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Asset Tag: Whole Genome Sequencing,

April 21, 2020

Complete Nucleotide Sequences of mcr-4.3-Carrying Plasmids in Acinetobacter baumannii Sequence Type 345 of Human and Food Origin from the Czech Republic, the First Case in Europe.

Here, we describe two plasmids carrying mcr-4.3 in two Acinetobacter baumannii strains isolated from imported food and a clinical sample. The comparative analysis of these plasmids, with two other plasmids reported in the NCBI database, highlighted the common origin of the plasmidic structure carrying mcr-4.3 This is the first case of the mcr-4.3 gene in a A. baumannii strain isolated from a clinical case in Europe. We hypothesize that food import is initiating the spread in Czech Republic.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Streptococcus oralis subsp. dentisani Produces Monolateral Serine-Rich Repeat Protein Fibrils, One of Which Contributes to Saliva Binding via Sialic Acid.

Our studies reveal that the oral colonizer and cause of infective endocarditis Streptococcus oralis subsp. dentisani displays a striking monolateral distribution of surface fibrils. Furthermore, our data suggest that these fibrils impact the structure of adherent bacterial chains. Mutagenesis studies indicate that these fibrils are dependent on three serine-rich repeat proteins (SRRPs), here named fibril-associated protein A (FapA), FapB, and FapC, and that each SRRP forms a different fibril with a distinct distribution. SRRPs are a family of bacterial adhesins that have diverse roles in adhesion and that can bind to different receptors through modular nonrepeat region domains. Amino acid sequence and predicted structural similarity searches using the nonrepeat regions suggested that FapA may contribute to interspecies interactions, that FapA and FapB may contribute to intraspecies interactions, and that FapC may contribute to sialic acid binding. We demonstrate that a fapC mutant was significantly reduced in binding to saliva. We confirmed a role for FapC in sialic acid binding by demonstrating that the parental strain was significantly reduced in adhesion upon addition of a recombinantly expressed, sialic acid-specific, carbohydrate binding module, while the fapC mutant was not reduced. However, mutation of a residue previously shown to be essential for sialic acid binding did not decrease bacterial adhesion, leaving the precise mechanism of FapC-mediated adhesion to sialic acid to be defined. We also demonstrate that the presence of any one of the SRRPs is sufficient for efficient biofilm formation. Similar structures were observed on all infective endocarditis isolates examined, suggesting that this distribution is a conserved feature of this S. oralis subspecies.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Advantage of the F2:A1:B- IncF Pandemic Plasmid over IncC Plasmids in In Vitro Acquisition and Evolution of blaCTX-M Gene-Bearing Plasmids in Escherichia coli.

Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum ß-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2:A1:B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25b:H4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2:A1:B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed a significant fitness cost to the bacterial host immediately after conjugation, we show, using an experimental-evolution approach, that a negative impact on the fitness of the host strain was maintained throughout 1,120 generations with the IncC-IncR plasmid, regardless of the presence or absence of cefotaxime, in contrast to the F2:A1:B- IncF plasmid, whose cost was alleviated. Many chromosomal and plasmid rearrangements were detected after conjugation in transconjugants carrying the IncC plasmids but not in transconjugants carrying the F2:A1:B- IncF plasmid, except for insertion sequence (IS) mobilization from the fliM gene leading to the restoration of motility of the recipient strains. Only a few mutations occurred on the chromosome of each transconjugant throughout the experimental-evolution assay. Our findings indicate that the F2:A1:B- IncF CTX-M-15 plasmid is well adapted to the E. coli strain studied, contrary to the IncC-IncR CTX-M-15 plasmid, and that such plasmid-host adaptation could participate in the evolutionary success of the CTX-M-15-producing pandemic E. coli ST131-O25b:H4 lineage.Copyright © 2019 Mahérault et al.

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April 21, 2020

Light Modulates the Physiology of Nonphototrophic Actinobacteria.

