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April 21, 2020

Identification of Diverse Integron and Plasmid Structures Carrying a Novel Carbapenemase Among Pseudomonas Species.

A novel carbapenem-hydrolyzing beta-lactamase, called IMP-63, was identified in three clonally distinct strains of Pseudomonas aeruginosa and two strains of Pseudomonas putida isolated within a 4 year timeframe in three French hospitals. The blaIMP-63 gene that encodes this carbapenemase turned out to be located in the variable region of four integrons (In1297, In1574, In1573, and In1572) and to coexist with novel or rare gene cassettes (fosM, gcu170, gcuF1) and insertion elements (ISPsp7v, ISPa16v). All these integrons except one (In1574) were flanked by a copy of insertion sequence ISPa17 next to the orf6 putative gene, and were carried by non-conjugative plasmids (pNECK1, pROUSS1, pROUSS2, pROUE1). These plasmids exhibit unique modular structures and partial sequence homologies with plasmids previously identified in various non-fermenting environmental Gram-negative species. Lines of evidence suggest that ISPa17 promoted en bloc the transposition of IMP-63-encoding integrons on these different plasmids. As demonstrated by genotyping experiments, isolates of P. aeruginosa harboring the 28.9-kb plasmid pNECK1 and belonging to international “high-risk” clone ST308 were responsible for an outbreak in one hospital. Collectively, these data provide an insight into the complex and unpredictable routes of diffusion of some resistance determinants, here blaIMP-63, among Pseudomonas species.


April 21, 2020

Exploring the landscape of focal amplifications in cancer using AmpliconArchitect.

Focal oncogene amplification and rearrangements drive tumor growth and evolution in multiple cancer types. We present AmpliconArchitect (AA), a tool to reconstruct the fine structure of focally amplified regions using whole genome sequencing (WGS) and validate it extensively on multiple simulated and real datasets, across a wide range of coverage and copy numbers. Analysis of AA-reconstructed amplicons in a pan-cancer dataset reveals many novel properties of copy number amplifications in cancer. These findings support a model in which focal amplifications arise due to the formation and replication of extrachromosomal DNA. Applying AA to 68 viral-mediated cancer samples, we identify a large fraction of amplicons with specific structural signatures suggestive of hybrid, human-viral extrachromosomal DNA. AA reconstruction, integrated with metaphase fluorescence in situ hybridization (FISH) and PacBio sequencing on the cell-line UPCI:SCC090 confirm the extrachromosomal origin and fine structure of a Forkhead box E1 (FOXE1)-containing hybrid amplicon.


April 21, 2020

Genomics-driven discovery of a biosynthetic gene cluster required for the synthesis of BII-Rafflesfungin from the fungus Phoma sp. F3723.

Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis.Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined.We describe the NRPS-t1PKS cluster ‘BIIRfg’ compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene.


April 21, 2020

Divergent evolutionary trajectories following speciation in two ectoparasitic honey bee mites.

Multispecies host-parasite evolution is common, but how parasites evolve after speciating remains poorly understood. Shared evolutionary history and physiology may propel species along similar evolutionary trajectories whereas pursuing different strategies can reduce competition. We test these scenarios in the economically important association between honey bees and ectoparasitic mites by sequencing the genomes of the sister mite species Varroa destructor and Varroa jacobsoni. These genomes were closely related, with 99.7% sequence identity. Among the 9,628 orthologous genes, 4.8% showed signs of positive selection in at least one species. Divergent selective trajectories were discovered in conserved chemosensory gene families (IGR, SNMP), and Halloween genes (CYP) involved in moulting and reproduction. However, there was little overlap in these gene sets and associated GO terms, indicating different selective regimes operating on each of the parasites. Based on our findings, we suggest that species-specific strategies may be needed to combat evolving parasite communities. © The Author(s) 2019.


April 21, 2020

Penaeid shrimp genome provides insights into benthic adaptation and frequent molting.

Crustacea, the subphylum of Arthropoda which dominates the aquatic environment, is of major importance in ecology and fisheries. Here we report the genome sequence of the Pacific white shrimp Litopenaeus vannamei, covering ~1.66?Gb (scaffold N50 605.56?Kb) with 25,596 protein-coding genes and a high proportion of simple sequence repeats (>23.93%). The expansion of genes related to vision and locomotion is probably central to its benthic adaptation. Frequent molting of the shrimp may be explained by an intensified ecdysone signal pathway through gene expansion and positive selection. As an important aquaculture organism, L. vannamei has been subjected to high selection pressure during the past 30 years of breeding, and this has had a considerable impact on its genome. Decoding the L. vannamei genome not only provides an insight into the genetic underpinnings of specific biological processes, but also provides valuable information for enhancing crustacean aquaculture.


