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September 22, 2019

Complete nucleotide sequences of two KPC-2-encoding plasmids from the same Citrobacter freundii isolate.

Large amounts of antibiotics are released from humans and animals into aquatic environments and lead to an increased abundance of environmental MDR bacteria, which pose a potential threat to public health. It is worrisome that the entry of carbapenemase-producing Enterobacteriaceae (CPE) into the environment is increasingly reported; these carbapenem-resistant bacteria pose a severe health threat as few therapeutic options are available for such pathogens. Although culture-independent approaches are capable of revealing the vast genetic diversity of the environmental resistome, there are few data regarding deeper characterization of mechanisms of environmental CPE isolates. Here, we describe the complete sequences of two blaKPC-2-containing plasmids present in the same Citrobacter freundii isolated from river sediment.

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September 22, 2019

Functional characterization of the mucus barrier on the Xenopus tropicalis skin surface.

Mucosal surfaces represent critical routes for entry and exit of pathogens. As such, animals have evolved strategies to combat infection at these sites, in particular the production of mucus to prevent attachment and to promote subsequent movement of the mucus/microbe away from the underlying epithelial surface. Using biochemical, biophysical, and infection studies, we have investigated the host protective properties of the skin mucus barrier of the Xenopus tropicalis tadpole. Specifically, we have characterized the major structural component of the barrier and shown that it is a mucin glycoprotein (Otogelin-like or Otogl) with similar sequence, domain organization, and structural properties to human gel-forming mucins. This mucin forms the structural basis of a surface barrier (~6 µm thick), which is depleted through knockdown of Otogl. Crucially, Otogl knockdown leads to susceptibility to infection by the opportunistic pathogen Aeromonas hydrophila To more accurately reflect its structure, tissue localization, and function, we have renamed Otogl as Xenopus Skin Mucin, or MucXS. Our findings characterize an accessible and tractable model system to define mucus barrier function and host-microbe interactions. Copyright © 2018 the Author(s). Published by PNAS.

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September 22, 2019

Genomic diversity in the endosymbiotic bacterium Rhizobium leguminosarum.

Rhizobium leguminosarum bv. viciae is a soil a-proteobacterium that establishes a diazotrophic symbiosis with different legumes of the Fabeae tribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome of R. leguminosarum bv. viciae UPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains of R. leguminosarum bv. viciae, 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.

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September 22, 2019

Multi-heme cytochromes provide a pathway for survival in energy-limited environments.

Bacterial reduction of oxidized sulfur species (OSS) is critical for energy production in anaerobic marine subsurfaces. In organic-poor sediments, H2 has been considered as a major energy source for bacterial respiration. We identified outer-membrane cytochromes (OMCs) that are broadly conserved in sediment OSS-respiring bacteria and enable cells to directly use electrons from insoluble minerals via extracellular electron transport. Biochemical, transcriptomic, and microscopic analyses revealed that the identified OMCs were highly expressed on the surface of cells and nanofilaments in response to electron donor limitation. This electron uptake mechanism provides sufficient but minimum energy to drive the reduction of sulfate and other OSS. These results suggest a widespread mechanism for survival of OSS-respiring bacteria via electron uptake from solid minerals in energy-poor marine sediments.

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September 22, 2019

High-quality assembly of Dermatophagoides pteronyssinus genome and transcriptome reveals a wide range of novel allergens.

House dust mites (HDM) are a predominant source of inhalant allergens that attribute to over 50% of worldwide allergy cases, while the full spectrum of HDM allergens remains unknown. Here we sequenced a high-quality genome of Dermatophagoides (D.) pteronyssinus to find known canonical allergens and allergen orthologs inferred from D. farinae genome.

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September 22, 2019

Aberration or analogy? The atypical plastomes of Geraniaceae

A number of plant groups have been proposed as ideal systems to explore plastid inheritance, plastome evolution and plastome-nuclear genome coevolution. Quick generation times and a compact nuclear genome in Arabidopsis thaliana, the relative ease of plastid isolation from Spinacia oleracea and the tractability of plastid transformation in Nicotiana tabacum are all desirable attributes in a model system; however, these and most other groups all lack novelty in terms of plastome structure and nucleotide sequence evolution. Contemporary sequencing and assembly technologies have facilitated analyses of atypical plastomes and, as predicted by early investigations, Geraniaceae plastomes have experienced unprecedented rearrangements relative to the canonical structure and exhibit remarkably high rates of synonymous and nonsynonymous nucleotide substitutions. While not the only lineage with unusual plastome features, likely no other group represents the array of aberrant phenomena recorded for the family. In this chapter, Geraniaceae plastomes will be discussed and, where possible, compared with other taxa.