Light is a source of energy and an environmental cue that is available in excess in most surface environments. In prokaryotic systems, conversion of light to energy by photoautotrophs and photoheterotrophs is well understood, but the conversion of light to information and the cellular response to that information have been characterized in only a few species. Our goal was to explore the response of freshwater Actinobacteria, which are ubiquitous in illuminated aquatic environments, to light. We found that Actinobacteria without functional photosystems grow faster in the light, likely because sugar transport and metabolism are upregulated in the light. Based on the action spectrum of the growth effect and comparisons of the genomes of three Actinobacteria with this growth rate phenotype, we propose that the photosensor in these strains is a putative CryB-type cryptochrome. The ability to sense light and upregulate carbohydrate transport during the day could allow these cells to coordinate their time of maximum organic carbon uptake with the time of maximum organic carbon release by primary producers.IMPORTANCE Sunlight provides information about both place and time. In sunlit aquatic environments, primary producers release organic carbon and nitrogen along with other growth factors during the day. The ability of Actinobacteria to coordinate organic carbon uptake and utilization with production of photosynthate enables them to grow more efficiently in the daytime, and it potentially gives them a competitive advantage over heterotrophs that constitutively produce carbohydrate transporters, which is energetically costly, or produce transporters only after detection of the substrate(s), which delays their response. Understanding how light cues the transport of organic carbon and its conversion to biomass is key to understanding biochemical mechanisms within the carbon cycle, the fluxes through it, and the variety of mechanisms by which light enhances growth.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Hybrid de novo genome assembly of Chinese chestnut (Castanea mollissima).

The Chinese chestnut (Castanea mollissima) is widely cultivated in China for nut production. This plant also plays an important ecological role in afforestation and ecosystem services. To facilitate and expand the use of C. mollissima for breeding and its genetic improvement, we report here the whole-genome sequence of C. mollissima.We produced a high-quality assembly of the C. mollissima genome using Pacific Biosciences single-molecule sequencing. The final draft genome is ~785.53 Mb long, with a contig N50 size of 944 kb, and we further annotated 36,479 protein-coding genes in the genome. Phylogenetic analysis showed that C. mollissima diverged from Quercus robur, a member of the Fagaceae family, ~13.62 million years ago.The high-quality whole-genome assembly of C. mollissima will be a valuable resource for further genetic improvement and breeding for disease resistance and nut quality. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

Genome sequence of the Chinese white wax scale insect Ericerus pela: the first draft genome for the Coccidae family of scale insects.

The Chinese white wax scale insect, Ericerus pela, is best known for producing wax, which has been widely used in candle production, casting, Chinese medicine, and wax printing products for thousands of years. The secretion of wax, and other unusual features of scale insects, is thought to be an adaptation to their change from an ancestral ground-dwelling lifestyle to a sedentary lifestyle on the higher parts of plants. As well as helping to improve its economic value, studies of E. pela might also help to explain the adaptation of scale insects. However, no genomic data are currently available for E. pela.To assemble the E. pela genome, 303.92 Gb of data were generated using Illumina and Pacific Biosciences sequencing, producing 277.22 Gb of clean data for assembly. The assembled genome size was 0.66 Gb, with 1,979 scaffolds and a scaffold N50 of 735 kb. The guanine + cytosine content was 33.80%. A total of 12,022 protein-coding genes were predicted, with a mean coding sequence length of 1,370 bp. Twenty-six fatty acyl-CoA reductase genes and 35 acyltransferase genes were identified. Evolutionary analysis revealed that E. pela and aphids formed a sister group and split ~241.1 million years ago. There were 214 expanded gene families and 2,219 contracted gene families in E. pela.We present the first genome sequence from the Coccidae family. These results will help to increase our understanding of the evolution of unique features in scale insects, and provide important genetic information for further research. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

Whole Genome Sequencing of the Giant Grouper (Epinephelus lanceolatus) and High-Throughput Screening of Putative Antimicrobial Peptide Genes.

Giant groupers, the largest grouper type in the world, are of economic importance in marine aquaculture for their rapid growth. At the same time, bacterial and viral diseases have become the main threats to the grouper industry. Here, we report a high-quality genome of a giant grouper sequenced by an Illumina HiSeq X-Ten and PacBio Bioscience Sequel platform. A total of 254 putative antimicrobial peptide (AMP) genes were identified, which can be divided into 34 classes according to the annotation of the Antimicrobial Peptides Database (APD3). Their locations in pseudochromosomes were also determined. Thrombin-, lectin-, and scolopendin-derived putative AMPs were the three largest parts. In addition, expressions of putative AMPs were measured by our transcriptome data. Two putative AMP genes (gapdh1 and gapdh2) were involved in glycolysis, which had extremely high expression levels in giant grouper muscle. As it has been reported that AMPs inhibit the growth of a broad spectrum of microbes and participate in regulating innate and adaptive immune responses, genome sequencing of this study provides a comprehensive cataloging of putative AMPs of groupers, supporting antimicrobial research and aquaculture therapy. These genomic resources will be beneficial to further molecular breeding of this economically important fish.

