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September 22, 2019

Genomic Tandem Quadruplication is Associated with Ketoconazole Resistance in Malassezia pachydermatis.

Malassezia pachydermatis is a commensal yeast found on the skin of dogs. However, M. pachydermatis is also considered an opportunistic pathogen and is associated with various canine skin diseases including otitis externa and atopic dermatitis, which usually require treatment using an azole antifungal drug, such as ketoconazole. In this study, we isolated a ketoconazole-resistant strain of M. pachydermatis, designated “KCTC 27587,” from the external ear canal of a dog with otitis externa and analyzed its resistance mechanism. To understand the mechanism underlying ketoconazole resistance of the clinical isolate M. pachydermatis KCTC 27587, the whole genome of the yeast was sequenced using the PacBio platform and was compared with M. pachydermatis type strain CBS 1879. We found that a ~84-kb region in chromosome 4 of M. pachydermatis KCTC 27587 was tandemly quadruplicated. The quadruplicated region contains 52 protein coding genes, including the homologs of ERG4 and ERG11, whose overexpression is known to be associated with azole resistance. Our data suggest that the quadruplication of the ~84-kb region may be the cause of the ketoconazole resistance in M. pachydermatis KCTC 27587.

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September 22, 2019

Analysis of structural variants in four African cichlids highlights an association with developmental and immune related genes

African Lakes Cichlids are one of the most impressive example of adaptive radiation. Independently in Lake Victoria, Tanganyika, and Malawi, several hundreds of species arose within the last 10 million to 100,000 years. Whereas most analyses in cichlids focused on nucleotide substitutions across species to investigate the genetic bases of this explosive radiation, to date, no study has investigated the contribution of structural variants (SVs) to speciation events (through a reduction of gene flow) and adaptation to different ecological niches. Here, we annotate and characterize the repertoires and evolutionary potential of different SV classes (deletion, duplication, inversion, insertions and translocations) in five cichlid species (Astatotilapia burtoni, Metriaclima zebra, Neolamprologus brichardi, Pundamilia nyererei and Oreochromis niloticus). We investigate the patterns of gain/loss evolution across the phylogeny for each SV type enabling the identification of both lineage specific events and a set of conserved SVs, common to all four species in the radiation. Both deletion and inversion events show a significant overlap with SINE elements, while inversions additionally show a limited, but significant association with DNA transposons. Genes lying inside inverted regions are enriched for genes regulating behaviour, or involved in skeletal and visual system development. Moreover, we find that duplicated genes show enrichment for textquoterightantigen processing and presentationtextquoteright (GO:0019882) and other immune related categories. Altogether, we provide the first, comprehensive overview of rearrangement evolution in East African Cichlids, and some initial insights into their possible contribution to adaptation.

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September 22, 2019

Genome mining for fungal polyketide-diterpenoid hybrids: discovery of key terpene cyclases and multifunctional P450s for structural diversification

A biosynthetic gene cluster for chevalone E (1) and its oxidized derivatives have been identified within the genome of the endophytic fungus Aspergillus versicolor 0312, by a mining strategy targeting a polyke- tide-diterpenoid hybrid molecule. The biosynthetic pathway has been successfully reconstituted in the heterologous fungus Aspergillus oryzae. Interestingly, two P450 monooxygenases, Cle2 and Cle4, were found to transform 1 into seven new analogues including 7 and 8 that possess a unique five-membered lactone ring. Furthermore, the replacement of the terpene cyclase gene with that from another fungus led to the production of sartorypyrone D (11), which has a monocyclic terpenoid moiety. Finally, some of the compounds obtained in this study synergistically enhanced the cytotoxicity of doxorubicin (DOX) in breast cancer cells.

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September 22, 2019

Complete Genome Sequence of Massilia oculi sp. nov. CCUG 43427T (=DSM 26321T), the Type Strain of M. oculi, and Comparison with Genome Sequences of Other Massilia Strains.

