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April 21, 2020

De novo genome assembly of the endangered Acer yangbiense, a plant species with extremely small populations endemic to Yunnan Province, China.

Acer yangbiense is a newly described critically endangered endemic maple tree confined to Yangbi County in Yunnan Province in Southwest China. It was included in a programme for rescuing the most threatened species in China, focusing on “plant species with extremely small populations (PSESP)”.We generated 64, 94, and 110 Gb of raw DNA sequences and obtained a chromosome-level genome assembly of A. yangbiense through a combination of Pacific Biosciences Single-molecule Real-time, Illumina HiSeq X, and Hi-C mapping, respectively. The final genome assembly is ~666 Mb, with 13 chromosomes covering ~97% of the genome and scaffold N50 sizes of 45 Mb. Further, BUSCO analysis recovered 95.5% complete BUSCO genes. The total number of repetitive elements account for 68.0% of the A. yangbiense genome. Genome annotation generated 28,320 protein-coding genes, assisted by a combination of prediction and transcriptome sequencing. In addition, a nearly 1:1 orthology ratio of dot plots of longer syntenic blocks revealed a similar evolutionary history between A. yangbiense and grape, indicating that the genome has not undergone a whole-genome duplication event after the core eudicot common hexaploidization.Here, we report a high-quality de novo genome assembly of A. yangbiense, the first genome for the genus Acer and the family Aceraceae. This will provide fundamental conservation genomics resources, as well as representing a new high-quality reference genome for the economically important Acer lineage and the wider order of Sapindales. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

Survey of the Bradysia odoriphaga Transcriptome Using PacBio Single-Molecule Long-Read Sequencing.

The damage caused by Bradysia odoriphaga is the main factor threatening the production of vegetables in the Liliaceae family. However, few genetic studies of B. odoriphaga have been conducted because of a lack of genomic resources. Many long-read sequencing technologies have been developed in the last decade; therefore, in this study, the transcriptome including all development stages of B. odoriphaga was sequenced for the first time by Pacific single-molecule long-read sequencing. Here, 39,129 isoforms were generated, and 35,645 were found to have annotation results when checked against sequences available in different databases. Overall, 18,473 isoforms were distributed in 25 various Clusters of Orthologous Groups, and 11,880 isoforms were categorized into 60 functional groups that belonged to the three main Gene Ontology classifications. Moreover, 30,610 isoforms were assigned into 44 functional categories belonging to six main Kyoto Encyclopedia of Genes and Genomes functional categories. Coding DNA sequence (CDS) prediction showed that 36,419 out of 39,129 isoforms were predicted to have CDS, and 4319 simple sequence repeats were detected in total. Finally, 266 insecticide resistance and metabolism-related isoforms were identified as candidate genes for further investigation of insecticide resistance and metabolism in B. odoriphaga.

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April 21, 2020

hicap: In Silico Serotyping of the Haemophilus influenzae Capsule Locus.

Haemophilus influenzae exclusively colonizes the human nasopharynx and can cause a variety of respiratory infections as well as invasive diseases, including meningitis and sepsis. A key virulence determinant of H. influenzae is the polysaccharide capsule, of which six serotypes are known, each encoded by a distinct variation of the capsule biosynthesis locus (cap-a to cap-f). H. influenzae type b (Hib) was historically responsible for the majority of invasive H. influenzae disease, and its prevalence has been markedly reduced in countries that have implemented vaccination programs targeting this serotype. In the postvaccine era, nontypeable H. influenzae emerged as the most dominant group causing disease, but in recent years a resurgence of encapsulated H. influenzae strains has also been observed, most notably serotype a. Given the increasing incidence of encapsulated strains and the high frequency of Hib in countries without vaccination programs, there is growing interest in genomic epidemiology of H. influenzae Here we present hicap, a software tool for rapid in silico serotype prediction from H. influenzae genome sequences. hicap is written using Python3 and is freely available at https://github.com/scwatts/hicap under the GNU General Public License v3 (GPL3). To demonstrate the utility of hicap, we used it to investigate the cap locus diversity and distribution in 691 high-quality H. influenzae genomes from GenBank. These analyses identified cap loci in 95 genomes and confirmed the general association of each serotype with a unique clonal lineage, and they also identified occasional recombination between lineages that gave rise to hybrid cap loci (2% of encapsulated strains).Copyright © 2019 Watts and Holt.