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April 21, 2020

Salmonella Genomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance of Salmonella enterica.

Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81?kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genes pheV or pheR The length of the target site was 52 or 55?bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Tn6674 Is a Novel Enterococcal optrA-Carrying Multiresistance Transposon of the Tn554 Family.

The novel 12,932-bp nonconjugative multiresistance transposon Tn6674 was identified in the chromosomal DNA of a porcine Enterococcus faecalis strain. Tn6674 belongs to the Tn554 family of transposons. It shares the same arrangement of the transposase genes tnpA, tnpB, and tnpC with Tn554 However, in addition to the Tn554-associated resistance genes spc and erm(A), Tn6674 harbored the resistance genes fexA and optrA Circular forms of Tn6674 were detected and suggest the functional activity of this transposon.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Outcomes and characterization of chromosomal self-targeting by native CRISPR-Cas systems in Streptococcus thermophilus.

CRISPR-Cas systems provide adaptive immunity against phages in prokaryotes via DNA-encoded, RNA-mediated, nuclease-dependent targeting and cleavage. Due to inefficient and relatively limited DNA repair pathways in bacteria, CRISPR-Cas systems can be repurposed for lethal DNA targeting that selects for sequence variants. In this study, the relative killing efficiencies of endogenous Type I and Type II CRISPR-Cas systems in the model organism Streptococcus thermophilus DGCC7710 were assessed. Additionally, the genetic and phenotypic outcomes of chromosomal targeting by plasmid-programmed Type I-E or Type II-A systems were analyzed. Efficient killing was observed using both systems, in a dose-dependent manner when delivering 0.4-400 ng of plasmid DNA. Targeted PCR screening and genome sequencing were used to determine the genetic basis enabling survival, showing that evasion of Type I-E self-targeting was primarily the result of low-frequency defective plasmids that excised the targeting spacer. The most notable genotype recovered from Type II-A targeting of genomic locus, lacZ, was a 34 kb-deletion derived from homologous recombination (HR) between identical conserved sequences in two separate galE coding regions, resulting in 2% loss of the genome. Collectively, these results suggest that HR contributes to the plasticity and remodeling of bacterial genomes, leading to evasion of genome targeting by CRISPR-Cas systems. © FEMS 2019.

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April 21, 2020

Complete Genome Sequence of the Silicimonas algicola Type Strain, a Representative of the Marine Roseobacter Group Isolated from the Cell Surface of the Marine Diatom Thalassiosira delicatula.

Silicimonas algicola strain KC90BT is an alphaproteobacterium of the Roseobacter clade that was isolated from a culture of the marine diatom Thalassiosira delicatula. Here, we report the complete genome sequence of this type strain, which is 4,351,658?bp in size with 4,272 coding sequences and an average G+C content of 65.2%.

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April 21, 2020

A chromosomal-level genome assembly for the insect vector for Chagas disease, Triatoma rubrofasciata.

Triatoma rubrofasciata is a widespread pathogen vector for Chagas disease, an illness that affects approximately 7 million people worldwide. Despite its importance to human health, its evolutionary origin has not been conclusively determined. A reference genome for T. rubrofasciata is not yet available.We have sequenced the genome of a female individual with T. rubrofasciatausing a single molecular DNA sequencing technology (i.e., PacBio Sequel platform) and have successfully reconstructed a whole-genome (680-Mb) assembly that covers 90% of the nuclear genome (757 Mb). Through Hi-C analysis, we have reconstructed full-length chromosomes of this female individual that has 13 unique chromosomes (2n = 24 = 22 + X1 + X2) with a contig N50 of 2.72 Mb and a scaffold N50 of 50.7 Mb. This genome has achieved a high base-level accuracy of 99.99%. This platinum-grade genome assembly has 12,691 annotated protein-coding genes. More than 95.1% of BUSCO genes were single-copy completed, indicating a high level of completeness of the genome.The platinum-grade genome assembly and its annotation provide valuable information for future in-depth comparative genomics studies, including sexual determination analysis in T. rubrofasciata and the pathogenesis of Chagas disease. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

A chromosome-level draft genome of the grain aphid Sitobion miscanthi.