Massilia oculi sp. nov. of type strain CCUG 43427T is a Gram-negative, rod-shaped, nonspore-forming bacterium, which was recently isolated from the eye of a patient suffering from endophthalmitis and was described as novel species in Massilia genus. In this study, we present the complete genome sequence of this strain by using Pacbio SMRT cell platform and compare this sequence with the genomes of 30 Massilia representative strains. Also, a comprehensive search was conducted for genes and proteins involved in antibiotic resistance and pathogenicity. The genome of CCUG 43427T is 5,844,653 bp with 65.55% GC content. This genome contains four prophages and four genomic islands (GIs). The cobalt/zinc/cadmium transporter locus CzcABCD is included in these GIs. This GI was predicted to play important role in bacterial heavy-metal tolerance. The in silico genome analysis also revealed that this strain contains a lot of antibiotic resistance and pathogenicity related genes. This result suggested that this strain may has evolved a wide arsenal of weapons for pathogenicity and survival. Genome comparison among CCUG 43427T and other 30 Massilia strains revealed that more than 400 genes are unique in CCUG 43427T. Among these, one gene cluster, which was annotated to be important for LOS biosynthesis, catalytic mechanism and the substrate specificity of the enzyme, was predicted to be horizontally transferred by using phylogenies and biased GC content.

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September 22, 2019

Genome sequences of two diploid wild relatives of cultivated sweetpotato reveal targets for genetic improvement

Sweetpotato [Ipomoea batatas (L.) Lam.] is a globally important staple food crop, especially for sub-Saharan Africa. Agronomic improvement of sweetpotato has lagged behind other major food crops due to a lack of genomic and genetic resources and inherent challenges in breeding a heterozygous, clonally propagated polyploid. Here, we report the genome sequences of its two diploid relatives, I. trifida and I. triloba, and show that these high-quality genome assemblies are robust references for hexaploid sweetpotato. Comparative and phylogenetic analyses reveal insights into the ancient whole-genome triplication history of Ipomoea and evolutionary relationships within the Batatas complex. Using resequencing data from 16 genotypes widely used in African breeding programs, genes and alleles associated with carotenoid biosynthesis in storage roots are identified, which may enable efficient breeding of varieties with high provitamin A content. These resources will facilitate genome-enabled breeding in this important food security crop.

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September 22, 2019

Molecular characteristics and comparative genomics analysis of a clinical Enterococcus casseliflavus with a resistance plasmid.

The aim of this work was to investigate the molecular characterization of a clinical Enterococcus casseliflavus strain with a resistance plasmid.En. casseliflavus EC369 was isolated from a patient in a hospital in southern China. The minimum inhibitory concentration was found by means of the agar dilution method to determine the antimicrobial susceptibilities of the strains. Whole-genome sequencing and comparative genomics analysis were performed to analyze the mechanism of antibiotic resistance and the horizontal gene transfer of the resistance gene-related mobile genetic elements.En. casseliflavus EC369 showed resistance to erythromycin, kanamycin, and streptomycin, but was susceptible to vancomycin, ampicillin, and streptothricin and other antimicrobials. There were six resistance genes (aph3′, ant6, bla, sat4, and two ermBs) carried by a transposon identified on the plasmid pEC369 and a complete resistance gene cluster of vancomycin and a tet (M) gene encoded on the chromosome. This is the first complete plasmid sequence reported in clinically isolated En. casseliflavus. The plasmid with the greatest sequence identity with pEC369 was the plasmid of Enterococcus sp. FDAARGOS_375, followed by the plasmids of Enterococcus faecium strains F12085 and pRE25, whereas the sequence with the greatest identity to the resistance genes carrying a transposon of pEC369 was on the chromosome of Staphylococcus aureus strain GD1677.The resistance profiles of En. casseliflavus EC369 might contribute to the resistance genes encoded on the plasmid. The fact that the most similar sequence to the transposon carrying resistance genes of pEC369 was encoded in the chromosome of a S. aureus strain provides insights into the mechanism of dissemination of multidrug resistance between bacteria of different species or genera through horizontal gene transfer.

