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April 21, 2020  |  

Chromulinavorax destructans, a pathogen of microzooplankton that provides a window into the enigmatic candidate phylum Dependentiae.

Members of the major candidate phylum Dependentiae (a.k.a. TM6) are widespread across diverse environments from showerheads to peat bogs; yet, with the exception of two isolates infecting amoebae, they are only known from metagenomic data. The limited knowledge of their biology indicates that they have a long evolutionary history of parasitism. Here, we present Chromulinavorax destructans (Strain SeV1) the first isolate of this phylum to infect a representative from a widespread and ecologically significant group of heterotrophic flagellates, the microzooplankter Spumella elongata (Strain CCAP 955/1). Chromulinavorax destructans has a reduced 1.2 Mb genome that is so specialized for infection that it shows no evidence of complete metabolic pathways, but encodes an extensive transporter system for importing nutrients and energy in the form of ATP from the host. Its replication causes extensive reorganization and expansion of the mitochondrion, effectively surrounding the pathogen, consistent with its dependency on the host for energy. Nearly half (44%) of the inferred proteins contain signal sequences for secretion, including many without recognizable similarity to proteins of known function, as well as 98 copies of proteins with an ankyrin-repeat domain; ankyrin-repeats are known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus. These observations help to cement members of this phylum as widespread and diverse parasites infecting a broad range of eukaryotic microbes.

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April 21, 2020  |  

Comparative genomic analysis of eight novel haloalkaliphilic bacteriophages from Lake Elmenteita, Kenya.

We report complete genome sequences of eight bacteriophages isolated from Haloalkaline Lake Elmenteita found on the floor of Kenyan Rift Valley. The bacteriophages were sequenced, annotated and a comparative genomic analysis using various Bioinformatics tools carried out to determine relatedness of the bacteriophages to each other, and to those in public databases. Basic genome properties like genome size, percentage coding density, number of open reading frames, percentage GC content and gene organizations revealed the bacteriophages had no relationship to each other. Comparison to other nucleotide sequences in GenBank database showed no significant similarities hence novel. At the amino acid level, phages of our study revealed mosaicism to genes with conserved domains to already described phages. Phylogenetic analyses of large terminase gene responsible for DNA packaging and DNA polymerase gene for replication further showed diversity among the bacteriophages. Our results give insight into diversity of bacteriophages in Lake Elmenteita and provide information on their evolution. By providing primary sequence information, this study not only provides novel sequences for biotechnological exploitation, but also sets stage for future studies aimed at better understanding of virus diversity and genomes from haloalkaline lakes in the Rift Valley.

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April 21, 2020  |  

Information about variations in multiple copies of bacterial 16S rRNA genes may aid in species identification.

Variable region analysis of 16S rRNA gene sequences is the most common tool in bacterial taxonomic studies. Although used for distinguishing bacterial species, its use remains limited due to the presence of variable copy numbers with sequence variation in the genomes. In this study, 16S rRNA gene sequences, obtained from completely assembled whole genome and Sanger electrophoresis sequencing of cloned PCR products from Serratia fonticola GS2, were compared. Sanger sequencing produced a combination of sequences from multiple copies of 16S rRNA genes. To determine whether the variant copies of 16S rRNA genes affected Sanger sequencing, two ratios (5:5 and 8:2) with different concentrations of cloned 16S rRNA genes were used; it was observed that the greater the number of copies with similar sequences the higher its chance of amplification. Effect of multiple copies for taxonomic classification of 16S rRNA gene sequences was investigated using the strain GS2 as a model. 16S rRNA copies with the maximum variation had 99.42% minimum pairwise similarity and this did not have an effect on species identification. Thus, PCR products from genomes containing variable 16S rRNA gene copies can provide sufficient information for species identification except from species which have high similarity of sequences in their 16S rRNA gene copies like the case of Bacillus thuringiensis and Bacillus cereus. In silico analysis of 1,616 bacterial genomes from long-read sequencing was also done. The average minimum pairwise similarity for each phylum was reported with their average genome size and average “unique copies” of 16S rRNA genes and we found that the phyla Proteobacteria and Firmicutes showed the highest amount of variation in their copies of their 16S rRNA genes. Overall, our results shed light on how the variations in the multiple copies of the 16S rRNA genes of bacteria can aid in appropriate species identification.