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April 21, 2020

Klebsiella quasipneumoniae Provides a Window into Carbapenemase Gene Transfer, Plasmid Rearrangements, and Patient Interactions with the Hospital Environment.

Several emerging pathogens have arisen as a result of selection pressures exerted by modern health care. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intraspecies transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, blaKPC, between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10- and 5-year periods, respectively. There were 32 blaKPC-positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from 8 patients and 11 sink drains, with evidence for seven separate blaKPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subsp. quasipneumoniae (n?=?23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then possible transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique blaKPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases. Copyright © 2019 Mathers et al.

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April 21, 2020

Genomic and transcriptomic characterization of Pseudomonas aeruginosa small colony variants derived from a chronic infection model.

Phenotypic change is a hallmark of bacterial adaptation during chronic infection. In the case of chronic Pseudomonas aeruginosa lung infection in patients with cystic fibrosis, well-characterized phenotypic variants include mucoid and small colony variants (SCVs). It has previously been shown that SCVs can be reproducibly isolated from the murine lung following the establishment of chronic infection with mucoid P. aeruginosa strain NH57388A. Using a combination of single-molecule real-time (PacBio) and Illumina sequencing we identify a large genomic inversion in the SCV through recombination between homologous regions of two rRNA operons and an associated truncation of one of the 16S rRNA genes and suggest this may be the genetic switch for conversion to the SCV phenotype. This phenotypic conversion is associated with large-scale transcriptional changes distributed throughout the genome. This global rewiring of the cellular transcriptomic output results in changes to normally differentially regulated genes that modulate resistance to oxidative stress, central metabolism and virulence. These changes are of clinical relevance because the appearance of SCVs during chronic infection is associated with declining lung function.

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April 21, 2020

Dynamics of Resistance Plasmids in Extended-Spectrum-ß-Lactamase-Producing Enterobacteriaceae during Postinfection Colonization.

Extended-spectrum ß-lactamase-producing Enterobacteriaceae (EPE) are a major cause of bloodstream infections, and the colonization rate of EPE in the gut microbiota of individuals lacking prior hospitalization or comorbidities is increasing. In this study, we performed an in-depth investigation of the temporal dynamics of EPE and their plasmids during one year by collecting fecal samples from three patients initially seeking medical care for urinary tract infections. In two of the patients, the same strain that caused the urinary tract infection (UTI) was found at all consecutive samplings from the gut microbiota, and no other EPEs were detected, while in the third patient the UTI strain was only found in the initial UTI sample. Instead, this patient presented a complex situation where a mixed microbiota of different EPE strain types, including three different E. coli ST131 variants, as well as different bacterial species, was identified over the course of the study. Different plasmid dynamics were displayed in each of the patients, including the spread of plasmids between different strain types over time and the transposition of blaCTX-M-15 from the chromosome to a plasmid, followed by subsequent loss through homologous recombination. Small cryptic plasmids were found in all isolates from all patients, and they appear to move frequently between different strains in the microbiota. In conclusion, we could demonstrate an extensive variation of EPE strain types, plasmid composition, rearrangements, and horizontal gene transfer of genetic material illustrating the high dynamics nature and interactive environment of the gut microbiota during post-UTI carriage.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Chromosome-scale assemblies reveal the structural evolution of African cichlid genomes.

