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July 7, 2019

Determination of nucleopolyhedrovirus’ taxonomic position

To date , over 78 genomes of nucleopolyhedroviruses (NPVs) have been sequenced and deposited in NCBI. How to define a new virus from the infected larvae in the field is usually the first question. Two NPV strains, which were isolated from casuarina moth (L. xylina) and golden birdwing larvae (Troides aeacus), respectively, displayed the same question. Due to the identity of polyhedrin (polh) sequences of these two isolates to that of Lymantria dispar MNPV and Bombyx mori NPV, they are named LdMNPV-like virus and TraeNPV, provisionally. To further clarify the relationships of LdMNPV-like virus and TraeNPV to closely related NPVs, Kimura 2-parameter (K-2-P) analysis was performed. Apparently, the results of K-2-P analysis that showed LdMNPV-like virus is an LdMNPV isolate, while TraeNPV had an ambiguous relationship to BmNPV. Otherwise, MaviNPV, which is a mini-AcMNPV, also exhibited a different story by K-2-P analysis. Since K-2-P analysis could not cover all species determination issues, therefore, TraeNPV needs to be sequenced for defining its taxonomic position. For this purpose, different genomic sequencing technologies and bioinformatic analysis approaches will be discussed. We anticipated that these applications will help to exam nucleotide information of unknown species and give an insight and facilitate to this issue.

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July 7, 2019

Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli.

The mobile colistin resistance gene mcr-1 has attracted global attention, as it heralds the breach of polymyxins, one of the last-resort antibiotics for the treatment of severe clinical infections caused by multidrug-resistant Gram-negative bacteria. To date, six slightly different variants of mcr-1, and a second mobile colistin resistance gene, mcr-2, have been reported or annotated in the GenBank database. Here, we characterized a third mobile colistin resistance gene, mcr-3 The gene coexisted with 18 additional resistance determinants in the 261-kb IncHI2-type plasmid pWJ1 from porcine Escherichia colimcr-3 showed 45.0% and 47.0% nucleotide sequence identity to mcr-1 and mcr-2, respectively, while the deduced amino acid sequence of MCR-3 showed 99.8 to 100% and 75.6 to 94.8% identity to phosphoethanolamine transferases found in other Enterobacteriaceae species and in 10 Aeromonas species, respectively. pWJ1 was mobilized to an E. coli recipient by conjugation and contained a plasmid backbone similar to those of other mcr-1-carrying plasmids, such as pHNSHP45-2 from the original mcr-1-harboring E. coli strain. Moreover, a truncated transposon element, TnAs2, which was characterized only in Aeromonas salmonicida, was located upstream of mcr-3 in pWJ1. This ?TnAs2-mcr-3 element was also identified in a shotgun genome sequence of a porcine E. coli isolate from Malaysia, a human Klebsiella pneumoniae isolate from Thailand, and a human Salmonella enterica serovar Typhimurium isolate from the United States. These results suggest the likelihood of a wide dissemination of the novel mobile colistin resistance gene mcr-3 among Enterobacteriaceae and aeromonads; the latter may act as a potential reservoir for mcr-3IMPORTANCE The emergence of the plasmid-mediated colistin resistance gene mcr-1 has attracted substantial attention worldwide. Here, we examined a colistin-resistant Escherichia coli isolate that was negative for both mcr-1 and mcr-2 and discovered a novel mobile colistin resistance gene, mcr-3 The amino acid sequence of MCR-3 aligned closely with phosphoethanolamine transferases from Enterobacteriaceae and Aeromonas species originating from both clinical infections and environmental samples collected in 12 countries on four continents. Due to the ubiquitous profile of aeromonads in the environment and the potential transfer of mcr-3 between Enterobacteriaceae and Aeromonas species, the wide spread of mcr-3 may be largely underestimated. As colistin has been and still is widely used in veterinary medicine and used at increasing frequencies in human medicine, the continuous monitoring of mobile colistin resistance determinants in colistin-resistant Gram-negative bacteria is imperative for understanding and tackling the dissemination of mcr genes in both the agricultural and health care sectors. Copyright © 2017 Yin et al.