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April 21, 2020  |  

Reconstruction of the genomes of drug-resistant pathogens for outbreak investigation through metagenomic sequencing

Culture-independent methods that target genome fragments have shown promise in identifying certain pathogens, but the holy grail of comprehensive pathogen genome detection from microbiologically complex samples for subsequent forensic analyses remains a challenge. In the context of an investigation of a nosocomial outbreak, we used shotgun metagenomic sequencing of a human fecal sample and a neural network algorithm based on tetranucleotide frequency profiling to reconstruct microbial genomes and tested the same approach using rectal swabs from a second patient. The approach rapidly and readily detected the genome of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae in the patient fecal specimen and in the rectal swab sample, achieving a level of strain resolution that was sufficient for confident transmission inference during a highly clonal outbreak. The analysis also detected previously unrecognized colonization of the patient by vancomycin-resistant Enterococcus faecium, another multidrug-resistant bacterium.IMPORTANCE The study results reported here perfectly demonstrate the power and promise of clinical metagenomics to recover genome sequences of important drug-resistant bacteria and to rapidly provide rich data that inform outbreak investigations and treatment decisions, independently of the need to culture the organisms.

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April 21, 2020  |  

Genome mining identifies cepacin as a plant-protective metabolite of the biopesticidal bacterium Burkholderia ambifaria.

Beneficial microorganisms are widely used in agriculture for control of plant pathogens, but a lack of efficacy and safety information has limited the exploitation of multiple promising biopesticides. We applied phylogeny-led genome mining, metabolite analyses and biological control assays to define the efficacy of Burkholderia ambifaria, a naturally beneficial bacterium with proven biocontrol properties but potential pathogenic risk. A panel of 64 B.?ambifaria strains demonstrated significant antimicrobial activity against priority plant pathogens. Genome sequencing, specialized metabolite biosynthetic gene cluster mining and metabolite analysis revealed an armoury of known and unknown pathways within B.?ambifaria. The biosynthetic gene cluster responsible for the production of the metabolite cepacin was identified and directly shown to mediate protection of germinating crops against Pythium damping-off disease. B.?ambifaria maintained biopesticidal protection and overall fitness in the soil after deletion of its third replicon, a non-essential plasmid associated with virulence in Burkholderia?cepacia complex bacteria. Removal of the third replicon reduced B.?ambifaria persistence in a murine respiratory infection model. Here, we show that by using interdisciplinary phylogenomic, metabolomic and functional approaches, the mode of action of natural biological control agents related to pathogens can be systematically established to facilitate their future exploitation.

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April 21, 2020  |  

Genome Analyses of a New Mycoplasma Species from the Scorpion Centruroides vittatus.

Arthropod Mycoplasma are little known endosymbionts in insects, primarily known as plant disease vectors. Mycoplasma in other arthropods such as arachnids are unknown. We report the first complete Mycoplasma genome sequenced, identified, and annotated from a scorpion, Centruroides vittatus, and designate it as Mycoplasma vittatus We find the genome is at least a 683,827 bp single circular chromosome with a GC content of 42.7% and with 987 protein-coding genes. The putative virulence determinants include 11 genes associated with the virulence operon associated with protein synthesis or DNA transcription and ten genes with antibiotic and toxic compound resistance. Comparative analysis revealed that the M. vittatus genome is smaller than other Mycoplasma genomes and exhibits a higher GC content. Phylogenetic analysis shows M. vittatus as part of the Hominis group of Mycoplasma As arthropod genomes accumulate, further novel Mycoplasma genomes may be identified and characterized. Copyright © 2019 Yamashita et al.

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April 21, 2020  |  

Parallels between natural selection in the cold-adapted crop-wild relative Tripsacum dactyloides and artificial selection in temperate adapted maize.

Artificial selection has produced varieties of domesticated maize that thrive in temperate climates around the world. However, the direct progenitor of maize, teosinte, is indigenous only to a relatively small range of tropical and subtropical latitudes and grows poorly or not at all outside of this region. Tripsacum, a sister genus to maize and teosinte, is naturally endemic to the majority of areas in the western hemisphere where maize is cultivated. A full-length reference transcriptome for Tripsacum dactyloides generated using long-read Iso-Seq data was used to characterize independent adaptation to temperate climates in this clade. Genes related to phospholipid biosynthesis, a critical component of cold acclimation in other cold-adapted plant lineages, were enriched among those genes experiencing more rapid rates of protein sequence evolution in T. dactyloides. In contrast with previous studies of parallel selection, we find that there is a significant overlap between the genes that were targets of artificial selection during the adaptation of maize to temperate climates and those that were targets of natural selection in temperate-adapted T. dactyloides. Genes related to growth, development, response to stimulus, signaling, and organelles were enriched in the set of genes identified as both targets of natural and artificial selection. © 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.

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April 21, 2020  |  

Whole genome sequencing of NDM-1-producing serotype K1 ST23 hypervirulent Klebsiella pneumoniae in China.