African cichlid fishes are well known for their rapid radiations and are a model system for studying evolutionary processes. Here we compare multiple, high-quality, chromosome-scale genome assemblies to elucidate the genetic mechanisms underlying cichlid diversification and study how genome structure evolves in rapidly radiating lineages.We re-anchored our recent assembly of the Nile tilapia (Oreochromis niloticus) genome using a new high-density genetic map. We also developed a new de novo genome assembly of the Lake Malawi cichlid, Metriaclima zebra, using high-coverage Pacific Biosciences sequencing, and anchored contigs to linkage groups (LGs) using 4 different genetic maps. These new anchored assemblies allow the first chromosome-scale comparisons of African cichlid genomes. Large intra-chromosomal structural differences (~2-28 megabase pairs) among species are common, while inter-chromosomal differences are rare (<10 megabase pairs total). Placement of the centromeres within the chromosome-scale assemblies identifies large structural differences that explain many of the karyotype differences among species. Structural differences are also associated with unique patterns of recombination on sex chromosomes. Structural differences on LG9, LG11, and LG20 are associated with reduced recombination, indicative of inversions between the rock- and sand-dwelling clades of Lake Malawi cichlids. M. zebra has a larger number of recent transposable element insertions compared with O. niloticus, suggesting that several transposable element families have a higher rate of insertion in the haplochromine cichlid lineage.This study identifies novel structural variation among East African cichlid genomes and provides a new set of genomic resources to support research on the mechanisms driving cichlid adaptation and speciation. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

Detection of VIM-1-Producing Enterobacter cloacae and Salmonella enterica Serovars Infantis and Goldcoast at a Breeding Pig Farm in Germany in 2017 and Their Molecular Relationship to Former VIM-1-Producing S. Infantis Isolates in German Livestock Production.

In 2011, VIM-1-producing Salmonella enterica serovar Infantis and Escherichia coli were isolated for the first time in four German livestock farms. In 2015/2016, highly related isolates were identified in German pig production. This raised the issue of potential reservoirs for these isolates, the relation of their mobile genetic elements, and potential links between the different affected farms/facilities. In a piglet-producing farm suspicious for being linked to some blaVIM-1 findings in Germany, fecal and environmental samples were examined for the presence of carbapenemase-producing Enterobacteriaceae and Salmonella spp. Newly discovered isolates were subjected to Illumina whole-genome sequencing (WGS) and S1 pulsed-field gel electrophoresis (PFGE) hybridization experiments. WGS data of these isolates were compared with those for the previously isolated VIM-1-producing Salmonella Infantis isolates from pigs and poultry. Among 103 samples, one Salmonella Goldcoast isolate, one Salmonella Infantis isolate, and one Enterobacter cloacae isolate carrying the blaVIM-1 gene were detected. Comparative WGS analysis revealed that the blaVIM-1 gene was part of a particular Tn21-like transposable element in all isolates. It was located on IncHI2 (ST1) plasmids of ~290 to 300?kb with a backbone highly similar (98 to 100%) to that of reference pSE15-SA01028. SNP analysis revealed a close relationship of all VIM-1-positive S Infantis isolates described since 2011. The findings of this study demonstrate that the occurrence of the blaVIM-1 gene in German livestock is restricted neither to a certain bacterial species nor to a certain Salmonella serovar but is linked to a particular Tn21-like transposable element located on transferable pSE15-SA01028-like IncHI2 (ST1) plasmids, being present in all of the investigated isolates from 2011 to 2017.IMPORTANCE Carbapenems are considered one of few remaining treatment options against multidrug-resistant Gram-negative pathogens in human clinical settings. The occurrence of carbapenemase-producing Enterobacteriaceae in livestock and food is a major public health concern. Particularly the occurrence of VIM-1-producing Salmonella Infantis in livestock farms is worrisome, as this zoonotic pathogen is one of the main causes for human salmonellosis in Europe. Investigations on the epidemiology of those carbapenemase-producing isolates and associated mobile genetic elements through an in-depth molecular characterization are indispensable to understand the transmission of carbapenemase-producing Enterobacteriaceae along the food chain and between different populations to develop strategies to prevent their further spread.Copyright © 2019 Roschanski et al.

