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September 22, 2019

The genomes of Crithidia bombi and C. expoeki, common parasites of bumblebees.

Trypanosomatids (Trypanosomatidae, Kinetoplastida) are flagellated protozoa containing many parasites of medical or agricultural importance. Among those, Crithidia bombi and C. expoeki, are common parasites in bumble bees around the world, and phylogenetically close to Leishmania and Leptomonas. They have a simple and direct life cycle with one host, and partially castrate the founding queens greatly reducing their fitness. Here, we report the nuclear genome sequences of one clone of each species, extracted from a field-collected infection. Using a combination of Roche 454 FLX Titanium, Pacific Biosciences PacBio RS, and Illumina GA2 instruments for C. bombi, and PacBio for C. expoeki, we could produce high-quality and well resolved sequences. We find that these genomes are around 32 and 34 MB, with 7,808 and 7,851 annotated genes for C. bombi and C. expoeki, respectively-which is somewhat less than reported from other trypanosomatids, with few introns, and organized in polycistronic units. A large fraction of genes received plausible functional support in comparison primarily with Leishmania and Trypanosoma. Comparing the annotated genes of the two species with those of six other trypanosomatids (C. fasciculata, L. pyrrhocoris, L. seymouri, B. ayalai, L. major, and T. brucei) shows similar gene repertoires and many orthologs. Similar to other trypanosomatids, we also find signs of concerted evolution in genes putatively involved in the interaction with the host, a high degree of synteny between C. bombi and C. expoeki, and considerable overlap with several other species in the set. A total of 86 orthologous gene groups show signatures of positive selection in the branch leading to the two Crithidia under study, mostly of unknown function. As an example, we examined the initiating glycosylation pathway of surface components in C. bombi, finding it deviates from most other eukaryotes and also from other kinetoplastids, which may indicate rapid evolution in the extracellular matrix that is involved in interactions with the host. Bumble bees are important pollinators and Crithidia-infections are suspected to cause substantial selection pressure on their host populations. These newly sequenced genomes provide tools that should help better understand host-parasite interactions in these pollinator pathogens.

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September 22, 2019

Functional metagenomics reveals a novel carbapenem-hydrolyzing mobile beta-lactamase from Indian river sediments contaminated with antibiotic production waste.

Evolution has provided environmental bacteria with a plethora of genes that give resistance to antibiotic compounds. Under anthropogenic selection pressures, some of these genes are believed to be recruited over time into pathogens by horizontal gene transfer. River sediment polluted with fluoroquinolones and other drugs discharged from bulk drug production in India constitute an environment with unprecedented, long-term antibiotic selection pressures. It is therefore plausible that previously unknown resistance genes have evolved and/or are promoted here. In order to search for novel resistance genes, we therefore analyzed such river sediments by a functional metagenomics approach. DNA fragments providing resistance to different antibiotics in E. coli were sequenced using Sanger and PacBio RSII platforms. We recaptured the majority of known antibiotic resistance genes previously identified by open shot-gun metagenomics sequencing of the same samples. In addition, seven novel resistance gene candidates (six beta-lactamases and one amikacin resistance gene) were identified. Two class A beta-lactamases, blaRSA1 and blaRSA2, were phylogenetically close to clinically important ESBLs like blaGES, blaBEL and blaL2, and were further characterized for their substrate spectra. The blaRSA1 protein, encoded as an integron gene cassette, efficiently hydrolysed penicillins, first generation cephalosporins and cefotaxime, while blaRSA2 was an inducible class A beta-lactamase, capable of hydrolyzing carbapenems albeit with limited efficiency, similar to the L2 beta-lactamase from Stenotrophomonas maltophilia. All detected novel genes were associated with plasmid mobilization proteins, integrons, and/or other resistance genes, suggesting a potential for mobility. This study provides insight into a resistome shaped by an exceptionally strong and long-term antibiotic selection pressure. An improved knowledge of mobilized resistance factors in the external environment may make us better prepared for the resistance challenges that we may face in clinics in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

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September 22, 2019

Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza.

The genus Oryza is a model system for the study of molecular evolution over time scales ranging from a few thousand to 15 million years. Using 13 reference genomes spanning the Oryza species tree, we show that despite few large-scale chromosomal rearrangements rapid species diversification is mirrored by lineage-specific emergence and turnover of many novel elements, including transposons, and potential new coding and noncoding genes. Our study resolves controversial areas of the Oryza phylogeny, showing a complex history of introgression among different chromosomes in the young ‘AA’ subclade containing the two domesticated species. This study highlights the prevalence of functionally coupled disease resistance genes and identifies many new haplotypes of potential use for future crop protection. Finally, this study marks a milestone in modern rice research with the release of a complete long-read assembly of IR 8 ‘Miracle Rice’, which relieved famine and drove the Green Revolution in Asia 50 years ago.