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July 7, 2019

Population genomics of picophytoplankton unveils novel chromosome hypervariability.

Tiny photosynthetic microorganisms that form the picoplankton (between 0.3 and 3 µm in diameter) are at the base of the food web in many marine ecosystems, and their adaptability to environmental change hinges on standing genetic variation. Although the genomic and phenotypic diversity of the bacterial component of the oceans has been intensively studied, little is known about the genomic and phenotypic diversity within each of the diverse eukaryotic species present. We report the level of genomic diversity in a natural population of Ostreococcus tauri (Chlorophyta, Mamiellophyceae), the smallest photosynthetic eukaryote. Contrary to the expectations of clonal evolution or cryptic species, the spectrum of genomic polymorphism observed suggests a large panmictic population (an effective population size of 1.2 × 10(7)) with pervasive evidence of sexual reproduction. De novo assemblies of low-coverage chromosomes reveal two large candidate mating-type loci with suppressed recombination, whose origin may pre-date the speciation events in the class Mamiellophyceae. This high genetic diversity is associated with large phenotypic differences between strains. Strikingly, resistance of isolates to large double-stranded DNA viruses, which abound in their natural environment, is positively correlated with the size of a single hypervariable chromosome, which contains 44 to 156 kb of strain-specific sequences. Our findings highlight the role of viruses in shaping genome diversity in marine picoeukaryotes.

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July 7, 2019

Genomic characterization of a large plasmid containing a bla NDM-1 gene carried on Salmonella enterica serovar Indiana C629 isolate from China.

The bla NDM-1 gene in Salmonella species is mostly reported in clinical cases, but is rarely isolated from red and white meat in China.A Salmonella Indiana (S. Indiana) isolate was cultured from a chicken carcass procured from a slaughterhouse in China. Antimicrobial susceptibility was tested against a panel of agents. Whole-genome sequencing of the isolate was carried out and data was analyzed.A large plasmid, denoted as plasmid pC629 (210,106 bp), containing a composite cassette, consisting of IS26-bla NDM-1-ble MBL -?trpF-tat-cutA-ISCR1-sul1-qacE?1-aadA2-dfrA12-intI1-IS26 was identified. The latter locus was physically linked with bla OXA-1, bla CTX-M-65, bla TEM-1-encoding genes. A mercury resistance operon merACDEPTR was also identified; it was flanked on the proximal side, among IS26 element and the distally located on the bla NDM-1 gene. Plasmid pC629 also contained 21 other antimicrobial resistance-encoding genes, such as aac(6′)-Ib-cr, aac(3)-VI, aadA5, aph(4)-Ia, arr-3, blmS, brp, catB3, dfrA17, floR, fosA, mph(A), mphR, mrx, nimC/nimA, oqxA, oqxB, oqxR, rmtB, sul1, sul2. Two virulence genes were also identified on plasmid pC629.To the best of our knowledge, this is the first report of bla NDM-1 gene being identified from a plasmid in a S. Indiana isolate cultured from chicken carcass in China.

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July 7, 2019

Detection of an Escherichia coli sequence type 167 strain with two tandem copies of blaNDM-1 in the chromosome.