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April 21, 2020

Spreading Patterns of NDM-Producing Enterobacteriaceae in Clinical and Environmental Settings in Yangon, Myanmar.

The spread of carbapenemase-producing Enterobacteriaceae (CPE), contributing to widespread carbapenem resistance, has become a global concern. However, the specific dissemination patterns of carbapenemase genes have not been intensively investigated in developing countries, including Myanmar, where NDM-type carbapenemases are spreading in clinical settings. In the present study, we phenotypically and genetically characterized 91 CPE isolates obtained from clinical (n = 77) and environmental (n = 14) samples in Yangon, Myanmar. We determined the dissemination of plasmids harboring genes encoding NDM-1 and its variants using whole-genome sequencing and plasmid analysis. IncFII plasmids harboring blaNDM-5 and IncX3 plasmids harboring blaNDM-4 or blaNDM-7 were the most prevalent plasmid types identified among the isolates. The IncFII plasmids were predominantly carried by clinical isolates of Escherichia coli, and their clonal expansion was observed within the same ward of a hospital. In contrast, the IncX3 plasmids were found in phylogenetically divergent isolates from clinical and environmental samples classified into nine species, suggesting widespread dissemination of plasmids via horizontal transfer. Half of the environmental isolates were found to possess IncX3 plasmids, and this type of plasmid was confirmed to transfer more effectively to recipient organisms at a relatively low temperature (25°C) compared to the IncFII plasmid. Moreover, various other plasmid types were identified harboring blaNDM-1, including IncFIB, IncFII, IncL/M, and IncA/C2, among clinical isolates of Klebsiella pneumoniae or Enterobacter cloacae complex. Overall, our results highlight three distinct patterns of the dissemination of blaNDM-harboring plasmids among CPE isolates in Myanmar, contributing to a better understanding of their molecular epidemiology and dissemination in a setting of endemicity.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

The Modern View of B Chromosomes Under the Impact of High Scale Omics Analyses.

Supernumerary B chromosomes (Bs) are extra karyotype units in addition to A chromosomes, and are found in some fungi and thousands of animals and plant species. Bs are uniquely characterized due to their non-Mendelian inheritance, and represent one of the best examples of genomic conflict. Over the last decades, their genetic composition, function and evolution have remained an unresolved query, although a few successful attempts have been made to address these phenomena. A classical concept based on cytogenetics and genetics is that Bs are selfish and abundant with DNA repeats and transposons, and in most cases, they do not carry any function. However, recently, the modern quantum development of high scale multi-omics techniques has shifted B research towards a new-born field that we call “B-omics”. We review the recent literature and add novel perspectives to the B research, discussing the role of new technologies to understand the mechanistic perspectives of the molecular evolution and function of Bs. The modern view states that B chromosomes are enriched with genes for many significant biological functions, including but not limited to the interesting set of genes related to cell cycle and chromosome structure. Furthermore, the presence of B chromosomes could favor genomic rearrangements and influence the nuclear environment affecting the function of other chromatin regions. We hypothesize that B chromosomes might play a key function in driving their transmission and maintenance inside the cell, as well as offer an extra genomic compartment for evolution.

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April 21, 2020

Divergent evolution in the genomes of closely related lacertids, Lacerta viridis and L. bilineata, and implications for speciation.