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September 22, 2019

Characterization of the SN35N strain-specific exopolysaccharide encoded in the whole circular genome of a plant-derived Lactobacillus plantarum.

Lactobacillus plantarum SN35N, which has been previously isolated from pear, secretes exopolysaccharide (EPS). The aim of the present study is to characterize the EPS chemically and to find the EPS-biosynthesizing gene cluster. The present study demonstrates that the strain produces an acidic EPS carrying phosphate residue, which is composed of glucose, galactose, and mannose at a molecular ratio of 15.0?:?5.7?:?1.0. We also show that acidic EPS strongly inhibits the catalytic activity of hyaluronidase (EC 3.2.1.35), promoting an inflammatory reaction. In the present study, we also determined the complete genome sequence of the SN35N strain, demonstrating that the genome is a circular DNA with 3267626?bp, and the number of predicted coding genes is 3146, with a GC content of 44.51%. In addition, the strain harbors four plasmids, designated pSN35N-1, -2, -3, and -4. Although four EPS-biosynthesizing genes, designated lpe1, lpe2, lpe3, and lpe4, are present in the SN35N chromosomal DNA, another EPS gene cluster, lpe5, is located in the pSN35N-3 plasmid, composed of 35425?bp. EPS low-producing mutants, which were obtained by treating SN35N cells with novobiocin, lost the lpe5 gene cluster in the plasmid-curing experiment, suggesting that the gene cluster for the biosynthesis of acidic EPS is present in the plasmid. The present study shows the chemical characterization of the acidic EPS and its inhibitory effect to the hyaluronidase.

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September 22, 2019

Re-classification of Clavibacter michiganensis subspecies on the basis of whole-genome and multi-locus sequence analyses.

Although the genus Clavibacter was originally proposed to accommodate all phytopathogenic coryneform bacteria containing B2? diaminobutyrate in the peptidoglycan, reclassification of all but one species into other genera has resulted in the current monospecific status of the genus. The single species in the genus, Clavibacter michiganensis, has multiple subspecies, which are all highly host-specific plant pathogens. Whole genome analysis based on average nucleotide identity and digital DNA-DNA hybridization as well as multi-locus sequence analysis (MLSA) of seven housekeeping genes support raising each of the C. michiganensis subspecies to species status. On the basis of whole genome and MLSA data, we propose the establishment of two new species and three new combinations: Clavibacter capsici sp. nov., comb. nov. and Clavibacter tessellarius sp. nov., comb. nov., and Clavibacter insidiosus comb. nov., Clavibacter nebraskensis comb. nov. and Clavibacter sepedonicus comb. nov.

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September 22, 2019

Whole-genome-sequencing characterization of bloodstream infection-causing hypervirulent Klebsiella pneumoniae of capsular serotype K2 and ST374.

Hypervirulent K. pneumoniae variants (hvKP) have been increasingly reported worldwide, causing metastasis of severe infections such as liver abscesses and bacteremia. The capsular serotype K2 hvKP strains show diverse multi-locus sequence types (MLSTs), but with limited genetics and virulence information. In this study, we report a hypermucoviscous K. pneumoniae strain, RJF293, isolated from a human bloodstream sample in a Chinese hospital. It caused a metastatic infection and fatal septic shock in a critical patient. The microbiological features and genetic background were investigated with multiple approaches. The Strain RJF293 was determined to be multilocis sequence type (ST) 374 and serotype K2, displayed a median lethal dose (LD50) of 1.5 × 102 CFU in BALB/c mice and was as virulent as the ST23 K1 serotype hvKP strain NTUH-K2044 in a mouse lethality assay. Whole genome sequencing revealed that the RJF293 genome codes for 32 putative virulence factors and exhibits a unique presence/absence pattern in comparison to the other 105 completely sequenced K. pneumoniae genomes. Whole genome SNP-based phylogenetic analysis revealed that strain RJF293 formed a single clade, distant from those containing either ST66 or ST86 hvKP. Compared to the other sequenced hvKP chromosomes, RJF293 contains several strain-variable regions, including one prophage, one ICEKp1 family integrative and conjugative element and six large genomic islands. The sequencing of the first complete genome of an ST374 K2 hvKP clinical strain should reinforce our understanding of the epidemiology and virulence mechanisms of this bloodstream infection-causing hvKP with clinical significance.

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September 22, 2019

Comparative genomic analyses reveal the features for adaptation to nematodes in fungi.