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September 22, 2019

Genomic and metatranscriptomic analyses of Weissella koreensis reveal its metabolic and fermentative features during kimchi fermentation

The genomic and metabolic features of Weissella koreensis, one of the major lactic acid bacteria in kimchi, were investigated through genomic, metabolic, and transcriptomic analyses for the genomes of strains KCTC 3621T, KACC 15510, and WiKim0080. W. koreensis strains were intrinsically vancomycin-resistant and harbored potential hemolysin genes that were actively transcribed although no hemolysin activity was detected. KEGG and reconstructed fermentative metabolic pathways displayed that W. koreensis strains commonly employ the heterolactic pathway to produce d-lactate, ethanol, acetate, CO2, d-sorbitol, thiamine, and folate from various carbohydrates including d-glucose, d-mannose, d-lactose, l-malate, d-xylose, l-arabinose, d-ribose, N-acetyl-glucosamine, and gluconate, and strains KCTC 3621T and WiKim0080 additionally have metabolic pathways of d-galacturonate and d-glucoronate. Phenotypic analyses showed that all strains did not ferment d-galactose, probably due to the lack of d-galactose transporting system, and strains KCTC 3621T and WiKim0080 fermented d-fructose, indicating the presence of d-fructose transporting system. Fermentative features of W. koreensis were investigated through kimchi transcriptional analysis, suggesting that W. koreensis is mainly responsible for kimchi fermentation with the production of various fermentative metabolites during late fermentation period. This was the first study to investigate the genomic and metabolic features of W. koreensis, which may provide better understandings on kimchi fermentation.

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September 22, 2019

Extensive and deep sequencing of the Venter/HuRef genome for developing and benchmarking genome analysis tools.

We produced an extensive collection of deep re-sequencing datasets for the Venter/HuRef genome using the Illumina massively-parallel DNA sequencing platform. The original Venter genome sequence is a very-high quality phased assembly based on Sanger sequencing. Therefore, researchers developing novel computational tools for the analysis of human genome sequence variation for the dominant Illumina sequencing technology can test and hone their algorithms by making variant calls from these Venter/HuRef datasets and then immediately confirm the detected variants in the Sanger assembly, freeing them of the need for further experimental validation. This process also applies to implementing and benchmarking existing genome analysis pipelines. We prepared and sequenced 200?bp and 350?bp short-insert whole-genome sequencing libraries (sequenced to 100x and 40x genomic coverages respectively) as well as 2?kb, 5?kb, and 12?kb mate-pair libraries (49x, 122x, and 145x physical coverages respectively). Lastly, we produced a linked-read library (128x physical coverage) from which we also performed haplotype phasing.

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September 22, 2019

Emergence of pathogenic and multiple-antibiotic-resistant Macrococcus caseolyticus in commercial broiler chickens.

Macrococcus caseolyticus is generally considered to be a non-pathogenic bacterium that does not cause human or animal diseases. However, recently, a strain of M. caseolyticus (SDLY strain) that causes high mortality rates was isolated from commercial broiler chickens in China. The main pathological changes caused by SDLY included caseous exudation in cranial cavities, inflammatory infiltration, haemorrhages and multifocal necrosis in various organs. The whole genome of the SDLY strain was sequenced and was compared with that of the non-pathogenic JCSC5402 strain of M. caseolyticus. The results showed that the SDLY strain harboured a large quantity of mutations, antibiotic resistance genes and numerous insertions and deletions of virulence genes. In particular, among the inserted genes, there is a cluster of eight connected genes associated with the synthesis of capsular polysaccharide. This cluster encodes a transferase and capsular polysaccharide synthase, promotes the formation of capsules and causes changes in pathogenicity. Electron microscopy revealed a distinct capsule surrounding the SDLY strain. The pathogenicity test showed that the SDLY strain could cause significant clinical symptoms and pathological changes in both SPF chickens and mice. In addition, these clinical symptoms and pathological changes were the same as those observed in field cases. Furthermore, the anti-microbial susceptibility test demonstrated that the SDLY strain exhibits multiple-antibiotic resistance. The emergence of pathogenic M. caseolyticus indicates that more attention should be paid to the effects of this micro-organism on both poultry and public health.© 2018 Blackwell Verlag GmbH.

