develop a strategy called Multiplex SCRaMbLE Iterative Cycling (MuSIC) to increase the production of carotenoids up to 38.8-fold through 5 iterative cycles of SCRaMbLE. This strategy is potentially a powerful tool for increasing the production of bio-based chemicals and for mining deep knowledge.

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September 22, 2019

The evolution of genomic and epigenomic features in two Pleurotus fungi.

Pleurotus tuoliensis (Bailinggu, designated Pt) and P. eryngii var. eryngii (Xingbaogu, designated Pe) are highly valued edible mushrooms. We report de novo assemblies of high-quality genomes for both mushrooms based on PacBio RS II sequencing and annotation of all identified genes. A comparative genomics analysis between Pt and Pe with P. ostreatus as an outgroup taxon revealed extensive genomic divergence between the two mushroom genomes primarily due to the rapid gain of taxon-specific genes and disruption of synteny in either taxon. The re-appraised phylogenetic relationship between Pt and Pe at the genome-wide level validates earlier proposals to designate Pt as an independent species. Variation of the identified wood-decay-related gene content can largely explain the variable adaptation and host specificity of the two mushrooms. On the basis of the two assembled genome sequences, methylomes and the regulatory roles of DNA methylation in gene expression were characterized and compared. The genome, methylome and transcriptome data of these two important mushrooms will provide valuable information for advancing our understanding of the evolution of Pleurotus and related genera and for facilitating genome- and epigenome-based strategies for mushroom breeding.

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September 22, 2019

Mutant phenotypes for thousands of bacterial genes of unknown function.

One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because they are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations.

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September 22, 2019

The FBT1 large serine recombinase catalyzes DNA integration at pseudo-attB sites in the genus Nocardia.

Plasmid vectors based on bacteriophage integrases are important tools in molecular microbiology for the introduction of foreign DNA, especially into bacterial species where other systems for genetic manipulation are limited. Site specific integrases catalyze recombination between phage and bacterial attachment sites (attP and attB, respectively) and the best studied integrases in the actinomycetes are the serine integrases from the Streptomyces bacteriophages FC31 and FBT1. As this reaction is unidirectional and highly stable, vectors containing phage integrase systems have been used in a number of genetic engineering applications. Plasmids bearing the FBT1 integrase have been used to introduce DNA into Streptomyces and Amycolatopsis strains; however, they have not been widely studied in other actinobacterial genera. Here, we show that vectors based on FBT1 integrase can stably integrate into the chromosomes of a range of Nocardia species, and that this integration occurs despite the absence of canonical attB sites in these genomes. Furthermore, we show that a FBT1 integrase-based vector can insert at multiple pseudo-attB sites within a single strain and we determine the sequence of a pseudo-attB motif. These data suggest that FBT1 integrase-based vectors can be used to readily and semi-randomly introduce foreign DNA into the genomes of a range of Nocardia species. However, the precise site of insertion will likely require empirical determination in each species to avoid unexpected off-target effects.

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September 22, 2019

Whole genome analysis reveals the diversity and evolutionary relationships between necrotic enteritis-causing strains of Clostridium perfringens.

Clostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C. perfringens isolates, from around the world and from different years. The genomes were sequenced and compared with 20 genomes available from public databases, which were from a diverse collection of isolates from chickens, other animals, and humans. We used a distance based phylogeny that was constructed based on gene content rather than sequence identity. Similarity between strains was defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. We also compared these methods to the sequence-based phylogeny of the core genome.Distinct pathogenic clades of necrotic enteritis-causing C. perfringens were identified. They were characterised by variable regions encoded on the chromosome, with predicted roles in capsule production, adhesion, inhibition of related strains, phage integration, and metabolism. Some strains have almost identical genomes, even though they were isolated from different geographic regions at various times, while other highly distant genomes appear to result in similar outcomes with regard to virulence and pathogenesis.The high level of diversity in chicken isolates suggests there is no reliable factor that defines a chicken strain of C. perfringens, however, disease-causing strains can be defined by the presence of netB-encoding plasmids. This study reveals that horizontal gene transfer appears to play a significant role in genetic variation of the C. perfringens chromosome as well as the plasmid content within strains.

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September 22, 2019

Whole-genome analysis of three yeast strains used for production of sherry-like wines revealed genetic traits specific to Flor yeasts.