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September 22, 2019

The putative functions of lysogeny in mediating the survivorship of Escherichia coli in seawater and marine sediment.

Escherichia coli colonizes various body parts of animal hosts as a commensal and a pathogen. It can also persist in the external environment in the absence of fecal pollution. It remains unclear how this species has evolved to adapt to such contrasting habitats. Lysogeny plays pivotal roles in the diversification of the phenotypic and ecologic characters of E. coli as a symbiont. We hypothesized that lysogeny could also confer fitness to survival in the external environment. To test this hypothesis, we used the induced phages of an E. coli strain originating from marine sediment to infect a fecal E. coli strain to obtain an isogenic lysogen of the latter. The three strains were tested for survivorship in microcosms of seawater, marine sediment and sediment interstitial water as well as swimming motility, glycogen accumulation, biofilm formation, substrate utilization and stress resistance. The results indicate that lysogenic infection led to tractable changes in many of the ecophysiological attributes tested. Particularly, the lysogen had better survivorship in the microcosms and had a substrate utilization profile resembling the sediment strain more than the wild type fecal strain. Our findings provide new insights into the understanding of how E. coli survives in the natural environment.© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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September 22, 2019

Bat biology, genomes, and the Bat1K project: To generate chromosome-level genomes for all living bat species.

Bats are unique among mammals, possessing some of the rarest mammalian adaptations, including true self-powered flight, laryngeal echolocation, exceptional longevity, unique immunity, contracted genomes, and vocal learning. They provide key ecosystem services, pollinating tropical plants, dispersing seeds, and controlling insect pest populations, thus driving healthy ecosystems. They account for more than 20% of all living mammalian diversity, and their crown-group evolutionary history dates back to the Eocene. Despite their great numbers and diversity, many species are threatened and endangered. Here we announce Bat1K, an initiative to sequence the genomes of all living bat species (n~1,300) to chromosome-level assembly. The Bat1K genome consortium unites bat biologists (>148 members as of writing), computational scientists, conservation organizations, genome technologists, and any interested individuals committed to a better understanding of the genetic and evolutionary mechanisms that underlie the unique adaptations of bats. Our aim is to catalog the unique genetic diversity present in all living bats to better understand the molecular basis of their unique adaptations; uncover their evolutionary history; link genotype with phenotype; and ultimately better understand, promote, and conserve bats. Here we review the unique adaptations of bats and highlight how chromosome-level genome assemblies can uncover the molecular basis of these traits. We present a novel sequencing and assembly strategy and review the striking societal and scientific benefits that will result from the Bat1K initiative.

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September 22, 2019

Blood CXCR3+CD4 T cells are enriched in inducible replication competent HIV in aviremic antiretroviral therapy-treated individuals.

We recently demonstrated that lymph nodes (LNs) PD-1+/T follicular helper (Tfh) cells from antiretroviral therapy (ART)-treated HIV-infected individuals were enriched in cells containing replication competent virus. However, the distribution of cells containing inducible replication competent virus has been only partially elucidated in blood memory CD4 T-cell populations including the Tfh cell counterpart circulating in blood (cTfh). In this context, we have investigated the distribution of (1) total HIV-infected cells and (2) cells containing replication competent and infectious virus within various blood and LN memory CD4 T-cell populations of conventional antiretroviral therapy (cART)-treated HIV-infected individuals. In the present study, we show that blood CXCR3-expressing memory CD4 T cells are enriched in cells containing inducible replication competent virus and contributed the most to the total pool of cells containing replication competent and infectious virus in blood. Interestingly, subsequent proviral sequence analysis did not indicate virus compartmentalization between blood and LN CD4 T-cell populations, suggesting dynamic interchanges between the two compartments. We then investigated whether the composition of blood HIV reservoir may reflect the polarization of LN CD4 T cells at the time of reservoir seeding and showed that LN PD-1+CD4 T cells of viremic untreated HIV-infected individuals expressed significantly higher levels of CXCR3 as compared to CCR4 and/or CCR6, suggesting that blood CXCR3-expressing CD4 T cells may originate from LN PD-1+CD4 T cells. Taken together, these results indicate that blood CXCR3-expressing CD4 T cells represent the major blood compartment containing inducible replication competent virus in treated aviremic HIV-infected individuals.