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September 22, 2019

Enterobacter cloacae Complex Sequence Type 171 Isolates Expressing KPC-4 Carbapenemase Recovered from Canine Patients in Ohio.

Companion animals are likely relevant in the transmission of antimicrobial-resistant bacteria. Enterobacter xiangfangensis sequence type 171 (ST171), a clone that has been implicated in clusters of infections in humans, was isolated from two dogs with clinical disease in Ohio. The canine isolates contained IncHI2 plasmids encoding blaKPC-4 Whole-genome sequencing was used to put the canine isolates in phylogenetic context with available human ST171 sequences, as well as to characterize their blaKPC-4 plasmids. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Quaternary ammonium compounds with multiple cationic moieties (multiQACs) provide antimicrobial activity against Campylobacter jejuni

Recently developed quaternary ammonium compounds (QACs) possessing multiple cationic moieties, referred to as multiQACs, were tested with strains of Campylobacter jejuni to determine their potential as antimicrobial compounds against this important foodborne pathogen. Eight multiQACs were tested against a cocktail of six C. jejuni strains isolated from environmental and clinical sources. The resulting reductions in C. jejuni numbers mediated by the multiQACs were compared to the reductions produced by the application of four commercially available QACs, each of which bears a single cation. Multiple concentrations and exposure times were utilized for all compounds. The compounds which yielded the maximum C. jejuni reductions at the lowest concentrations and applied over the shortest exposure times were judged to be the most successful. Of the eight multiQACs investigated, four demonstrated reductions in C. jejuni numbers superior to the commercial QACs; these four are biscationic, and two of them bear an additional uncharged nitrogen atom. The remaining four multiQACs, which contain three or four cations, did not produce reductions in bacterial numbers comparable to commercial QACs in the timeframes tested. At the intermediary compound concentration (0.05?mM) and exposure time (5?min) the most effective multiQACs (PQ-12,12 and 12(3)0(3)12) on average killed over 99% of the Campylobacter cells present while the best commercial compound at those parameters (cetyl pyridinium chloride, CPC) only killed on average 84.56% of the Campylobacter cells. At the highest compound concentration tested (0.1?mM) and shortest exposure time (1?min), the same two biscationic multiQACs averaged mean percent reductions of Campylobacter cell numbers around 99.5% while CPC at the same concentration/exposure only managed a percent reduction of 91.3%. The biscationic multiQACs demonstrate the potential for providing a new group of antimicrobial compounds superior to current commercially available QACs in their effectiveness against C. jejuni.

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September 22, 2019

DNA Methylation by Restriction Modification Systems Affects the Global Transcriptome Profile in Borrelia burgdorferi.

Prokaryote restriction modification (RM) systems serve to protect bacteria from potentially detrimental foreign DNA. Recent evidence suggests that DNA methylation by the methyltransferase (MTase) components of RM systems can also have effects on transcriptome profiles. The type strain of the causative agent of Lyme disease, Borrelia burgdorferi B31, possesses two RM systems with N6-methyladenosine (m6A) MTase activity, which are encoded by the bbe02 gene located on linear plasmid lp25 and bbq67 on lp56. The specific recognition and/or methylation sequences had not been identified for either of these B. burgdorferi MTases, and it was not previously known whether these RM systems influence transcript levels. In the current study, single-molecule real-time sequencing was utilized to map genome-wide m6A sites and to identify consensus modified motifs in wild-type B. burgdorferi as well as MTase mutants lacking either the bbe02 gene alone or both bbe02 and bbq67 genes. Four novel conserved m6A motifs were identified and were fully attributable to the presence of specific MTases. Whole-genome transcriptome changes were observed in conjunction with the loss of MTase enzymes, indicating that DNA methylation by the RM systems has effects on gene expression. Genes with altered transcription in MTase mutants include those involved in vertebrate host colonization (e.g., rpoS regulon) and acquisition by/transmission from the tick vector (e.g., rrp1 and pdeB). The results of this study provide a comprehensive view of the DNA methylation pattern in B. burgdorferi, and the accompanying gene expression profiles add to the emerging body of research on RM systems and gene regulation in bacteria.IMPORTANCE Lyme disease is the most prevalent vector-borne disease in North America and is classified by the Centers for Disease Control and Prevention (CDC) as an emerging infectious disease with an expanding geographical area of occurrence. Previous studies have shown that the causative bacterium, Borrelia burgdorferi, methylates its genome using restriction modification systems that enable the distinction from foreign DNA. Although much research has focused on the regulation of gene expression in B. burgdorferi, the effect of DNA methylation on gene regulation has not been evaluated. The current study characterizes the patterns of DNA methylation by restriction modification systems in B. burgdorferi and evaluates the resulting effects on gene regulation in this important pathogen. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