April 21, 2020

Mitochondrial genome and transcriptome analysis of five alloplasmic male-sterile lines in Brassica juncea.

Alloplasmic lines, in which the nuclear genome is combined with wild cytoplasm, are often characterized by cytoplasmic male sterility (CMS), regardless of whether it was derived from sexual or somatic hybridization with wild relatives. In this study, we sequenced and analyzed the mitochondrial genomes of five such alloplasmic lines in Brassica juncea.The assembled and annotated mitochondrial genomes of the five alloplasmic lines were found to have virtually identical gene contents. They preserved most of the ancestral mitochondrial segments, and the same candidate male sterility gene (orf108) was found harbored in mitotype-specific sequences. We also detected promiscuous sequences of chloroplast origin that were conserved among plants of the Brassicaceae, and found the RNA editing profiles to vary across the five mitochondrial genomes.On the basis of our characterization of the genetic nature of five alloplasmic mitochondrial genomes, we speculated that the putative candidate male sterility gene orf108 may not be responsible for the CMS observed in Brassica oxyrrhina and Diplotaxis catholica. Furthermore, we propose the potential coincidence of CMS in alloplasmic lines. Our findings lay the foundation for further elucidation of male sterility gene.


April 21, 2020

Characterization of mcr-1-Harboring Plasmids from Pan Drug-Resistant Escherichia coli Strains Isolated from Retail Raw Chicken in South Korea

A number of studies from different countries have characterized mcr-1-harboring plasmids isolated from food; however, nothing has been reported about it in South Korea. In this study, we report the characterization of mcr-1 plasmids from pan drug-resistant (PDR) Escherichia coli strains isolated from retail food in the country. Colistin-resistant E. coli strains were isolated from retail raw chicken, and PCR was carried out to detect the mcr-1 gene. Whole genome sequencing of the mcr-1-positive strains was performed for further characterization. The results of whole genome sequencing revealed that all mcr-1 plasmids belonged to the IncI2 type. In addition to the mcr-1 plasmids, all of the isolates also carried additional plasmids possessing multiple antibiotic resistance genes, and the PDR was mediated by resistant plasmids except for fluoroquinolone resistance resulting from mutations in gyrA and parC. Interestingly, the mcr-1 plasmids were transferred by conjugation to other pathogenic strains including enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), Salmonella, and Klebsiella at the frequencies of 10−3−10−6, 10−2−10−5, 10−4−10−5, 10−4−10−6, and 10−5−10−6, respectively. The results showed that mcr-1 plasmids can be easily transmitted to pathogenic bacteria by conjugation.


April 21, 2020

Proteomic Analysis of Lactobacillus nagelii in the Presence of Saccharomyces cerevisiae Isolated From Water Kefir and Comparison With Lactobacillus hordei.

Water kefir is a slightly alcoholic and traditionally fermented beverage, which is prepared from sucrose, water, kefir grains, and dried or fresh fruits (e.g., figs). Lactobacillus (L.) nagelii, L. hordei, and Saccharomyces (S.) cerevisiae are predominant and stable lactic acid bacteria and yeasts, respectively, isolated from water kefir consortia. The growth of L. nagelii and L. hordei are improved in the presence of S. cerevisiae. In this work we demonstrate that quantitative comparative proteomics enables the investigation of interactions between LAB and yeast to predict real-time metabolic exchange in water kefir. It revealed 73 differentially expressed (DE) in L. nagelii TMW 1.1827 in the presence of S. cerevisiae. The presence of the yeast induced changes in the changes in the carbohydrate metabolism of L. nagelii and affected reactions involved in NAD+/NADH homeostasis. Furthermore, the DE enzymes involved in amino acid biosynthesis or catabolism predict that S. cerevisiae releases glutamine, histidine, methionine, and arginine, which are subsequently used by L. nagelii to ensure its survival in the water kefir consortium. In co-culture with S. cerevisiae, L. nagelii profits from riboflavin, most likely secreted by the yeast. The reaction of L. nagelii to the presence of S. cerevisiae differs from that one of the previously studied L. hordei, which displays 233 differentially expressed proteins, changes in citrate metabolism and an antidromic strategy for NAD+/NADH homeostasis. So far, aggregation promotion factors, i.e., formation of a specific glucan and bifunctional enzymes were only detected in L. hordei.


April 21, 2020

Comprehensive characterization of T-DNA integration induced chromosomal rearrangement in a birch T-DNA mutant.