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September 22, 2019

Identification of the streptothricin and tunicamycin biosynthetic gene clusters by genome mining in Streptomyces sp. strain fd1-xmd.

The genus Streptomyces have been highly regarded for their important source of natural products. Combined with the technology of genome sequencing and mining, we could identify the active ingredients from fermentation broth quickly. Here, we report on Streptomyces sp. strain fd1-xmd, which was isolated from a soil sample collected in Shanghai. Interestingly, the fermentation broth derived from this strain demonstrated broad-spectrum antimicrobial activity against gram-positive bacteria, gram-negative bacteria, and eukaryotes. To identify the antimicrobial substances and their biosynthetic gene clusters, we sequenced the fd1-xmd strain and obtained a genome 7,929,999 bp in length. The average GC content of the chromosome was 72.5 mol%. Knockout experiments demonstrated that out of eight biosynthetic gene clusters we could identify, two are responsible for the biosynthesis of the antibiotics streptothricin (ST) and tunicamycin (TM). The ST biosynthetic gene cluster from fd1-xmd was verified via successful heterologous expression in Streptomyces coelicolor M1146. ST production had a yield of up to 0.5 g/L after the optimization of culture conditions. This study describes a novel producer of ST and TM and outlines the complete process undertaken for Streptomyces sp. strain fd1-xmd genome mining.

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September 22, 2019

A reference genome and methylome for the Plasmodium knowlesi A1-H.1 line.

Plasmodium knowlesi, a common parasite of macaques, is recognised as a significant cause of human malaria in Malaysia. The P. knowlesi A1H1 line has been adapted to continuous culture in human erythrocytes, successfully providing an in vitro model to study the parasite. We have assembled a reference genome for the PkA1-H.1 line using PacBio long read combined with Illumina short read sequence data. Compared with the H-strain reference, the new reference has improved genome coverage and a novel description of methylation sites. The PkA1-H.1 reference will enhance the capabilities of the in vitro model to improve the understanding of P. knowlesi infection in humans. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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September 22, 2019

Primordial origin and diversification of plasmids in Lyme disease agent bacteria.

With approximately one-third of their genomes consisting of linear and circular plasmids, the Lyme disease agent cluster of species has the most complex genomes among known bacteria. We report here a comparative analysis of plasmids in eleven Borreliella (also known as Borrelia burgdorferi sensu lato) species.We sequenced the complete genomes of two B. afzelii, two B. garinii, and individual B. spielmanii, B. bissettiae, B. valaisiana and B. finlandensis isolates. These individual isolates carry between seven and sixteen plasmids, and together harbor 99 plasmids. We report here a comparative analysis of these plasmids, along with 70 additional Borreliella plasmids available in the public sequence databases. We identify only one new putative plasmid compatibility type (the 30th) among these 169 plasmid sequences, suggesting that all or nearly all such types have now been discovered. We find that the linear plasmids in the non-B. burgdorferi species have undergone the same kinds of apparently random, chaotic rearrangements mediated by non-homologous recombination that we previously discovered in B. burgdorferi. These rearrangements occurred independently in the different species lineages, and they, along with an expanded chromosomal phylogeny reported here, allow the identification of several whole plasmid transfer events among these species. Phylogenetic analyses of the plasmid partition genes show that a majority of the plasmid compatibility types arose early, most likely before separation of the Lyme agent Borreliella and relapsing fever Borrelia clades, and this, with occasional cross species plasmid transfers, has resulted in few if any species-specific or geographic region-specific Borreliella plasmid types.The primordial origin and persistent maintenance of the Borreliella plasmid types support their functional indispensability as well as evolutionary roles in facilitating genome diversity. The improved resolution of Borreliella plasmid phylogeny based on conserved partition-gene clusters will lead to better determination of gene orthology which is essential for prediction of biological function, and it will provide a basis for inferring detailed evolutionary mechanisms of Borreliella genomic variability including homologous gene and plasmid exchanges as well as non-homologous rearrangements.

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September 22, 2019

Unique genetic cassettes in a Thermoanaerobacterium contribute to simultaneous conversion of cellulose and monosugars into butanol.