Lacerta viridis and Lacerta bilineata are sister species of European green lizards (eastern and western clades, respectively) that, until recently, were grouped together as the L. viridis complex. Genetic incompatibilities were observed between lacertid populations through crossing experiments, which led to the delineation of two separate species within the L. viridis complex. The population history of these sister species and processes driving divergence are unknown. We constructed the first high-quality de novo genome assemblies for both L. viridis and L. bilineata through Illumina and PacBio sequencing, with annotation support provided from transcriptome sequencing of several tissues. To estimate gene flow between the two species and identify factors involved in reproductive isolation, we studied their evolutionary history, identified genomic rearrangements, detected signatures of selection on non-coding RNA, and on protein-coding genes.Here we show that gene flow was primarily unidirectional from L. bilineata to L. viridis after their split at least 1.15 million years ago. We detected positive selection of the non-coding repertoire; mutations in transcription factors; accumulation of divergence through inversions; selection on genes involved in neural development, reproduction, and behavior, as well as in ultraviolet-response, possibly driven by sexual selection, whose contribution to reproductive isolation between these lacertid species needs to be further evaluated.The combination of short and long sequence reads resulted in one of the most complete lizard genome assemblies. The characterization of a diverse array of genomic features provided valuable insights into the demographic history of divergence among European green lizards, as well as key species differences, some of which are candidates that could have played a role in speciation. In addition, our study generated valuable genomic resources that can be used to address conservation-related issues in lacertids. © The Author(s) 2018. Published by Oxford University Press.

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April 21, 2020

Diverse Vectors and Mechanisms Spread New Delhi Metallo-ß-Lactamases among Carbapenem-Resistant Enterobacteriaceae in the Greater Boston Area.

New Delhi metallo-beta-lactamases (NDMs) are an uncommon but emerging cause of carbapenem resistance in the United States. Genomic factors promoting their domestic spread remain poorly characterized. A prospective genomic surveillance program among Boston-area hospitals identified multiple new occurrences of NDM-carrying strains of Escherichia coli and Enterobacter cloacae complex in inpatient and outpatient settings, representing the first occurrences of NDM-mediated resistance since initiating genomic surveillance in 2011. Cases included domestic patients with no international exposures. PacBio sequencing of isolates identified strain characteristics, resistance genes, and the complement of mobile vectors mediating spread. Analyses revealed a common 3,114-bp region containing the blaNDM gene, with carriage of this conserved region among unique strains by diverse transposon and plasmid backbones. Functional studies revealed a broad capacity for blaNDM transmission by conjugation, transposition, and complex interplasmid recombination events. NDMs represent a rapidly spreading form of drug resistance that can occur in inpatient and outpatient settings and in patients without international exposures. In contrast to Tn4401-based spread of Klebsiella pneumoniae carbapenemases (KPCs), diverse transposable elements mobilize NDM enzymes, commonly with other resistance genes, enabling naive strains to acquire multi- and extensively drug-resistant profiles with single transposition or plasmid conjugation events. Genomic surveillance provides effective means to rapidly identify these gene-level drivers of resistance and mobilization in order to inform clinical decisions to prevent further spread.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Evolution of a clade of Acinetobacter baumannii global clone 1, lineage 1 via acquisition of carbapenem- and aminoglycoside-resistance genes and dispersion of ISAba1.

Resistance to carbapenem and aminoglycoside antibiotics is a critical problem in Acinetobacter baumannii, particularly when genes conferring resistance are acquired by multiply or extensively resistant members of successful globally distributed clonal complexes, such as global clone 1 (GC1) . Here, we investigate the evolution of an expanding clade of lineage 1 of the GC1 complex via repeated acquisition of carbapenem- and aminoglycoside-resistance genes. Lineage 1 arose in the late 1970s and the Tn6168/OCL3 clade arose in the late 1990s from an ancestor that had already acquired resistance to third-generation cephalosporins and fluoroquinolones. Between 2000 and 2002, two distinct subclades have emerged, and they are distinguishable via the presence of an integrated phage genome in subclade 1 and AbaR4 (carrying the oxa23 carbapenem-resistance gene in Tn2006) at a specific chromosomal location in subclade 2. Part or all of the original resistance gene cluster in the chromosomally located AbaR3 has been lost from some isolates, but plasmids carrying alternate resistance genes have been gained. In one group in subclade 2, the chromosomally located AbGRI3, carrying the armA aminoglycoside-resistance gene, has been acquired from a GC2 isolate and incorporated via homologous recombination. ISAba1 entered the common ancestor of this clade as part of the cephalosporin-resistance transposon Tn6168 and has dispersed differently in each subclade. Members of subclade 1 share an ISAba1 in one specific position in the chromosome and in subclade 2 two different ISAba1 locations are shared. Further shared ISAba1 locations distinguish further divisions, potentially providing simple markers for epidemiological studies.