Integration of T-DNA into plant genomes via Agrobacterium may interrupt gene structure and generate numerous mutants. The T-DNA caused mutants are valuable materials for understanding T-DNA integration model in plant research. T-DNA integration in plants is complex and still largely unknown. In this work, we reported that multiple T-DNA fragments caused chromosomal translocation and deletion in a birch (Betula platyphylla × B. pendula) T-DNA mutant yl.We performed PacBio genome resequencing for yl and the result revealed that two ends of a T-DNA can be integrated into plant genome independently because the two ends can be linked to different chromosomes and cause chromosomal translocation. We also found that these T-DNA were connected into tandem fragment regardless of direction before integrating into plant genome. In addition, the integration of T-DNA in yl genome also caused several chromosomal fragments deletion. We then summarized three cases for T-DNA integration model in the yl genome. (1) A T-DNA fragment is linked to the two ends of a double-stranded break (DSB); (2) Only one end of a T-DNA fragment is linked to a DSB; (3) A T-DNA fragment is linked to the ends of different DSBs. All the observations in the yl genome supported the DSB repair model.In this study, we showed a comprehensive genome analysis of a T-DNA mutant and provide a new insight into T-DNA integration in plants. These findings would be helpful for the analysis of T-DNA mutants with special phenotypes.


April 21, 2020

Illumina error correction near highly repetitive DNA regions improves de novo genome assembly.

Several standalone error correction tools have been proposed to correct sequencing errors in Illumina data in order to facilitate de novo genome assembly. However, in a recent survey, we showed that state-of-the-art assemblers often did not benefit from this pre-correction step. We found that many error correction tools introduce new errors in reads that overlap highly repetitive DNA regions such as low-complexity patterns or short homopolymers, ultimately leading to a more fragmented assembly.We propose BrownieCorrector, an error correction tool for Illumina sequencing data that focuses on the correction of only those reads that overlap short DNA patterns that are highly repetitive in the genome. BrownieCorrector extracts all reads that contain such a pattern and clusters them into different groups using a community detection algorithm that takes into account both the sequence similarity between overlapping reads and their respective paired-end reads. Each cluster holds reads that originate from the same genomic region and hence each cluster can be corrected individually, thus providing a consistent correction for all reads within that cluster.BrownieCorrector is benchmarked using six real Illumina datasets for different eukaryotic genomes. The prior use of BrownieCorrector improves assembly results over the use of uncorrected reads in all cases. In comparison with other error correction tools, BrownieCorrector leads to the best assembly results in most cases even though less than 2% of the reads within a dataset are corrected. Additionally, we investigate the impact of error correction on hybrid assembly where the corrected Illumina reads are supplemented with PacBio data. Our results confirm that BrownieCorrector improves the quality of hybrid genome assembly as well. BrownieCorrector is written in standard C++11 and released under GPL license. BrownieCorrector relies on multithreading to take advantage of multi-core/multi-CPU systems. The source code is available at https://github.com/biointec/browniecorrector .


April 21, 2020

A hybrid de novo genome assembly of the honeybee, Apis mellifera, with chromosome-length scaffolds.

The ability to generate long sequencing reads and access long-range linkage information is revolutionizing the quality and completeness of genome assemblies. Here we use a hybrid approach that combines data from four genome sequencing and mapping technologies to generate a new genome assembly of the honeybee Apis mellifera. We first generated contigs based on PacBio sequencing libraries, which were then merged with linked-read 10x Chromium data followed by scaffolding using a BioNano optical genome map and a Hi-C chromatin interaction map, complemented by a genetic linkage map.Each of the assembly steps reduced the number of gaps and incorporated a substantial amount of additional sequence into scaffolds. The new assembly (Amel_HAv3) is significantly more contiguous and complete than the previous one (Amel_4.5), based mainly on Sanger sequencing reads. N50 of contigs is 120-fold higher (5.381 Mbp compared to 0.053 Mbp) and we anchor >?98% of the sequence to chromosomes. All of the 16 chromosomes are represented as single scaffolds with an average of three sequence gaps per chromosome. The improvements are largely due to the inclusion of repetitive sequence that was unplaced in previous assemblies. In particular, our assembly is highly contiguous across centromeres and telomeres and includes hundreds of AvaI and AluI repeats associated with these features.The improved assembly will be of utility for refining gene models, studying genome function, mapping functional genetic variation, identification of structural variants, and comparative genomics.


April 21, 2020

ICESsuHN105, a Novel Multiple Antibiotic Resistant ICE in Streptococcus suis Serotype 5 Strain HN105.