The demand for cellulosic biofuels is on the rise because of the anticipation for sustainable energy and less greenhouse gas emissions in the future. However, production of cellulosic biofuels, especially cellulosic butanol, has been hampered by the lack of potent microbes that are capable of converting cellulosic biomass into biofuels. We report a wild-type Thermoanaerobacterium thermosaccharolyticum strain TG57, which is capable of using microcrystalline cellulose directly to produce butanol (1.93 g/liter) as the only final product (without any acetone or ethanol produced), comparable to that of engineered microbes thus far. Strain TG57 exhibits significant advances including unique genes responsible for a new butyrate synthesis pathway, no carbon catabolite repression, and the absence of genes responsible for acetone synthesis (which is observed as the main by-product in most Clostridium strains known today). Furthermore, the use of glucose analog 2-deoxyglucose posed a selection pressure to facilitate isolation of strain TG57 with deletion/silencing of carbon catabolite repressor genes-the ccr and xylR genes-and thus is able to simultaneously ferment glucose, xylose, and arabinose to produce butanol (7.33 g/liter) as the sole solvent. Combined analysis of genomic and transcriptomic data revealed unusual aspects of genome organization, numerous determinants for unique bioconversions, regulation of central metabolic pathways, and distinct transcriptomic profiles. This study provides a genome-level understanding of how cellulose is metabolized by T. thermosaccharolyticum and sheds light on the potential of competitive and sustainable biofuel production.

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September 22, 2019

The genome sequence of a new strain of Mycobacterium ulcerans ecovar Liflandii, emerging as a sturgeon pathogen

Mycobacterium ulcerans ecovar Liflandii (MuLiflandii) is emerging as a non-mycobacterial pathogen in amphibians. Here, we make the first report on the prevalence of a new strain of MuLiflandii infection in Chinese sturgeon. All the diseased fish showed the classic clinical symptoms of ascites and/or muscle ulceration. A new slow-growing and acid-fast bacillus ASM001 strain was obtained from the ascites of infected fish; this strain demonstrated pathogenicity when tested in hybrid sturgeon. The complete genome sequence of MuLiflandii ASM001 is a circular chromosome of 6,167,296?bp, with a G?+?C content of 65.57%, containing 4518 predicted coding DNA sequences and 999 pseudo-genes, 3 rRNA operons, and 47 transfer RNA sequences. In addition, we found 245 copies of IS2404, 34 microsatellites, and 36 CRISPR sequences in the whole MuLiflandii ASM001 genome. Among the predicted genes of MuLiflandii ASM001, we found orthologs of 203 virulence factors of clinical MuLiflandii 128FXT operating in host cell invasion, modulation of phagocyte function, and survival inside the macrophages. These virulence factor candidates provide a key basis for understanding their pathogenic mechanisms at the molecular level. A comparative analysis that used complete, existing genomes showed that MuLiflandii ASM001 has high synteny with MuLiflandii 128FXT. We anticipate the availability of the complete MuLiflandii ASM001 genome sequence will provide a valuable resource for comparative genomic studies of MuLiflandii isolates, as well as provide new insights into the host, ecological, and functional diversity of the genus Mycobacterium.

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September 22, 2019

New Delhi metallo-beta-lactamase-producing Enterobacteriaceae in South Korea between 2010 and 2015.

This study was carried out to investigate the epidemiological time-course of New Delhi metallo-beta-lactamase- (NDM-) mediated carbapenem resistance in Enterobacteriaceae in South Korea. A total of 146 non-duplicate NDM-producing Enterobacteriaceae recovered between 2010 and 2015 were voluntarily collected from 33 general hospitals and confirmed by PCR. The species were identified by sequences of the 16S rDNA. Antimicrobial susceptibility was determined either by the disk diffusion method or by broth microdilution, and the carbapenem MICs were determined by agar dilution. Then, multilocus sequence typing and PCR-based replicon typing was carried out. Co-carried genes for drug resistance were identified by PCR and sequencing. The entire genomes of eight random selected NDM producers were sequenced. A total of 69 Klebsiella pneumoniae of 12 sequence types (STs), 34 Escherichia coli of 15 STs, 28 Enterobacter spp. (including one Enterobacter aerogenes), nine Citrobacter freundii, four Raoultella spp., and two Klebsiella oxytoca isolates produced either NDM-1 (n = 126), NDM-5 (n = 18), or NDM-7 (n = 2). The isolates co-produced CTX-M-type ESBL (52.1%), AmpCs (27.4%), additional carbapenemases (7.1%), and/or 16S rRNA methyltransferases (4.8%), resulting in multidrug-resistance (47.9%) or extensively drug-resistance (52.1%). Among plasmids harboring blaNDM, IncX3 was predominant (77.4%), followed by the IncFII type (5.8%). Genome analysis revealed inter-species and inter-strain horizontal gene transfer of the plasmid. Both clonal dissemination and plasmid transfer contributed to the wide dissemination of NDM producers in South Korea.

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September 22, 2019

Genetic characterization of a VanG-type vancomycin-resistant Enterococcus faecium clinical isolate.