The Genome of Opium Poppy Reveals Evolutionary History of Morphinan Pathway.

Plants, as primary producers, have been playing an indispensable role in other organisms’ survival and the balance of whole ecosystem on Earth. Especially, they provide the main source of energy, food, and medicine for human beings, some of which are derived from the primary or secondary metabolites [1]. Angiosperms, with more than 300,000 species on Earth, are the largest group of land plants by far. Most agricultural crops, fruits, ornamental plants, and medicinal herbs belong to this group. The medicinal herbs are usually rich in specialized metabolites that could provide safe and valuable resources for pharmaceutical development.

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September 22, 2019

Cloning and characterization of short-chain N-acyl homoserine lactone-producing Enterobacter asburiae strain L1 from lettuce leaves.

In gram-negative bacteria, bacterial communication or quorum sensing (QS) is achieved using common signaling molecules known as N-acyl homoserine lactones (AHL). We have previously reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. In silico analysis of the strain L1 genome revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easIR. In this work, the 639 bp luxI homolog, encoding 212 amino acids, have been cloned and overexpressed in Escherichia coli BL21 (DE3)pLysS. The purified protein (~25 kDa) shares high similarity to several members of the LuxI family among different E asburiae strains. Our findings showed that the heterologously expressed EasI protein has activated violacein production by AHL biosensor Chromobacterium violaceum CV026 as the wild-type E. asburiae. The mass spectrometry analysis showed the production of N-butanoyl homoserine lactone and N-hexanoyl homoserine lactone from induced E. coli harboring the recombinant EasI, suggesting that EasI is a functional AHL synthase. E. asburiae strain L1 was also shown to possess biofilm-forming characteristic activity using crystal violet binding assay. This is the first report on cloning and characterization of the luxI homolog from E. asburiae.© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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September 22, 2019

Complete genome sequence of Leuconostoc citreum EFEL2700, a host strain for transformation of pCB vectors.

Leuconostoc citreum is an important lactic acid bacterium used as a starter culture for producing kimchi, the traditional Korean fermented vegetables. An efficient host strain for plasmid transformation, L. citreum EFEL2700, was isolated from kimchi, and it has been frequently used for genetic engineering of L. citreum. In this study, we report the whole genome sequence of the strain and its genetic characteristics. Genome assembly yielded 5 contigs (1 chromosome and 4 plasmids), and the complete genome contained 1,923,830 base pairs (bp) with a G?+?C content of 39.0%. Average nucleotide identity analysis showed high homology (= 99%) to the reference strain L. citreum KM 20. The smallest plasmid (4.3 kbp) was used as an Escherichia coli shuttle vector (pCB) for heterologous gene expression, and L. citreum EFEL2700 showed the highest transformation efficiency, 6.7?×?104 CFU µg-1 DNA. Genetic analysis of the genome enabled the construction of primary metabolic pathway showing a typical hetero-type lactic acid fermentation. Notably, no core genes for primary metabolism were observed in plasmid 4 and it could be eliminated to create an efficient host for gene transformation. This report will facilitate the understanding and application of L. citreum EFEL2700 as a food-grade microbial cell factory.Copyright © 2018. Published by Elsevier B.V.

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