Sitobion miscanthi is an ideal model for studying host plant specificity, parthenogenesis-based phenotypic plasticity, and interactions between insects and other species of various trophic levels, such as viruses, bacteria, plants, and natural enemies. However, the genome information for this species has not yet to be sequenced and published. Here, we analyzed the entire genome of a parthenogenetic female aphid colony using Pacific Biosciences long-read sequencing and Hi-C data to generate chromosome-length scaffolds and a highly contiguous genome assembly.The final draft genome assembly from 33.88 Gb of raw data was ~397.90 Mb in size, with a 2.05 Mb contig N50. Nine chromosomes were further assembled based on Hi-C data to a 377.19 Mb final size with a 36.26 Mb scaffold N50. The identified repeat sequences accounted for 26.41% of the genome, and 16,006 protein-coding genes were annotated. According to the phylogenetic analysis, S. miscanthi is closely related to Acyrthosiphon pisum, with S. miscanthi diverging from their common ancestor ~25.0-44.9 million years ago.We generated a high-quality draft of the S. miscanthi genome. This genome assembly should help promote research on the lifestyle and feeding specificity of aphids and their interactions with each other and species at other trophic levels. It can serve as a resource for accelerating genome-assisted improvements in insecticide-resistant management and environmentally safe aphid management. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

Intercellular Transfer of Chromosomal Antimicrobial Resistance Genes between Acinetobacter baumannii Strains Mediated by Prophages.

The spread of antimicrobial resistance genes (ARGs) among Gram-negative pathogens, including Acinetobacter baumannii, is primarily mediated by transferable plasmids; however, ARGs are frequently integrated into its chromosome. How ARG gets horizontally incorporated into the chromosome of A. baumannii, and whether it functions as a cause for further spread of ARG, remains unknown. Here, we demonstrated intercellular prophage-mediated transfer of chromosomal ARGs without direct cell-cell interaction in A. baumannii We prepared ARG-harboring extracellular DNA (eDNA) components from the culture supernatant of a multidrug-resistant (MDR) A. baumannii NU-60 strain and exposed an antimicrobial-susceptible (AS) A. baumannii ATCC 17978 strain to the eDNA components. The antimicrobial-resistant (AR) A. baumannii ATCC 17978 derivatives appeared to acquire various ARGs, originating from dispersed loci of the MDR A. baumannii chromosome, along with their surrounding regions, by homologous recombination, with the ARGs including armA (aminoglycoside resistance), blaTEM-1 (ß-lactam resistance), tet(B) (tetracycline resistance), and gyrA-81L (nalidixic acid resistance) genes. Notably, the eDNAs conferring antimicrobial resistance were enveloped in specific capsid proteins consisting of phage particles, thereby protecting the eDNAs from detergent and DNase treatments. The phages containing ARGs were likely released into the extracellular space from MDR A. baumannii, thereby transducing ARGs into AS A. baumannii, resulting in the acquisition of AR properties by the recipient. We concluded that the generalized transduction, in which phages were capable of carrying random pieces of A. baumannii genomic DNAs, enabled efficacious intercellular transfer of chromosomal ARGs between A. baumannii strains without direct cell-cell interaction. Copyright © 2019 American Society for Microbiology.

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April 21, 2020

The blaNDM-1-Carrying IncA/C2 Plasmid Underlies Structural Alterations and Cointegrate Formation In Vivo.

In 2012, a carbapenemase-producing Salmonella enterica serovar Corvallis isolate carrying a blaNDM-1 multiresistance IncA/C2 plasmid, apart from IncHI2 and ColE-like plasmids, was detected in a wild bird in Germany. In a recent broiler chicken infection study, we observed transfer of this blaNDM-1-carrying IncA/C2 plasmid to other Enterobacteriaceae Here, we focused on the stability of this plasmid and gained insight into the type and frequency of its structural alterations after an in vivo passage in a broiler chicken infection study.Copyright © 2019 Hadziabdic et al.

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