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July 7, 2019

Suppressed recombination and unique candidate genes in the divergent haplotype encoding Fhb1, a major Fusarium head blight resistance locus in wheat.

Fine mapping and sequencing revealed 28 genes in the non-recombining haplotype containing Fhb1 . Of these, only a GDSL lipase gene shows a pathogen-dependent expression pattern. Fhb1 is a prominent Fusarium head blight resistance locus of wheat, which has been successfully introgressed in adapted breeding material, where it confers a significant increase in overall resistance to the causal pathogen Fusarium graminearum and the fungal virulence factor and mycotoxin deoxynivalenol. The Fhb1 region has been resolved for the susceptible wheat reference genotype Chinese Spring, yet the causal gene itself has not been identified in resistant cultivars. Here, we report the establishment of a 1 Mb contig embracing Fhb1 in the donor line CM-82036. Sequencing revealed that the region of Fhb1 deviates from the Chinese Spring reference in DNA size and gene content, which explains the repressed recombination at the locus in the performed fine mapping. Differences in genes expression between near-isogenic lines segregating for Fhb1 challenged with F. graminearum or treated with mock were investigated in a time-course experiment by RNA sequencing. Several candidate genes were identified, including a pathogen-responsive GDSL lipase absent in susceptible lines. The sequence of the Fhb1 region, the resulting list of candidate genes, and near-diagnostic KASP markers for Fhb1 constitute a valuable resource for breeding and further studies aiming to identify the gene(s) responsible for F. graminearum and deoxynivalenol resistance.

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July 7, 2019

Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasite Plasmodium knowlesi.

The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

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July 7, 2019

Chromosome and plasmids of the tick-borne relapsing fever agent Borrelia hermsii.

The zoonotic pathogen Borrelia hermsii bears its multiple paralogous genes for variable antigens on several linear plasmids. Application of combined long-read and short-read next-generation sequencing provided complete sequences for antigen-encoding plasmids as well as other linear and circular plasmids and the linear chromosome of the genome. Copyright © 2016 Barbour.

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July 7, 2019

Complete genome sequence of a multidrug-resistant Acinetobacter baumannii isolate obtained from a Mexican hospital (sequence type 422).

Acinetobacter baumannii has emerged as a dangerous nosocomial pathogen, particularly for severely ill patients in intensive care units and patients with hematologic malignancies. Here, we present the complete genome sequence of a multidrug-resistant A. baumannii isolate, recovered from a Mexican hospital and classified as sequence type 422 according to the multilocus sequence typing Pasteur scheme. Copyright © 2016 Castro-Jaimes et al.

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July 7, 2019

Plasmid dynamics in KPC-positive Klebsiella pneumoniae during long-term patient colonization.

Carbapenem-resistant Klebsiella pneumoniae strains are formidable hospital pathogens that pose a serious threat to patients around the globe due to a rising incidence in health care facilities, high mortality rates associated with infection, and potential to spread antibiotic resistance to other bacterial species, such as Escherichia coli Over 6 months in 2011, 17 patients at the National Institutes of Health (NIH) Clinical Center became colonized with a highly virulent, transmissible carbapenem-resistant strain of K. pneumoniae Our real-time genomic sequencing tracked patient-to-patient routes of transmission and informed epidemiologists’ actions to monitor and control this outbreak. Two of these patients remained colonized with carbapenemase-producing organisms for at least 2 to 4 years, providing the opportunity to undertake a focused genomic study of long-term colonization with antibiotic-resistant bacteria. Whole-genome sequencing studies shed light on the underlying complex microbial colonization, including mixed or evolving bacterial populations and gain or loss of plasmids. Isolates from NIH patient 15 showed complex plasmid rearrangements, leaving the chromosome and the blaKPC-carrying plasmid intact but rearranging the two other plasmids of this outbreak strain. NIH patient 16 has shown continuous colonization with blaKPC-positive organisms across multiple time points spanning 2011 to 2015. Genomic studies defined a complex pattern of succession and plasmid transmission across two different K. pneumoniae sequence types and an E. coli isolate. These findings demonstrate the utility of genomic methods for understanding strain succession, genome plasticity, and long-term carriage of antibiotic-resistant organisms.In 2011, the NIH Clinical Center had a nosocomial outbreak involving 19 patients who became colonized or infected with blaKPC-positive Klebsiella pneumoniae Patients who have intestinal colonization with blaKPC-positive K. pneumoniae are at risk for developing infections that are difficult or nearly impossible to treat with existing antibiotic options. Two of those patients remained colonized with blaKPC-positive Klebsiella pneumoniae for over a year, leading to the initiation of a detailed genomic analysis exploring mixed colonization, plasmid recombination, and plasmid diversification. Whole-genome sequence analysis identified a variety of changes, both subtle and large, in the blaKPC-positive organisms. Long-term colonization of patients with blaKPC-positive Klebsiella pneumoniae creates new opportunities for horizontal gene transfer of plasmids encoding antibiotic resistance genes and poses complications for the delivery of health care. Copyright © 2016 Conlan et al.