New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Enterobacteriaceae has disseminated rapidly throughout the world and poses an urgent threat to public health. Previous studies confirmed that the blaNDM-1 gene is typically carried in plasmids but rarely in chromosome. We discovered a multidrug-resistant Escherichia coli strain Y5, originating from a urine sample and containing the blaNDM-1 gene, which did not transfer by either conjugation or electrotransformation. We confirmed the possibility of its chromosome location by S1-pulsed-field gel electrophoresis (PFGE) and XbaI-PFGE, followed by Southern blotting. To determine the genomic background of blaNDM-1, the genome of Y5 was completely sequenced and compared to other reference genomes. The results of our study revealed that this isolate consists of a 4.8-Mbp chromosome and three plasmids, it is an epidemic clone of sequence type (ST) 167, and it shows 99% identity with Escherichia coli 6409 (GenBank accession no. CP010371), which lacks the same blaNDM-1 gene-surrounding structure as Y5. The blaNDM-1 gene is embedded in the chromosome along with two tandem copies of an insertion sequence common region 1 (ISCR1) element (sul1-ARR-3-cat-blaNDM-1-bleo-ISCR1), which appears intact in the plasmid from Proteus mirabilis (GenBank accession no. KP662515). The genomic context indicates that the ISCR1 element mediated the blaNDM-1 transposition from a single source plasmid to the chromosome. Our study is the first report of an Enterobacteriaceae strain harboring a chromosomally integrated blaNDM-1, which directly reveals the vertical spreading pattern of the gene. Close surveillance is urgently needed to monitor the emergence and potential spread of ST167 strains that harbor blaNDM-1. Copyright © 2016 American Society for Microbiology.

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July 7, 2019

Expanding landscapes of the diversified mcr-1-bearing plasmid reservoirs.

Polymyxin is a cationic polypeptide antibiotic that can disrupt bacterial cell membrane by interacting with its lipopolysaccharide molecules and is used as a last resort drug against lethal infections by the carbapenem-resistant superbugs (like NDM-1). However, global discovery of the MCR-1 colistin resistance dramatically challenges the newly renewed interest in colistin for clinical use.The mcr-1-harboring plasmids were acquired from swine and human Escherichia coli isolated in China, from 2015 to 2016, and subjected to Illumina PacBio RSII and Hi-Seq2000 for full genome sequencing. PCR was applied to close the gap of the assembled contigs. Ori-Finder was employed to predict the replication origin (oriC) in plasmids. The phenotype of MCR-1-producing isolates was evaluated on the LBA plates with various level of colistin. Genetic deletion was used to test the requirement of the initial “ATG” codon for the MCR-1 function.Here, we report full genomes of over 10 mcr-1-harboring plasmids with diversified replication incompatibilities. A novel hybrid IncI2/IncFIB plasmid pGD17-2 was discovered and characterized from a swine isolate with colistin resistance. Intriguingly, co-occurrence of two unique mcr-1-bearing plasmids (pGD65-3, IncI2, and pGD65-5, IncX4) was detected in a single isolate GD65, which might accelerate dissemination of the mcr-1 under environmental selection pressure. Genetic analyses of these plasmids mapped mobile elements in the context of antibiotic resistance and determined two insertion sequences (ISEcp1 and ISApl1) that are responsible for the mobilization of mcr-1. Gene deletion also proved that the first ATG codon is redundant in the mcr-1 gene.Collectively, our results extend landscapes of the diversified mcr-1-bearing plasmid reservoirs.

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July 7, 2019

Genome evolution to penicillin resistance in serotype 3 Streptococcus pneumoniae by capsular switching.