The emergence and spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) is causing worldwide concern, whereas NDM-producing hvKP is still rare. Here we report the complete genome sequence characteristics of an NDM-1-producing ST23 type clinical hvKP in PR China.Capsular polysaccharide serotyping was performed by PCR. The complete genome sequence of isolate 3214 was obtained using both the Illumina Hiseq platform and Pacbio RS platform. Multilocus sequence type was identified by submitting the genome sequence to mlst 2.0 and the antimicrobial resistance genes and plasmid replicons were identified using ResFinder and PlasmidFinder, respectively. Transferability of the blaNDM-1-bearing plasmid was determined by conjugation experiment, S1 pulsed-field gel electrophoresis and Southern hybridization.Isolate 3214 was classified to ST23 and belonged to the K1 capsular serotype. The isolate’s total genome size was 6 171 644?bp with a G+C content of 56.39 %, consisting of a 5 448 209?bp chromosome and seven plasmids. The resistome included 18 types of antibiotic resistance genes. Fourteen resistance genes including blaNDM-1 and blaCTX-M-14 were located on plasmids and five also including blaCTX-M-14 were in the chromosome. Plasmid pNDM_3214 carrying blaNDM-1 harboured six types of resistance genes surrounded by insertion sequences and was conjugative. The worldwide pLVPK-like virulence plasmid harbouring rmpA2 and rmpA was also found in this isolate.This study provides basic information of phenotypic and genomic features of ST23 CR-hvKP isolate 3214. Our data highlights the potential risk of spread of NDM-1-producing ST23 hvKP.

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April 21, 2020  |  

Expedited assessment of terrestrial arthropod diversity by coupling Malaise traps with DNA barcoding 1.

Monitoring changes in terrestrial arthropod communities over space and time requires a dramatic increase in the speed and accuracy of processing samples that cannot be achieved with morphological approaches. The combination of DNA barcoding and Malaise traps allows expedited, comprehensive inventories of species abundance whose cost will rapidly decline as high-throughput sequencing technologies advance. Aside from detailing protocols from specimen sorting to data release, this paper describes their use in a survey of arthropod diversity in a national park that examined 21?194 specimens representing 2255 species. These protocols can support arthropod monitoring programs at regional, national, and continental scales.

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April 21, 2020  |  

Streptococcus periodonticum sp. nov., Isolated from Human Subgingival Dental Plaque of Periodontitis Lesion.

A novel facultative anaerobic and Gram-stain-positive coccus, designated strain ChDC F135T, was isolated from human subgingival dental plaque of periodontitis lesion and was characterized by polyphasic taxonomic analysis. The 16S rRNA gene (16S rDNA) sequence of strain ChDC F135T was closest to that of Streptococcus sinensis HKU4T (98.2%), followed by Streptococcus intermedia SK54T (97.0%), Streptococcus constellatus NCTC11325T (96.0%), and Streptococcus anginosus NCTC 10713T (95.7%). In contrast, phylogenetic analysis based on the superoxide dismutase gene (sodA) and the RNA polymerase beta-subunit gene (rpoB) showed that the nucleotide sequence similarities of strain ChDC F135T were highly similar to the corresponding genes of S. anginosus NCTC 10713T (99.2% and 97.6%, respectively), S. constellatus NCTC11325T (87.8% and 91.4%, respectively), and S. intermedia SK54T (85.8% and 91.2%, respectively) rather than those of S. sinensis HKU4T (80.5% and 82.6%). The complete genome of strain ChDC F135T consisted of 1,901,251 bp and the G+C content was 38.9 mol %. Average nucleotide identity value between strain ChDC F135T and S. sinensis HKU4T or S. anginosus NCTC 10713T were 75.7% and 95.6%, respectively. The C14:0 composition of the cellular fatty acids of strain ChDC F135T (32.8%) was different from that of S. intermedia (6-8%), S. constellatus (6-13%), and S. anginosus (13-20%). Based on the results of phylogenetic and phenotypic analysis, strain ChDC F135T (=?KCOM 2412T?=?JCM 33300T) was classified as a type strain of a novel species of the genus Streptococcus, for which we proposed the name Streptococcus periodonticum sp. nov.