Flor yeast strains represent a specialized group of Saccharomyces cerevisiae yeasts used for biological wine aging. We have sequenced the genomes of three flor strains originated from different geographic regions and used for production of sherry-like wines in Russia. According to the obtained phylogeny of 118 yeast strains, flor strains form very tight cluster adjacent to the main wine clade. SNP analysis versus available genomes of wine and flor strains revealed 2,270 genetic variants in 1,337 loci specific to flor strains. Gene ontology analysis in combination with gene content evaluation revealed a complex landscape of possibly adaptive genetic changes in flor yeast, related to genes associated with cell morphology, mitotic cell cycle, ion homeostasis, DNA repair, carbohydrate metabolism, lipid metabolism, and cell wall biogenesis. Pangenomic analysis discovered the presence of several well-known “non-reference” loci of potential industrial importance. Events of gene loss included deletions of asparaginase genes, maltose utilization locus, and FRE-FIT locus involved in iron transport. The latter in combination with a flor-yeast-specific mutation in the Aft1 transcription factor gene is likely to be responsible for the discovered phenotype of increased iron sensitivity and improved iron uptake of analyzed strains. Expansion of the coding region of the FLO11 flocullin gene and alteration of the balance between members of the FLO gene family are likely to positively affect the well-known propensity of flor strains for velum formation. Our study provides new insights in the nature of genetic variation in flor yeast strains and demonstrates that different adaptive properties of flor yeast strains could have evolved through different mechanisms of genetic variation.

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September 22, 2019

Convergent loss of ABC transporter genes from Clostridioides difficile genomes is associated with impaired tyrosine uptake and p-cresol production.

We report the frequent, convergent loss of two genes encoding the substrate-binding protein and the ATP-binding protein of an ATP-binding cassette (ABC) transporter from the genomes of unrelated Clostridioides difficile strains. This specific genomic deletion was strongly associated with the reduced uptake of tyrosine and phenylalanine and production of derived Stickland fermentation products, including p-cresol, suggesting that the affected ABC transporter had been responsible for the import of aromatic amino acids. In contrast, the transporter gene loss did not measurably affect bacterial growth or production of enterotoxins. Phylogenomic analysis of publically available genome sequences indicated that this transporter gene deletion had occurred multiple times in diverse clonal lineages of C. difficile, with a particularly high prevalence in ribotype 027 isolates, where 48 of 195 genomes (25%) were affected. The transporter gene deletion likely was facilitated by the repetitive structure of its genomic location. While at least some of the observed transporter gene deletions are likely to have occurred during the natural life cycle of C. difficile, we also provide evidence for the emergence of this mutation during long-term laboratory cultivation of reference strain R20291.

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September 22, 2019

Isolation, functional characterization and transmissibility of p3PS10, a multidrug resistance plasmid of the fish pathogen Piscirickettsia salmonis.

Antibiotic resistance is a major public health concern due to its association with the loss of efficacy of antimicrobial therapies. Horizontal transfer events may play a significant role in the dissemination of resistant bacterial phenotypes, being mobilizable plasmids a well-known mechanism. In this study, we aimed to gain insights into the genetics underlying the development of antibiotic resistance by Piscirickettsia salmonis isolates, a bacterial fish pathogen and causative agent of salmonid piscirickettsiosis, and the main target of antibiotics used in Chilean salmon farming. We provide experimental evidence that the plasmid p3PS10, which harbors multidrug resistance genes for chloramphenicol (cat2), tetracyclines [tet(31)], aminoglycosides (sat1 and aadA1), and sulfonamides (sul2), is carried by a group of P. salmonis isolates exhibiting a markedly reduced susceptibility to oxytetracycline in vitro (128-256 µg/mL of minimal inhibitory concentration, MIC). Antibiotic susceptibility analysis extended to those antibiotics showed that MIC of chloramphenicol, streptomycin, and sulfamethoxazole/trimethoprim were high, but the MIC of florfenicol remained at the wild-type level. By means of molecular cloning, we demonstrate that those genes encoding putative resistance markers are indeed functional. Interestingly, mating assays clearly show that p3PS10 is able to be transferred into and replicate in different hosts, thereby conferring phenotypes similar to those found in the original host. According to epidemiological data, this strain is distributed across aquaculture settings in southern Chile and is likely to be responsible for oxytetracycline treatment failures. This work demonstrates that P. salmonis is more versatile than it was thought, capable of horizontally transferring DNA, and probably playing a role as a vector of resistance traits among the seawater bacterial population. However, the low transmission frequency of p3PS10 suggests a negligible chance of resistance markers being spread to human pathogens.

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September 22, 2019

Biosynthesis of the 15-membered ring depsipeptide neoantimycin.