Streptococcus pneumoniae isolates of serotype 3 were collected from cases of invasive pneumococcal disease (n= 124) throughout Japan between April 2010 and March 2013. A penicillin-resistantS. pneumoniae(PRSP) isolate from an adult patient, strain KK0981 of serotype 3, was identified among these strains. Whole-genome analysis characterized this PRSP as a recombinant strain derived from PRSP of serotype 23F with thecpslocus (20.3 kb) replaced by that of a penicillin-susceptible strain of serotype 3. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Phenotypic and genomic comparison of Mycobacterium aurum and surrogate model species to Mycobacterium tuberculosis: implications for drug discovery.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis and represents one of the major challenges facing drug discovery initiatives worldwide. The considerable rise in bacterial drug resistance in recent years has led to the need of new drugs and drug regimens. Model systems are regularly used to speed-up the drug discovery process and circumvent biosafety issues associated with manipulating M. tuberculosis. These include the use of strains such as Mycobacterium smegmatis and Mycobacterium marinum that can be handled in biosafety level 2 facilities, making high-throughput screening feasible. However, each of these model species have their own limitations.We report and describe the first complete genome sequence of Mycobacterium aurum ATCC23366, an environmental mycobacterium that can also grow in the gut of humans and animals as part of the microbiota. This species shows a comparable resistance profile to that of M. tuberculosis for several anti-TB drugs. The aims of this study were to (i) determine the drug resistance profile of a recently proposed model species, Mycobacterium aurum, strain ATCC23366, for anti-TB drug discovery as well as Mycobacterium smegmatis and Mycobacterium marinum (ii) sequence and annotate the complete genome sequence of this species obtained using Pacific Bioscience technology (iii) perform comparative genomics analyses of the various surrogate strains with M. tuberculosis (iv) discuss how the choice of the surrogate model used for drug screening can affect the drug discovery process.We describe the complete genome sequence of M. aurum, a surrogate model for anti-tuberculosis drug discovery. Most of the genes already reported to be associated with drug resistance are shared between all the surrogate strains and M. tuberculosis. We consider that M. aurum might be used in high-throughput screening for tuberculosis drug discovery. We also highly recommend the use of different model species during the drug discovery screening process.

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July 7, 2019

Comparative genomic analysis of Acinetobacter strains isolated from murine colonic crypts.

A restricted set of aerobic bacteria dominated by the Acinetobacter genus was identified in murine intestinal colonic crypts. The vicinity of such bacteria with intestinal stem cells could indicate that they protect the crypt against cytotoxic and genotoxic signals. Genome analyses of these bacteria were performed to better appreciate their biodegradative capacities.Two taxonomically different clusters of Acinetobacter were isolated from murine proximal colonic crypts, one was identified as A. modestus and the other as A. radioresistens. Their identification was performed through biochemical parameters and housekeeping gene sequencing. After selection of one strain of each cluster (A. modestus CM11G and A. radioresistens CM38.2), comparative genomic analysis was performed on whole-genome sequencing data. The antibiotic resistance pattern of these two strains is different, in line with the many genes involved in resistance to heavy metals identified in both genomes. Moreover whereas the operon benABCDE involved in benzoate metabolism is encoded by the two genomes, the operon antABC encoding the anthranilate dioxygenase, and the phenol hydroxylase gene cluster are absent in the A. modestus genomic sequence, indicating that the two strains have different capacities to metabolize xenobiotics. A common feature of the two strains is the presence of a type IV pili system, and the presence of genes encoding proteins pertaining to secretion systems such as Type I and Type II secretion systems.Our comparative genomic analysis revealed that different Acinetobacter isolated from the same biological niche, even if they share a large majority of genes, possess unique features that could play a specific role in the protection of the intestinal crypt.

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July 7, 2019

Adaptive evolution of a hyperthermophilic archaeon pinpoints a formate transporter as a critical factor for the growth enhancement on formate.

Previously, we reported that the hyperthermophilic archaeon Thermococcus onnurineus NA1 could grow on formate and produce H2. Formate conversion to hydrogen was mediated by a formate-hydrogen lyase complex and was indeed a part of chemiosmotic coupling to ATP generation. In this study, we employed an adaptation approach to enhance the cell growth on formate and investigated molecular changes. As serial transfer continued on formate-containing medium at the serum vial, cell growth, H2 production and formate consumption increased remarkably. The 156 times transferred-strain, WTF-156T, was demonstrated to enhance H2 production using formate in a bioreactor. The whole-genome sequencing of the WTF-156T strain revealed eleven mutations. While no mutation was found among the genes encoding formate hydrogen lyase, a point mutation (G154A) was identified in a formate transporter (TON_1573). The TON_1573 (A52T) mutation, when introduced into the parent strain, conferred increase in formate consumption and H2 production. Another adaptive passage, carried out by culturing repeatedly in a bioreactor, resulted in a strain, which has a mutation in TON_1573 (C155A) causing amino acid change, A52E. These results implicate that substitution of A52 residue of a formate transporter might be a critical factor to ensure the increase in formate uptake and cell growth.