Nematophagous (NP) fungi are ecologically important components of the soil microbiome in natural ecosystems. Esteya vermicola (Ev) has been reported as a NP fungus with a poorly understood evolutionary history and mechanism of adaptation to parasitism. Furthermore, NP fungal genomic basis of lifestyle was still unclear. We sequenced and annotated the Ev genome (34.2 Mbp) and integrated genetic makeup and evolution of pathogenic genes to investigate NP fungi. The results revealed that NP fungi had some abundant pathogenic genes corresponding to their niche. A number of gene families involved in pathogenicity were expanded, and some pathogenic orthologous genes underwent positive selection. NP fungi with diverse morphological features exhibit similarities of evolutionary convergence in attacking nematodes, but their genetic makeup and microscopic mechanism are different. Endoparasitic NP fungi showed similarity in large number of transporters and secondary metabolite coding genes. Noteworthy, expanded families of transporters and endo-beta-glucanase implied great genetic potential of Ev in quickly perturbing nematode metabolism and parasitic behavior. These results facilitate our understanding of NP fungal genomic features for adaptation to nematodes and lay a solid theoretical foundation for further research and application.© The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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September 22, 2019

The genome sequence of the commercially cultivated mushroom Agrocybe aegerita reveals a conserved repertoire of fruiting-related genes and a versatile suite of biopolymer-degrading enzymes.

Agrocybe aegerita is an agaricomycete fungus with typical mushroom features, which is commercially cultivated for its culinary use. In nature, it is a saprotrophic or facultative pathogenic fungus causing a white-rot of hardwood in forests of warm and mild climate. The ease of cultivation and fructification on solidified media as well as its archetypal mushroom fruit body morphology render A. aegerita a well-suited model for investigating mushroom developmental biology.Here, the genome of the species is reported and analysed with respect to carbohydrate active genes and genes known to play a role during fruit body formation. In terms of fruit body development, our analyses revealed a conserved repertoire of fruiting-related genes, which corresponds well to the archetypal fruit body morphology of this mushroom. For some genes involved in fruit body formation, paralogisation was observed, but not all fruit body maturation-associated genes known from other agaricomycetes seem to be conserved in the genome sequence of A. aegerita. In terms of lytic enzymes, our analyses suggest a versatile arsenal of biopolymer-degrading enzymes that likely account for the flexible life style of this species. Regarding the amount of genes encoding CAZymes relevant for lignin degradation, A. aegerita shows more similarity to white-rot fungi than to litter decomposers, including 18 genes coding for unspecific peroxygenases and three dye-decolourising peroxidase genes expanding its lignocellulolytic machinery.The genome resource will be useful for developing strategies towards genetic manipulation of A. aegerita, which will subsequently allow functional genetics approaches to elucidate fundamentals of fruiting and vegetative growth including lignocellulolysis.

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September 22, 2019

Translating genomics into practice for real-time surveillance and response to carbapenemase-producing Enterobacteriaceae: evidence from a complex multi-institutional KPC outbreak.

Until recently, Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae were rarely identified in Australia. Following an increase in the number of incident cases across the state of Victoria, we undertook a real-time combined genomic and epidemiological investigation. The scope of this study included identifying risk factors and routes of transmission, and investigating the utility of genomics to enhance traditional field epidemiology for informing management of established widespread outbreaks.All KPC-producing Enterobacteriaceae isolates referred to the state reference laboratory from 2012 onwards were included. Whole-genome sequencing was performed in parallel with a detailed descriptive epidemiological investigation of each case, using Illumina sequencing on each isolate. This was complemented with PacBio long-read sequencing on selected isolates to establish high-quality reference sequences and interrogate characteristics of KPC-encoding plasmids.Initial investigations indicated that the outbreak was widespread, with 86 KPC-producing Enterobacteriaceae isolates (K. pneumoniae 92%) identified from 35 different locations across metropolitan and rural Victoria between 2012 and 2015. Initial combined analyses of the epidemiological and genomic data resolved the outbreak into distinct nosocomial transmission networks, and identified healthcare facilities at the epicentre of KPC transmission. New cases were assigned to transmission networks in real-time, allowing focussed infection control efforts. PacBio sequencing confirmed a secondary transmission network arising from inter-species plasmid transmission. Insights from Bayesian transmission inference and analyses of within-host diversity informed the development of state-wide public health and infection control guidelines, including interventions such as an intensive approach to screening contacts following new case detection to minimise unrecognised colonisation.A real-time combined epidemiological and genomic investigation proved critical to identifying and defining multiple transmission networks of KPC Enterobacteriaceae, while data from either investigation alone were inconclusive. The investigation was fundamental to informing infection control measures in real-time and the development of state-wide public health guidelines on carbapenemase-producing Enterobacteriaceae surveillance and management.