To characterize, phenotypically and genotypically, the first Enterococcus faecium clinical isolate harbouring a vanG operon.The antibiotic resistance profile of E. faecium 16-346 was determined and its whole genome sequenced using PacBio technology. Attempts to transfer vancomycin resistance by filter mating were performed and the inducibility of expression of the vanG operon was studied by reverse-transcription quantitative PCR (RT-qPCR) in the presence or absence of subinhibitory concentrations of vancomycin.E. faecium 16-346 was resistant to rifampicin (MIC >4?mg/L), erythromycin (MIC >4?mg/L), tetracycline (MIC >16?mg/L) and vancomycin (MIC 8?mg/L), but susceptible to teicoplanin (MIC 0.5?mg/L). The strain harboured the vanG operon in its chromosome, integrated in a 45.5?kb putative mobile genetic element, similar to that of Enterococcus faecalis BM4518. We were unable to transfer vancomycin resistance from E. faecium 16-346 to E. faecium BM4107 and E. faecalis JH2-2. Lastly, transcription of the vanG gene was inducible by vancomycin.This is, to the best of our knowledge, the first report of a VanG-type vancomycin-resistant strain of E. faecium. Despite the alarm pulled because of the therapeutic problems caused by VRE, our work shows that new resistant loci can still be found in E. faecium.

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September 22, 2019

Genetic basis of chromosomally-encoded mcr-1 gene.

Compared with plasmid-borne mcr-1, the occurrence of chromosomally-encoded mcr-1 is rare although it has been reported in several cases. This study aimed to investigate the genetic features of chromosomally-encoded mcr-1 among Escherichia coli strains as well as the potential genetic basis governing mobilisation of mcr-1 in bacterial chromosomes. The genome sequences of 16 E. coli strains containing a chromosomal mcr-1 gene were obtained and analysed. Phylogenetic and whole-genome sequencing (WGS) analysis demonstrated that mcr-1 was associated with four major types of genetic arrangements, namely ISApl1-mcr1-orf, Tn6330, complex Tn6330 and ?Tn6330 in chromosomes of genetically unrelated E. coli strains. The mcr-1-carrying mobile elements were shown to insert into the AT-rich region, which was also the case for ISApl1. Analysis of complete E. coli genome sequences showed that there were multiple copies of ISApl1 present in E. coli chromosomes that also carried mcr-1, whilst all mcr-1-negative chromosomes were absent of any copy of ISApl1, suggesting the strong association of ISApl1 and mcr-1. Insertion of ISApl1 into E. coli chromosomes may be a prerequisite for the insertion of mcr-1-carrying mobile elements. Insertion of mcr-1 into E. coli chromosomes would enable it to become intrinsically resistant, which is expected to become more prevalent. Policy on the prudent use of colistin both in veterinary and clinical settings should be imposed globally to further prevent dissemination of mcr-1 in E. coli and other bacterial pathogens. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

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September 22, 2019

A large-scale comparative metagenomic study reveals the functional interactions in six bloom-forming Microcystis-epibiont communities.

Cyanobacterial blooms are worldwide issues of societal concern and scientific interest. Lake Taihu and Lake Dianchi, two of the largest lakes in China, have been suffering from annual Microcystis-based blooms over the past two decades. These two eutrophic lakes differ in both nutrient load and environmental parameters, where Microcystis microbiota consisting of different Microcystis morphospecies and associated bacteria (epibionts) have dominated. We conducted a comprehensive metagenomic study that analyzed species diversity, community structure, functional components, metabolic pathways and networks to investigate functional interactions among the members of six Microcystis-epibiont communities in these two lakes. Our integrated metagenomic pipeline consisted of efficient assembly, binning, annotation, and quality assurance methods that ensured high-quality genome reconstruction. This study provides a total of 68 reconstructed genomes including six complete Microcystis genomes and 28 high quality bacterial genomes of epibionts belonging to 14 distinct taxa. This metagenomic dataset constitutes the largest reference genome catalog available for genome-centric studies of the Microcystis microbiome. Epibiont community composition appears to be dynamic rather than fixed, and the functional profiles of communities were related to the environment of origin. This study demonstrates mutualistic interactions between Microcystis and epibionts at genetic and metabolic levels. Metabolic pathway reconstruction provided evidence for functional complementation in nitrogen and sulfur cycles, fatty acid catabolism, vitamin synthesis, and aromatic compound degradation among community members. Thus, bacterial social interactions within Microcystis-epibiont communities not only shape species composition, but also stabilize the communities functional profiles. These interactions appear to play an important role in environmental adaptation of Microcystis colonies.

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