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July 7, 2019

ConcatSeq: A method for increasing throughput of single molecule sequencing by concatenating short DNA fragments.

Single molecule sequencing (SMS) platforms enable base sequences to be read directly from individual strands of DNA in real-time. Though capable of long read lengths, SMS platforms currently suffer from low throughput compared to competing short-read sequencing technologies. Here, we present a novel strategy for sequencing library preparation, dubbed ConcatSeq, which increases the throughput of SMS platforms by generating long concatenated templates from pools of short DNA molecules. We demonstrate adaptation of this technique to two target enrichment workflows, commonly used for oncology applications, and feasibility using PacBio single molecule real-time (SMRT) technology. Our approach is capable of increasing the sequencing throughput of the PacBio RSII platform by more than five-fold, while maintaining the ability to correctly call allele frequencies of known single nucleotide variants. ConcatSeq provides a versatile new sample preparation tool for long-read sequencing technologies.

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July 7, 2019

Discovery and genotyping of novel sequence insertions in many sequenced individuals

Motivation: Despite recent advances in algorithms design to characterize structural variation using high-throughput short read sequencing (HTS) data, characterization of novel sequence insertions longer than the average read length remains a challenging task. This is mainly due to both computational difficulties and the complexities imposed by genomic repeats in generating reliable assemblies to accurately detect both the sequence content and the exact location of such insertions. Additionally, de novo genome assembly algorithms typically require a very high depth of coverage, which may be a limiting factor for most genome studies. Therefore, characterization of novel sequence insertions is not a routine part of most sequencing projects. There are only a handful of algorithms that are specifically developed for novel sequence insertion discovery that can bypass the need for the whole genome de novo assembly. Still, most such algorithms rely on high depth of coverage, and to our knowledge there is only one method (PopIns) that can use multi-sample data to “collectively” obtain a very high coverage dataset to accurately find insertions common in a given population. Result: Here, we present Pamir, a new algorithm to efficiently and accurately discover and genotype novel sequence insertions using either single or multiple genome sequencing datasets. Pamir is able to detect breakpoint locations of the insertions and calculate their zygosity (i.e. heterozygous versus homozygous) by analyzing multiple sequence signatures, matching one-end-anchored sequences to small-scale de novo assemblies of unmapped reads, and conducting strand-aware local assembly. We test the efficacy of Pamir on both simulated and real data, and demonstrate its potential use in accurate and routine identification of novel sequence insertions in genome projects. Availability and implementation: Pamir is available at https://github.com/vpc-ccg/pamir. Contact:fhach@sfu.ca, prostatecentre.com or calkan@cs.bilkent.edu.tr Supplementary information:Supplementary data are available at Bioinformatics online.

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July 7, 2019

Whole-genome restriction mapping by “subhaploid”-based RAD sequencing: An efficient and flexible approach for physical mapping and genome scaffolding.

Assembly of complex genomes using short reads remains a major challenge, which usually yields highly fragmented assemblies. Generation of ultradense linkage maps is promising for anchoring such assemblies, but traditional linkage mapping methods are hindered by the infrequency and unevenness of meiotic recombination that limit attainable map resolution. Here we develop a sequencing-based “in vitro” linkage mapping approach (called RadMap), where chromosome breakage and segregation are realized by generating hundreds of “subhaploid” fosmid/bacterial-artificial-chromosome clone pools, and by restriction site-associated DNA sequencing of these clone pools to produce an ultradense whole-genome restriction map to facilitate genome scaffolding. A bootstrap-based minimum spanning tree algorithm is developed for grouping and ordering of genome-wide markers and is implemented in a user-friendly, integrated software package (AMMO). We perform extensive analyses to validate the power and accuracy of our approach in the model plant Arabidopsis thaliana and human. We also demonstrate the utility of RadMap for enhancing the contiguity of a variety of whole-genome shotgun assemblies generated using either short Illumina reads (300 bp) or long PacBio reads (6-14 kb), with up to 15-fold improvement of N50 (~816 kb-3.7 Mb) and high scaffolding accuracy (98.1-98.5%). RadMap outperforms BioNano and Hi-C when input assembly is highly fragmented (contig N50 = 54 kb). RadMap can capture wide-range contiguity information and provide an efficient and flexible tool for high-resolution physical mapping and scaffolding of highly fragmented assemblies. Copyright © 2017 Dou et al.

