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April 21, 2020  |  

Systematic evasion of the restriction-modification barrier in bacteria.

Bacteria that are recalcitrant to genetic manipulation using modern in vitro techniques are termed genetically intractable. Genetic intractability is a fundamental barrier to progress that hinders basic, synthetic, and translational microbiology research and development beyond a few model organisms. The most common underlying causes of genetic intractability are restriction-modification (RM) systems, ubiquitous defense mechanisms against xenogeneic DNA that hinder the use of genetic approaches in the vast majority of bacteria and exhibit strain-level variation. Here, we describe a systematic approach to overcome RM systems. Our approach was inspired by a simple hypothesis: if a synthetic piece of DNA lacks the highly specific target recognition motifs for a host’s RM systems, then it is invisible to these systems and will not be degraded during artificial transformation. Accordingly, in this process, we determine the genome and methylome of an individual bacterial strain and use this information to define the bacterium’s RM target motifs. We then synonymously eliminate RM targets from the nucleotide sequence of a genetic tool in silico, synthesize an RM-silent “SyngenicDNA” tool, and propagate the tool as minicircle plasmids, termed SyMPL (SyngenicDNA Minicircle Plasmid) tools, before transformation. In a proof-of-principle of our approach, we demonstrate a profound improvement (five orders of magnitude) in the transformation of a clinically relevant USA300 strain of Staphylococcus aureus This stealth-by-engineering SyngenicDNA approach is effective, flexible, and we expect in future applications could enable microbial genetics free of the restraints of restriction-modification barriers.Copyright © 2019 the Author(s). Published by PNAS.


April 21, 2020  |  

Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm.

Our ability to predict evolutionary trajectories of pathogens in response to antibiotic pressure is one of the promising leverage to fight against the present antibiotic resistance worldwide crisis. Yet, few studies tackled this question in situ at the outbreak level, due to the difficulty to link a given pathogenic clone evolution with its precise antibiotic exposure over time. In this study, we monitored the real-time evolution of an Aeromonas salmonicida clone in response to successive antibiotic and vaccine therapies in a commercial fish farm. The clone was responsible for a four-year outbreak of furunculosis within a Recirculating Aquaculture System Salmo salar farm in China, and we reconstructed the precise tempo of mobile genetic elements (MGEs) acquisition events during this period. The resistance profile provided by the acquired MGEs closely mirrored the antibiotics used to treat the outbreak, and we evidenced that two subclonal groups developed similar resistances although unrelated MGE acquisitions. Finally, we also demonstrated the efficiency of vaccination in outbreak management and its positive effect on antibiotic resistance prevalence. Our study provides unprecedented knowledge critical to understand evolutionary trajectories of resistant pathogens outside the laboratory. © 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.


April 21, 2020  |  

Mutation of a bHLH transcription factor allowed almond domestication.

Wild almond species accumulate the bitter and toxic cyanogenic diglucoside amygdalin. Almond domestication was enabled by the selection of genotypes harboring sweet kernels. We report the completion of the almond reference genome. Map-based cloning using an F1 population segregating for kernel taste led to the identification of a 46-kilobase gene cluster encoding five basic helix-loop-helix transcription factors, bHLH1 to bHLH5. Functional characterization demonstrated that bHLH2 controls transcription of the P450 monooxygenase-encoding genes PdCYP79D16 and PdCYP71AN24, which are involved in the amygdalin biosynthetic pathway. A nonsynonymous point mutation (Leu to Phe) in the dimerization domain of bHLH2 prevents transcription of the two cytochrome P450 genes, resulting in the sweet kernel trait. Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.


April 21, 2020  |  

Genome assembly of a tropical maize inbred line provides insights into structural variation and crop improvement.

Maize is one of the most important crops globally, and it shows remarkable genetic diversity. Knowledge of this diversity could help in crop improvement; however, gold-standard genomes have been elucidated only for modern temperate varieties. Here, we present a high-quality reference genome (contig N50 of 15.78?megabases) of the maize small-kernel inbred line, which is derived from a tropical landrace. Using haplotype maps derived from B73, Mo17 and SK, we identified 80,614 polymorphic structural variants across 521 diverse lines. Approximately 22% of these variants could not be detected by traditional single-nucleotide-polymorphism-based approaches, and some of them could affect gene expression and trait performance. To illustrate the utility of the diverse SK line, we used it to perform map-based cloning of a major effect quantitative trait locus controlling kernel weight-a key trait selected during maize improvement. The underlying candidate gene ZmBARELY ANY MERISTEM1d provides a target for increasing crop yields.


April 21, 2020  |  

Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race.

CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.


April 21, 2020  |  

The complete genome sequence of the denitrifying bacterium Marinobacter sp. Arc7-DN-1 isolated from Arctic Ocean sediment

The general features and genome characteristics of the denitrifying bacterium Marinobacter sp. Arc7-DN-1, isolated from Arctic Ocean sediment, are described. Marinobacter sp. Arc7-DN-1 uses NO3- or NH4+ as the sole nitrogen source to grow at low temperatures. The strain can grow at a wide range of temperatures (0–30?°C) and NaCl concentration (15–90‰). The genome has one circular chromosome of 4,300,456?bp (57.64?mol%?G?+?C content), consisting of 4012 coding genes, including 50 tRNAs and three rRNA operons as 16S-23S-5S rRNA. On the basis of the KEGG analysis, strain Arc7-DN-1 encodes 43 proteins related to nitrogen metabolism, including a complete denitrifying pathway and an assimilatory nitrate reduction pathway.