Antimycins are a family of natural products possessing outstanding biological activities and unique structures, which have intrigued chemists for over a half century. Of particular interest are the ring-expanded antimycins that show promising anticancer potential and whose biosynthesis remains uncharacterized. Specifically, neoantimycin and its analogs have been shown to be effective regulators of the oncogenic proteins GRP78/BiP and K-Ras. The neoantimycin structural skeleton is built on a 15-membered tetralactone ring containing one methyl, one hydroxy, one benzyl, and three alkyl moieties, as well as an amide linkage to a conserved 3-formamidosalicylic acid moiety. Although the biosynthetic gene cluster for neoantimycins was recently identified, the enzymatic logic that governs the synthesis of neoantimycins has not yet been revealed. In this work, the neoantimycin gene cluster is identified, and an updated sequence and annotation is provided delineating a nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) hybrid scaffold. Using cosmid expression and CRISPR/Cas-based genome editing, several heterologous expression strains for neoantimycin production are constructed in two separate Streptomyces species. A combination of in vivo and in vitro analysis is further used to completely characterize the biosynthesis of neoantimycins including the megasynthases and trans-acting domains. This work establishes a set of highly tractable hosts for producing and engineering neoantimycins and their C11 oxidized analogs, paving the way for neoantimycin-based drug discovery and development.

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September 22, 2019

Multiple large inversions and breakpoint rewiring of gene expression in the evolution of the fire ant social supergene.

Supergenes consist of co-adapted loci that segregate together and are associated with adaptive traits. In the fire ant Solenopsis invicta, two ‘social’ supergene variants regulate differences in colony queen number and other traits. Suppressed recombination in this system is maintained, in part, by a greater than 9 Mb inversion, but the supergene is larger. Has the supergene in S. invicta undergone multiple large inversions? The initial gene content of the inverted allele of a supergene would be the same as that of the wild-type allele. So, how did the inversion increase in frequency? To address these questions, we cloned one extreme breakpoint in the fire ant supergene. In doing so, we found a second large (greater than 800 Kb) rearrangement. Furthermore, we determined the temporal order of the two big inversions based on the translocation pattern of a third small fragment. Because the S. invicta supergene lacks evolutionary strata, our finding of multiple inversions may support an introgression model of the supergene. Finally, we showed that one of the inversions swapped the promoter of a breakpoint-adjacent gene, which might have conferred a selective advantage relative to the non-inverted allele. Our findings provide a rare example of gene alterations arising directly from an inversion event.© 2018 The Author(s).

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September 22, 2019

Size and content of the sex-determining region of the Y chromosome in dioecious Mercurialis annua, a plant with homomorphic sex chromosomes.

Dioecious plants vary in whether their sex chromosomes are heteromorphic or homomorphic, but even homomorphic sex chromosomes may show divergence between homologues in the non-recombining, sex-determining region (SDR). Very little is known about the SDR of these species, which might represent particularly early stages of sex-chromosome evolution. Here, we assess the size and content of the SDR of the diploid dioecious herb Mercurialis annua, a species with homomorphic sex chromosomes and mild Y-chromosome degeneration. We used RNA sequencing (RNAseq) to identify new Y-linked markers for M. annua. Twelve of 24 transcripts showing male-specific expression in a previous experiment could be amplified by polymerase chain reaction (PCR) only from males, and are thus likely to be Y-linked. Analysis of genome-capture data from multiple populations of M. annua pointed to an additional six male-limited (and thus Y-linked) sequences. We used these markers to identify and sequence 17 sex-linked bacterial artificial chromosomes (BACs), which form 11 groups of non-overlapping sequences, covering a total sequence length of about 1.5 Mb. Content analysis of this region suggests that it is enriched for repeats, has low gene density, and contains few candidate sex-determining genes. The BACs map to a subset of the sex-linked region of the genetic map, which we estimate to be at least 14.5 Mb. This is substantially larger than estimates for other dioecious plants with homomorphic sex chromosomes, both in absolute terms and relative to their genome sizes. Our data provide a rare, high-resolution view of the homomorphic Y chromosome of a dioecious plant.

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September 22, 2019

Comparative genomic analysis of Geosporobacter ferrireducens and its versatility of anaerobic energy metabolism.

Members of the family Clostridiaceae within phylum Firmicutes are ubiquitous in various iron-reducing environments. However, genomic data on iron-reducing bacteria of the family Clostridiaceae, particularly regarding their environmental distribution, are limited. Here, we report the analysis and comparison of the genomic properties of Geosporobacter ferrireducens IRF9, a strict anaerobe that ferments sugars and degrades toluene under iron-reducing conditions, with those of the closely related species, Geosporobacter subterraneus DSM 17957. Putative alkyl succinate synthase-encoding genes were observed in the genome of strain IRF9 instead of the typical benzyl succinate synthase-encoding genes. Canonical genes associated with iron reduction were not observed in either genome. The genomes of strains IRF9 and DMS 17957 harbored genes for acetogenesis, that encode two types of Rnf complexes mediating the translocation of H+ and Na+ ions, respectively. Strain IRF9 harbored two different types of ATPases (Na+-dependent F-type ATPase and H+-dependent V-type ATPase), which enable full exploitation of ion gradients. The versatile energy conservation potential of strain IRF9 promotes its survival in various environmental conditions.

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