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July 7, 2019

Reclassification of the specialized metabolite producer Pseudomonas mesoacidophila ATCC 31433 as a member of the Burkholderia cepacia complex.

Pseudomonas mesoacidophila ATCC 31433 is a Gram-negative bacterium, first isolated from Japanese soil samples, that produces the monobactam isosulfazecin and the ß-lactam-potentiating bulgecins. To characterize the biosynthetic potential of P. mesoacidophila ATCC 31433, its complete genome was determined using single-molecule real-time DNA sequence analysis. The 7.8-Mb genome comprised four replicons, three chromosomal (each encoding rRNA) and one plasmid. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 was misclassified at the time of its deposition and is a member of the Burkholderia cepacia complex, most closely related to Burkholderia ubonensis The sequenced genome shows considerable additional biosynthetic potential; known gene clusters for malleilactone, ornibactin, isosulfazecin, alkylhydroxyquinoline, and pyrrolnitrin biosynthesis and several uncharacterized biosynthetic gene clusters for polyketides, nonribosomal peptides, and other metabolites were identified. Furthermore, P. mesoacidophila ATCC 31433 harbors many genes associated with environmental resilience and antibiotic resistance and was resistant to a range of antibiotics and metal ions. In summary, this bioactive strain should be designated B. cepacia complex strain ATCC 31433, pending further detailed taxonomic characterization.IMPORTANCE This work reports the complete genome sequence of Pseudomonas mesoacidophila ATCC 31433, a known producer of bioactive compounds. Large numbers of both known and novel biosynthetic gene clusters were identified, indicating that P. mesoacidophila ATCC 31433 is an untapped resource for discovery of novel bioactive compounds. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 is in fact a member of the Burkholderia cepacia complex, most closely related to the species Burkholderia ubonensis Further investigation of the classification and biosynthetic potential of P. mesoacidophila ATCC 31433 is warranted. Copyright © 2017 Loveridge et al.

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July 7, 2019

Characterization of potential polysaccharide utilization systems in the marine bacteroidetes Gramella flava JLT2011 using a multi-omics approach.

Members of phylum Bacteroidetes are distributed across diverse marine niches and Flavobacteria is often the predominant bacterial class decomposing algae-derived polysaccharides. Here, we report the complete genome of Gramella flava JLT2011 (Flavobacteria) isolated from surface water of the southeastern Pacific. A remarkable genomic feature is that the number of glycoside hydrolase (GH) genes in the genome of G. flava JLT2011 is more than 2-fold higher than that of other Gramella species. The functional profiles of the GHs suggest extensive variation in Gramella species. Growth experiments revealed that G. flava JLT2011 has the ability to utilize a wide range of polysaccharides for growth such as xylan and homogalacturonan in pectin. Nearly half of all GH genes were located on the multi-gene polysaccharide utilization loci (PUL) or PUL-like systems in G. flava JLT2011. This species was also found to harbor the two xylan PULs and a pectin PUL, respectively. Gene expression data indicated that more GHs and sugar-specific outer-membrane susC-susD systems were found in the presence of xylan than in the presence of pectin, suggesting a different strategy for heteropolymeric xylan and homoglacturonan utilization. Multi-omics data (transcriptomics, proteomics, and metabolomics) indicated that xylan PULs and pectin PUL are respectively involved in the catabolism of their corresponding polysaccharides. This work presents a comparison of polysaccharide decomposition within a genus and expands current knowledge on the diversity and function of PULs in marine Bacteroidetes, thereby deepening our understanding of their ecological role in polysaccharide remineralization in the marine system.

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