April 21, 2020  |  

Development of CRISPR-Cas systems for genome editing and beyond

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotech- nology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstra- tion of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated a deeper examination of natural CRISPR-Cas systems, including the discovery of new types of CRISPR-Cas systems. These new discoveries in turn spurred further technological developments. I review these exciting discoveries and technologies as well as provide an overview of the broad array of applications of these technologies in basic research and in the improvement of human health. It is clear that we are only just beginning to unravel the potential within microbial diversity, and it is quite likely that we will continue to discover other exciting phenomena, some of which it may be possible to repurpose as molecular technologies. The transformation of mysterious natural phenomena to powerful tools, however, takes a collective effort to discover, characterize, and engineer them, and it has been a privilege to join the numerous researchers who have contributed to this transformation of CRISPR-Cas systems.


April 21, 2020  |  

Wild relatives of maize

Crop domestication changed the course of human evolution, and domestication of maize (Zea mays L. subspecies mays), today the world’s most important crop, enabled civilizations to flourish and has played a major role in shaping the world we know today. Archaeological and ethnobotanical research help us understand the development of the cultures and the movements of the peoples who carried maize to new areas where it continued to adapt. Ancient remains of maize cobs and kernels have been found in the place of domestication, the Balsas River Valley (~9,000 years before present era), and the cultivation center, the Tehuacan Valley (~5,000 years before present era), and have been used to study the process of domestication. Paleogenomic data showed that some of the genes controlling the stem and inflorescence architecture were comparable to modern maize, while other genes controlling ear shattering and starch biosynthesis retain high levels of variability, similar to those found in the wild relative teosinte. These results indicate that the domestication process was both gradual and complex, where different genetic loci were selected at different points in time, and that the transformation of teosinte to maize was completed in the last 5,000 years. Mesoamerican native cultures domesticated teosinte and developed maize from a 6 cm long, popping-kernel ear to what we now recognize as modern maize with its wide variety in ear size, kernel texture, color, size, and adequacy for diverse uses and also invented nixtamalization, a process key to maximizing its nutrition. Used directly for human and animal consumption, processed food products, bioenergy, and many cultural applications, it is now grown on six of the world’s seven continents. The study of its evolution and domestication from the wild grass teosinte helps us understand the nature of genetic diversity of maize and its wild relatives and gene expression. Genetic barriers to direct use of teosinte or Tripsacum in maize breeding have challenged our ability to identify valuable genes and traits, let alone incorporate them into elite, modern varieties. Genomic information and newer genetic technologies will facilitate the use of wild relatives in crop improvement; hence it is more important than ever to ensure their conservation and availability, fundamental to future food security. In situ conservation efforts dedicated to preserving remnant populations of wild relatives in Mexico are key to safeguarding the genetic diversity of maize and its genepool, as well as enabling these species to continue to adapt to dynamic climate and environmental changes. Genebank ex situ efforts are crucial to securely maintain collected wild relative resources and to provide them for gene discovery and other research efforts.


April 21, 2020  |  

Genome analysis and genetic transformation of a water surface-floating microalga Chlorococcum sp. FFG039.

Microalgal harvesting and dewatering are the main bottlenecks that need to be overcome to tap the potential of microalgae for production of valuable compounds. Water surface-floating microalgae form robust biofilms, float on the water surface along with gas bubbles entrapped under the biofilms, and have great potential to overcome these bottlenecks. However, little is known about the molecular mechanisms involved in the water surface-floating phenotype. In the present study, we analysed the genome sequence of a water surface-floating microalga Chlorococcum sp. FFG039, with a next generation sequencing technique to elucidate the underlying mechanisms. Comparative genomics study with Chlorococcum sp. FFG039 and other non-floating green microalgae revealed some of the unique gene families belonging to this floating microalga, which may be involved in biofilm formation. Furthermore, genetic transformation of this microalga was achieved with an electroporation method. The genome information and transformation techniques presented in this study will be useful to obtain molecular insights into the water surface-floating phenotype of Chlorococcum sp. FFG039.


April 21, 2020  |  

A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.


April 21, 2020  |  

Metatranscriptomic evidence for classical and RuBisCO-mediated CO2 reduction to methane facilitated by direct interspecies electron transfer in a methanogenic system.