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April 21, 2020

Gammaherpesvirus Readthrough Transcription Generates a Long Non-Coding RNA That Is Regulated by Antisense miRNAs and Correlates with Enhanced Lytic Replication In Vivo.

Gammaherpesviruses, including the human pathogens Epstein?Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) are oncogenic viruses that establish lifelong infections in hosts and are associated with the development of lymphoproliferative diseases and lymphomas. Recent studies have shown that the majority of the mammalian genome is transcribed and gives rise to numerous long non-coding RNAs (lncRNAs). Likewise, the large double-stranded DNA virus genomes of herpesviruses undergo pervasive transcription, including the expression of many as yet uncharacterized lncRNAs. Murine gammaperherpesvirus 68 (MHV68, MuHV-4, ?HV68) is a natural pathogen of rodents, and is genetically and pathogenically related to EBV and KSHV, providing a highly tractable model for studies of gammaherpesvirus biology and pathogenesis. Through the integrated use of parallel data sets from multiple sequencing platforms, we previously resolved transcripts throughout the MHV68 genome, including at least 144 novel transcript isoforms. Here, we sought to molecularly validate novel transcripts identified within the M3/M2 locus, which harbors genes that code for the chemokine binding protein M3, the latency B cell signaling protein M2, and 10 microRNAs (miRNAs). Using strand-specific northern blots, we validated the presence of M3-04, a 3.91 kb polyadenylated transcript that initiates at the M3 transcription start site and reads through the M3 open reading frame (ORF), the M3 poly(a) signal sequence, and the M2 ORF. This unexpected transcript was solely localized to the nucleus, strongly suggesting that it is not translated and instead may function as a lncRNA. Use of an MHV68 mutant lacking two M3-04-antisense pre-miRNA stem loops resulted in highly increased expression of M3-04 and increased virus replication in the lungs of infected mice, demonstrating a key role for these RNAs in regulation of lytic infection. Together these findings suggest the possibility of a tripartite regulatory relationship between the lncRNA M3-04, antisense miRNAs, and the latency gene M2.

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April 21, 2020

Into the Thermus Mobilome: Presence, Diversity and Recent Activities of Insertion Sequences Across Thermus spp.

A high level of transposon-mediated genome rearrangement is a common trait among microorganisms isolated from thermal environments, probably contributing to the extraordinary genomic plasticity and horizontal gene transfer (HGT) observed in these habitats. In this work, active and inactive insertion sequences (ISs) spanning the sequenced members of the genus Thermus were characterized, with special emphasis on three T. thermophilus strains: HB27, HB8, and NAR1. A large number of full ISs and fragments derived from different IS families were found, concentrating within megaplasmids present in most isolates. Potentially active ISs were identified through analysis of transposase integrity, and domestication-related transposition events of ISTth7 were identified in laboratory-adapted HB27 derivatives. Many partial copies of ISs appeared throughout the genome, which may serve as specific targets for homologous recombination contributing to genome rearrangement. Moreover, recruitment of IS1000 32 bp segments as spacers for CRISPR sequence was identified, pointing to the adaptability of these elements in the biology of these thermophiles. Further knowledge about the activity and functional diversity of ISs in this genus may contribute to the generation of engineered transposons as new genetic tools, and enrich our understanding of the outstanding plasticity shown by these thermophiles.

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