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July 7, 2019

Genomics-informed isolation and characterization of a symbiotic Nanoarchaeota system from a terrestrial geothermal environment.

Biological features can be inferred, based on genomic data, for many microbial lineages that remain uncultured. However, cultivation is important for characterizing an organism’s physiology and testing its genome-encoded potential. Here we use single-cell genomics to infer cultivation conditions for the isolation of an ectosymbiotic Nanoarchaeota (‘Nanopusillus acidilobi’) and its host (Acidilobus, a crenarchaeote) from a terrestrial geothermal environment. The cells of ‘Nanopusillus’ are among the smallest known cellular organisms (100-300?nm). They appear to have a complete genetic information processing machinery, but lack almost all primary biosynthetic functions as well as respiration and ATP synthesis. Genomic and proteomic comparison with its distant relative, the marine Nanoarchaeum equitans illustrate an ancient, common evolutionary history of adaptation of the Nanoarchaeota to ectosymbiosis, so far unique among the Archaea.

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July 7, 2019

Lactobacillus rhamnosus GG outcompetes Enterococcus faecium by mucus-binding pili – Evidence for a novel probiotic mechanism on a distance.

Vancomycin-resistant enterococci (VRE) have become a major nosocomial threat. Enterococcus faecium is of special concern, as it can easily acquire new antibiotic resistances and is an excellent colonizer of the human intestinal tract. Several clinical studies have explored the potential use of beneficial bacteria to weed out opportunistic pathogens. Specifically, the widely studied Lactobacillus rhamnosus strain GG has been applied successfully in the context of VRE infections. Here, we provide new insight into the molecular mechanism underlying the effects of this model probiotic on VRE decolonization. Both clinical VRE isolates and L. rhamnosus GG express pili on their cell walls, which are the key modulators of their highly efficient colonization of the intestinal mucosa. We found that one of the VRE pilus clusters shares considerable sequence similarity with the SpaCBA-SrtC1 pilus cluster of L. rhamnosus GG. Remarkable immunological and functional similarities were discovered between the mucus-binding pili of L. rhamnosus GG and those of the clinical E. faecium strain E1165, which was characterized at the genome level. Moreover, E. faecium strain E1165 bound efficiently to mucus, which may be prevented by the presence of the mucus-binding SpaC protein or antibodies against L. rhamnosus GG or SpaC. These results present experimental support for a novel probiotic mechanism, in which the mucus-binding pili of L. rhamnosus GG prevent the binding of a potential pathogen to the host. Hence, we provide a molecular basis for the further exploitation of L. rhamnosus GG and its pilins for prophylaxis and treatment of VRE infections. IMPORTANCE Concern about vancomycin-resistant Enterococcus faecium causing nosocomial infections is rising globally. The arsenal of antibiotic strategies to treat these infections is nearly exhausted, and hence, new treatment strategies are urgently needed. Here, we provide molecular evidence to underpin reports of the successful clinical application of Lactobacillus rhamnosus GG in VRE decolonization strategies. Our results provide support for a new molecular mechanism, in which probiotics can perform competitive exclusion and possibly immune interaction. Moreover, we spur further exploration of the potential of intact L. rhamnosus GG and purified SpaC pilin as prophylactic and curative agents of the VRE carrier state.

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July 7, 2019

Microsatellite length scoring by Single Molecule Real Time Sequencing – Effects of sequence structure and PCR regime.

Microsatellites are DNA sequences consisting of repeated, short (1-6 bp) sequence motifs that are highly mutable by enzymatic slippage during replication. Due to their high intrinsic variability, microsatellites have important applications in population genetics, forensics, genome mapping, as well as cancer diagnostics and prognosis. The current analytical standard for microsatellites is based on length scoring by high precision electrophoresis, but due to increasing efficiency next-generation sequencing techniques may provide a viable alternative. Here, we evaluated single molecule real time (SMRT) sequencing, implemented in the PacBio series of sequencing apparatuses, as a means of microsatellite length scoring. To this end we carried out multiplexed SMRT sequencing of plasmid-carried artificial microsatellites of varying structure under different pre-sequencing PCR regimes. For each repeat structure, reads corresponding to the target length dominated. We found that pre-sequencing amplification had large effects on scoring accuracy and error distribution relative to controls, but that the effects of the number of amplification cycles were generally weak. In line with expectations enzymatic slippage decreased proportionally with microsatellite repeat unit length and increased with repetition number. Finally, we determined directional mutation trends, showing that PCR and SMRT sequencing introduced consistent but opposing error patterns in contraction and expansion of the microsatellites on the repeat motif and single nucleotide level.