In a staged anaerobic fluidized-bed ceramic membrane bioreactor, metagenomic and metatranscriptomic analyses were performed to decipher the microbial interactions on the granular activated carbon. Metagenome bins, representing the predominating microbes in the bioreactor: syntrophic propionate-oxidizing bacteria (SPOB), acetoclastic Methanothrix concilii, and exoelectrogenic Geobacter lovleyi, were successfully recovered for the reconstruction and analysis of metabolic pathways involved in the transformation of fatty acids to methane. In particular, SPOB degraded propionate into acetate, which was further converted into methane and CO2 by M. concilii via the acetoclastic methanogenesis. Concurrently, G. lovleyi oxidized acetate into CO2, releasing electrons into the extracellular environment. By accepting these electrons through direct interspecies electron transfer (DIET), M. concilii was capable of performing CO2 reduction for further methane formation. Most notably, an alternative RuBisCO-mediated CO2 reduction (the reductive hexulose-phosphate (RHP) pathway) is transcriptionally-active in M. concilii. This RHP pathway enables M. concilii dominance and energy gain by carbon fixation and methanogenesis, respectively via a methyl-H4MPT intermediate, constituting the third methanogenesis route. The complete acetate reduction (2 mole methane formation/1 mole acetate consumption), coupling of acetoclastic methanogenesis and two CO2 reduction pathways, are thermodynamically favorable even under very low substrate condition (down to to 10-5?M level). Such tight interactions via both mediated and direct interspecies electron transfer (MIET and DIET), induced by the conductive GAC promote the overall efficiency of bioenergy processes.


April 21, 2020  |  

Genome-wide systematic identification of methyltransferase recognition and modification patterns.

Genome-wide analysis of DNA methylation patterns using single molecule real-time DNA sequencing has boosted the number of publicly available methylomes. However, there is a lack of tools coupling methylation patterns and the corresponding methyltransferase genes. Here we demonstrate a high-throughput method for coupling methyltransferases with their respective motifs, using automated cloning and analysing the methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites. To validate the method, we analyse the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes. Using our method, we characterize the 23 methyltransferases, assign motifs to the respective enzymes and verify activity for 11 of the 12 motifs.


April 21, 2020  |  

Extensive intraspecific gene order and gene structural variations in upland cotton cultivars.

Multiple cotton genomes (diploid and tetraploid) have been assembled. However, genomic variations between cultivars of allotetraploid upland cotton (Gossypium hirsutum L.), the most widely planted cotton species in the world, remain unexplored. Here, we use single-molecule long read and Hi-C sequencing technologies to assemble genomes of the two upland cotton cultivars TM-1 and zhongmiansuo24 (ZM24). Comparisons among TM-1 and ZM24 assemblies and the genomes of the diploid ancestors reveal a large amount of genetic variations. Among them, the top three longest structural variations are located on chromosome A08 of the tetraploid upland cotton, which account for ~30% total length of this chromosome. Haplotype analyses of the mapping population derived from these two cultivars and the germplasm panel show suppressed recombination rates in this region. This study provides additional genomic resources for the community, and the identified genetic variations, especially the reduced meiotic recombination on chromosome A08, will help future breeding.


April 21, 2020  |  

Urinary tract colonization is enhanced by a plasmid that regulates uropathogenic Acinetobacter baumannii chromosomal genes.

Multidrug resistant (MDR) Acinetobacter baumannii poses a growing threat to global health. Research on Acinetobacter pathogenesis has primarily focused on pneumonia and bloodstream infections, even though one in five A. baumannii strains are isolated from urinary sites. In this study, we highlight the role of A. baumannii as a uropathogen. We develop the first A. baumannii catheter-associated urinary tract infection (CAUTI) murine model using UPAB1, a recent MDR urinary isolate. UPAB1 carries the plasmid pAB5, a member of the family of large conjugative plasmids that represses the type VI secretion system (T6SS) in multiple Acinetobacter strains. pAB5 confers niche specificity, as its carriage improves UPAB1 survival in a CAUTI model and decreases virulence in a pneumonia model. Comparative proteomic and transcriptomic analyses show that pAB5 regulates the expression of multiple chromosomally-encoded virulence factors besides T6SS. Our results demonstrate that plasmids can impact bacterial infections by controlling the expression of chromosomal genes.


April 21, 2020  |  

Interspecies conservation of organisation and function between nonhomologous regional centromeres.

Despite the conserved essential function of centromeres, centromeric DNA itself is not conserved. The histone-H3 variant, CENP-A, is the epigenetic mark that specifies centromere identity. Paradoxically, CENP-A normally assembles on particular sequences at specific genomic locations. To gain insight into the specification of complex centromeres, here we take an evolutionary approach, fully assembling genomes and centromeres of related fission yeasts. Centromere domain organization, but not sequence, is conserved between Schizosaccharomyces pombe, S. octosporus and S. cryophilus with a central CENP-ACnp1 domain flanked by heterochromatic outer-repeat regions. Conserved syntenic clusters of tRNA genes and 5S rRNA genes occur across the centromeres of S. octosporus and S. cryophilus, suggesting conserved function. Interestingly, nonhomologous centromere central-core sequences from S. octosporus and S. cryophilus are recognized in S. pombe, resulting in cross-species establishment of CENP-ACnp1 chromatin and functional kinetochores. Therefore, despite the lack of sequence conservation, Schizosaccharomyces centromere DNA possesses intrinsic conserved properties that promote assembly of CENP-A chromatin.


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