Streptococcussuis serotype 5, an emerging zoonosis bacterial pathogen, has been isolated from infections in both pigs and humans. In this study, we sequenced the first complete genome of a virulent, multidrug-resistant SS5 strain HN105. The strain HN105 displayed enhanced pathogenicity in zebrafish and BABL/c mouse infection models. Comparative genome analysis identified a novel 80K integrative conjugative element (ICE), ICESsuHN105, as required for the multidrug resistance phenotype. Six corresponding antibiotic resistance genes in this ICE were identified, namely tet (O), tet (M), erm (two copies), aph, and spc. Phylogenetic analysis classified the element as a homolog of the ICESa2603 family, containing the typical family backbone and insertion DNA. DNA hybrids mediated by natural transformation between HN105 and ZY05719 verified the antibiotic resistant genes of ICESsuHN105 that could be transferred successfully, while they were dispersedly inserted with a single gene in different genomic locations of ZY05719(HN105) transformants. To further identify the horizontal transfer of ICESsuHN105 as a whole mobile genetic element, a circular intermediate form of ICESsuHN105 was detected by PCR. However, the effective conjugation using serotype 2 S. suis as recipients was not observed in current assays in vitro. Further studies confirmed the presence of the complete lantibiotic locus encoded in ICESsuHN105 that effectively inhibits the growth of other streptococci. In summary, this study demonstrated the presence of antibiotic resistance genes in ICE that are able to transfer between different clinical isolates and adapt to a broader range of Streptococcus serotype or species.


April 21, 2020

Comparative analysis of the chicken IFITM locus by targeted genome sequencing reveals evolution of the locus and positive selection in IFITM1 and IFITM3.

The interferon-induced transmembrane (IFITM) protein family comprises a class of restriction factors widely characterised in humans for their potent antiviral activity. Their biological activity is well documented in several animal species, but their genetic variation and biological mechanism is less well understood, particularly in avian species.Here we report the complete sequence of the domestic chicken Gallus gallus IFITM locus from a wide variety of chicken breeds to examine the detailed pattern of genetic variation of the locus on chromosome 5, including the flanking genes ATHL1 and B4GALNT4. We have generated chIFITM sequences from commercial breeds (supermarket-derived chicken breasts), indigenous chickens from Nigeria (Nsukka) and Ethiopia, European breeds and inbred chicken lines from the Pirbright Institute, totalling of 206 chickens. Through mapping of genetic variants to the latest chIFITM consensus sequence our data reveal that the chIFITM locus does not show structural variation in the locus across the populations analysed, despite spanning diverse breeds from different geographic locations. However, single nucleotide variants (SNVs) in functionally important regions of the proteins within certain groups of chickens were detected, in particular the European breeds and indigenous birds from Ethiopia and Nigeria. In addition, we also found that two out of four SNVs located in the chIFITM1 (Ser36 and Arg77) and chIFITM3 (Val103) proteins were simultaneously under positive selection.Together these data suggest that IFITM genetic variation may contribute to the capacities of different chicken populations to resist virus infection.


April 21, 2020

Large Plasmid Complement Resolved: Complete Genome Sequencing of Lactobacillus plantarum MF1298, a Candidate Probiotic Strain Associated with Unfavorable Effect.

Considerable attention has been given to the species Lactobacillus plantarum regarding its probiotic potential. L. plantarum strains have shown health benefits in several studies, and even nonstrain-specific claims are allowed in certain markets. L. plantarum strain MF1298 was considered a candidate probiotic, demonstrating in vitro probiotic properties and the ability to survive passage through the human intestinal tract. However, the strain showed an unfavorable effect on symptoms in subjects with irritable bowel syndrome in a clinical trial. The properties and the genome of this strain are thus of general interest. Obtaining the complete genome of strain MF1298 proved difficult due to its large plasmid complement. Here, we exploit a combination of sequencing approaches to obtain the complete chromosome and plasmid assemblies of MF1298. The Oxford Nanopore Technologies MinION long-read sequencer was particularly useful in resolving the unusually large number of plasmids in the strain, 14 in total. The complete genome sequence of 3,576,440 basepairs contains 3272 protein-encoding genes, of which 315 are located on plasmids. Few unique regions were found in comparison with other L. plantarum genomes. Notably, however, one of the plasmids contains genes related to vitamin B12 (cobalamin) turnover and genes encoding bacterial reverse transcriptases, features not previously reported for L. plantarum. The extensive plasmid information will be important for future studies with this strain.


April 21, 2020

Chromosome-level assembly of the water buffalo genome surpasses human and goat genomes in sequence contiguity.

Rapid innovation in sequencing technologies and improvement in assembly algorithms have enabled the creation of highly contiguous mammalian genomes. Here we report a chromosome-level assembly of the water buffalo (Bubalus bubalis) genome using single-molecule sequencing and chromatin conformation capture data. PacBio Sequel reads, with a mean length of 11.5?kb, helped to resolve repetitive elements and generate sequence contiguity. All five B. bubalis sub-metacentric chromosomes were correctly scaffolded with centromeres spanned. Although the index animal was partly inbred, 58% of the genome was haplotype-phased by FALCON-Unzip. This new reference genome improves the contig N50 of the previous short-read based buffalo assembly more than a thousand-fold and contains only 383 gaps. It surpasses the human and goat references in sequence contiguity and facilitates the annotation of hard to assemble gene clusters such as the major histocompatibility complex (MHC).


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