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September 22, 2019

Adaptive strategies of Bacillus thuringiensis isolated from acid mine drainage site in Sabah, Malaysia.

The adaptive process in bacteria is driven by specific genetic elements which regulate phenotypic characteristics such as tolerance to high metal ion concentrations and the secretion of protective biofilms. Extreme environments such as those associated with heavy metal pollution and extremes of acidity offer opportunities to study the adaptive mechanisms of microorganisms. This study focused on the genome analysis of Bacillus thuringiensis (Bt MCMY1), a gram positive rod shaped bacterium isolated from an acid mine drainage site in Sabah, Malaysia by using a combination of Single Molecule Real Time DNA Sequencing, Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). The genome size of Bt MCMY1 was determined to be 5,458,152 bases which was encoded on a single chromosome. Analysis of the genome revealed genes associated with resistance to Copper, Mercury, Arsenic, Cobalt, Zinc, Cadmium and Aluminum. Evidence from SEM and FTIR indicated that the bacterial colonies form distinct films which bear the signature of polyhydroxyalkanoates (PHA) and this finding was supported by the genome data indicating the presence of a genetic pathway associated with the biosynthesis of PHAs. This is the first report of a Bacillus sp. isolated from an acid mine drainage site in Sabah, Malaysia and the genome sequence will provide insights into the manner in which B. thuringiensis adapts to acid mine drainage.

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September 22, 2019

Complete genome sequence of Lactobacillus pentosus SLC13, isolated from mustard pickles, a potential probiotic strain with antimicrobial activity against foodborne pathogenic microorganisms.

Lactobacillus pentosus SLC13 is a high exopolysaccharide (EPS)-producing strain with broad-spectrum antimicrobial activity and the ability to grow in simulated gastrointestinal conditions. SLC13 was isolated from mustard pickles in Taiwan for potential probiotic applications. To better understand the molecular base for its antimicrobial activity and high EPS production, entire genome of SLC13 was determined by PacBio SMRT sequencing.L. pentosus SLC13 contains a genome with a 3,520,510-bp chromosome and a 62,498-bp plasmid. GC content of the complete genome was 46.5% and that of plasmid pSLC13 was 41.3%. Sequences were annotated at the RAST prokaryotic genome annotation server, and the results showed that the genome contained 3172 coding sequences and 82 RNA genes. Seventy-six protein-coding sequences were identified on the plasmid pSLC13. A plantaricin gene cluster, which is responsible for bacteriosins biosynthesis and could be associated with its broad-spectrum antimicrobial activity, was identified based on comparative genomic analysis. Two gene clusters involved in EPS production were also identified.This genomic sequence might contribute to a future application of this strain as probiotic in productive livestock potentially inhibiting competing and pathogenic organisms.

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September 22, 2019

Functional metagenomics reveals a novel carbapenem-hydrolyzing mobile beta-lactamase from Indian river sediments contaminated with antibiotic production waste.

Evolution has provided environmental bacteria with a plethora of genes that give resistance to antibiotic compounds. Under anthropogenic selection pressures, some of these genes are believed to be recruited over time into pathogens by horizontal gene transfer. River sediment polluted with fluoroquinolones and other drugs discharged from bulk drug production in India constitute an environment with unprecedented, long-term antibiotic selection pressures. It is therefore plausible that previously unknown resistance genes have evolved and/or are promoted here. In order to search for novel resistance genes, we therefore analyzed such river sediments by a functional metagenomics approach. DNA fragments providing resistance to different antibiotics in E. coli were sequenced using Sanger and PacBio RSII platforms. We recaptured the majority of known antibiotic resistance genes previously identified by open shot-gun metagenomics sequencing of the same samples. In addition, seven novel resistance gene candidates (six beta-lactamases and one amikacin resistance gene) were identified. Two class A beta-lactamases, blaRSA1 and blaRSA2, were phylogenetically close to clinically important ESBLs like blaGES, blaBEL and blaL2, and were further characterized for their substrate spectra. The blaRSA1 protein, encoded as an integron gene cassette, efficiently hydrolysed penicillins, first generation cephalosporins and cefotaxime, while blaRSA2 was an inducible class A beta-lactamase, capable of hydrolyzing carbapenems albeit with limited efficiency, similar to the L2 beta-lactamase from Stenotrophomonas maltophilia. All detected novel genes were associated with plasmid mobilization proteins, integrons, and/or other resistance genes, suggesting a potential for mobility. This study provides insight into a resistome shaped by an exceptionally strong and long-term antibiotic selection pressure. An improved knowledge of mobilized resistance factors in the external environment may make us better prepared for the resistance challenges that we may face in clinics in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

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