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July 7, 2019

Vibrio natriegens as a fast-growing host for molecular biology.

A rapidly growing bacterial host would be desirable for a range of routine applications in molecular biology and biotechnology. The bacterium Vibrio natriegens has the fastest growth rate of any known organism, with a reported doubling time of <10 min. We report the development of genetic tools and methods to engineer V. natriegens and demonstrate the advantages of using these engineered strains in common biotech processes.

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July 7, 2019

New Delhi metallo-ß-lactamase-1-producing Klebsiella pneumoniae, Florida, USA(1).

New Delhi metallo-ß-lactamase (NDM)–producing Enterobacteriaceae have swiftly spread worldwide since an initial report in 2008 from a patient who had been transferred from India back home to Sweden (1). Epidemiologically, the global diffusion of NDM-1 producers has been associated with the Indian subcontinent and the Balkan region, which are considered the primary and secondary reservoirs of these pathogens, respectively (1). However, recent reports suggest that countries in the Middle East may constitute another potential reservoir for NDM-1 producers (1). More than 100 NDM-producing isolates have been reported in the United States, most of which were associated with recent travel from the Indian subcontinent (2,3). We report an NDM-1–producing Klebsiella pneumoniae strain that was recovered from a patient who had been transferred from Iran to a hospital in Florida, United States.

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July 7, 2019

Isolation and genomic characterization of ‘Desulfuromonas soudanensis WTL’, a metal- and electrode-respiring bacterium from anoxic deep subsurface brine.

Reaching a depth of 713 m below the surface, the Soudan Underground Iron Mine (Soudan, MN, USA) transects a massive Archaean (2.7 Ga) banded iron formation, providing a remarkably accessible window into the terrestrial deep biosphere. Despite organic carbon limitation, metal-reducing microbial communities are present in potentially ancient anoxic brines continuously emanating from exploratory boreholes on Level 27. Using graphite electrodes deposited in situ as bait, we electrochemically enriched and isolated a novel halophilic iron-reducing Deltaproteobacterium, ‘Desulfuromonas soudanensis’ strain WTL, from an acetate-fed three-electrode bioreactor poised at +0.24 V (vs. standard hydrogen electrode). Cyclic voltammetry revealed that ‘D. soudanensis’ releases electrons at redox potentials approximately 100 mV more positive than the model freshwater surface isolate Geobacter sulfurreducens, suggesting that its extracellular respiration is tuned for higher potential electron acceptors. ‘D. soudanensis’ contains a 3,958,620-bp circular genome, assembled to completion using single-molecule real-time (SMRT) sequencing reads, which encodes a complete TCA cycle, 38 putative multiheme c-type cytochromes, one of which contains 69 heme-binding motifs, and a LuxI/LuxR quorum sensing cassette that produces an unidentified N-acyl homoserine lactone. Another cytochrome is predicted to lie within a putative prophage, suggesting that horizontal gene transfer plays a role in respiratory flexibility among metal reducers. Isolation of ‘D. soudanensis’ underscores the utility of electrode-based approaches for enriching rare metal reducers from a wide range of habitats.

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July 7, 2019

High-quality genome assembly and annotation for Plasmodium coatneyi, generated using single-molecule real-time PacBio technology.

Plasmodium coatneyi is a protozoan parasite species that causes simian malaria and is an excellent model for studying disease caused by the human malaria parasite, P. falciparum Here we report the complete (nontelomeric) genome sequence of P. coatneyi Hackeri generated by the application of only Pacific Biosciences RS II (PacBio RS II) single-molecule real-time (SMRT) high-resolution sequence technology and assembly using the Hierarchical Genome Assembly Process (HGAP). This is the first Plasmodium genome sequence reported to use only PacBio technology. This approach has proven to be superior to short-read only approaches for this species. Copyright © 2016 Chien et al.

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July 7, 2019

Building two indica rice reference genomes with PacBio long-read and Illumina paired-end sequencing data.

Over the past 30 years, we have performed many fundamental studies on two Oryza sativa subsp. indica varieties, Zhenshan 97 (ZS97) and Minghui 63 (MH63). To improve the resolution of many of these investigations, we generated two reference-quality reference genome assemblies using the most advanced sequencing technologies. Using PacBio SMRT technology, we produced over 108 (ZS97) and 174 (MH63) Gb of raw sequence data from 166 (ZS97) and 209 (MH63) pools of BAC clones, and generated ~97 (ZS97) and ~74 (MH63) Gb of paired-end whole-genome shotgun (WGS) sequence data with Illumina sequencing technology. With these data, we successfully assembled two platinum standard reference genomes that have been publicly released. Here we provide the full sets of raw data used to generate these two reference genome assemblies. These data sets can be used to test new programs for better genome assembly and annotation, aid in the discovery of new insights into genome structure, function, and evolution, and help to provide essential support to biological research in general.

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