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April 21, 2020  |  

Morphotypes of the common beadlet anemone Actinia equina (L.) are genetically distinct

Anemones of the genus Actinia are ecologically important and familiar organisms on many rocky shores. However, this genus is taxonomically problematical and prior evidence suggests that the North Atlantic beadlet anemone, Actinia equina, may actually consist of a number of cryptic species. Previous genetic work has been largely limited to allozyme electrophoresis and there remains a dearth of genetic resources with which to study this genus. Mitochondrial DNA sequencing may help to clarify the taxonomy of Actinia. Here, the complete mitochondrial genome of the beadlet anemone Actinia equina (Cnidaria: Anthozoa: Actinaria: Actiniidae) is shown to be 20,690?bp in length and to contain the standard complement of Cnidarian features including 13 protein coding genes, two rRNA genes, two tRNAs and two Group I introns, one with an in-frame truncated homing endonuclease gene open reading frame. However, amplification and sequencing of the standard mtDNA barcoding region of the cytochrome oxidase I gene revealed only two haplotypes, differing by a single base pair, in widely geographically separated A. equina and its congener A. prasina. COI barcoding shows that whilst A. equina and A. prasina share the common mtDNA haplotype, haplotype frequency differed significantly between A. equina with red/orange pedal discs and those with green pedal discs, consistent with the hypothesis that these morphotypes represent incipient species.

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April 21, 2020  |  

Conventional culture methods with commercially available media unveil the presence of novel culturable bacteria.

Recent metagenomic analysis has revealed that our gut microbiota plays an important role in not only the maintenance of our health but also various diseases such as obesity, diabetes, inflammatory bowel disease, and allergy. However, most intestinal bacteria are considered ‘unculturable’ bacteria, and their functions remain unknown. Although culture-independent genomic approaches have enabled us to gain insight into their potential roles, culture-based approaches are still required to understand their characteristic features and phenotypes. To date, various culturing methods have been attempted to obtain these ‘unculturable’ bacteria, but most such methods require advanced techniques. Here, we have tried to isolate possible unculturable bacteria from a healthy Japanese individual by using commercially available media. A 16S rRNA (ribosomal RNA) gene metagenomic analysis revealed that each culture medium showed bacterial growth depending on its selective features and a possibility of the presence of novel bacterial species. Whole genome sequencing of these candidate strains suggested the isolation of 8 novel bacterial species classified in the Actinobacteria and Firmicutes phyla. Our approach indicates that a number of intestinal bacteria hitherto considered unculturable are potentially culturable and can be cultured on commercially available media. We have obtained novel gut bacteria from a healthy Japanese individual using a combination of comprehensive genomics and conventional culturing methods. We would expect that the discovery of such novel bacteria could illuminate pivotal roles for the gut microbiota in association with human health.

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April 21, 2020  |  

Polysaccharide utilization loci of North Sea Flavobacteriia as basis for using SusC/D-protein expression for predicting major phytoplankton glycans.

Marine algae convert a substantial fraction of fixed carbon dioxide into various polysaccharides. Flavobacteriia that are specialized on algal polysaccharide degradation feature genomic clusters termed polysaccharide utilization loci (PULs). As knowledge on extant PUL diversity is sparse, we sequenced the genomes of 53 North Sea Flavobacteriia and obtained 400 PULs. Bioinformatic PUL annotations suggest usage of a large array of polysaccharides, including laminarin, a-glucans, and alginate as well as mannose-, fucose-, and xylose-rich substrates. Many of the PULs exhibit new genetic architectures and suggest substrates rarely described for marine environments. The isolates’ PUL repertoires often differed considerably within genera, corroborating ecological niche-associated glycan partitioning. Polysaccharide uptake in Flavobacteriia is mediated by SusCD-like transporter complexes. Respective protein trees revealed clustering according to polysaccharide specificities predicted by PUL annotations. Using the trees, we analyzed expression of SusC/D homologs in multiyear phytoplankton bloom-associated metaproteomes and found indications for profound changes in microbial utilization of laminarin, a-glucans, ß-mannan, and sulfated xylan. We hence suggest the suitability of SusC/D-like transporter protein expression within heterotrophic bacteria as a proxy for the temporal utilization of discrete polysaccharides.

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April 21, 2020  |  

Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Ka inhibitors.

Activating mutations in PIK3CA are frequent in human breast cancer, and phosphoinositide 3-kinase alpha (PI3Ka) inhibitors have been approved for therapy. To characterize determinants of sensitivity to these agents, we analyzed PIK3CA-mutant cancer genomes and observed the presence of multiple PIK3CA mutations in 12 to 15% of breast cancers and other tumor types, most of which (95%) are double mutations. Double PIK3CA mutations are in cis on the same allele and result in increased PI3K activity, enhanced downstream signaling, increased cell proliferation, and tumor growth. The biochemical mechanisms of dual mutations include increased disruption of p110a binding to the inhibitory subunit p85a, which relieves its catalytic inhibition, and increased p110a membrane lipid binding. Double PIK3CA mutations predict increased sensitivity to PI3Ka inhibitors compared with single-hotspot mutations.Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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April 21, 2020  |  

Genomic and Functional Characterization of the Endophytic Bacillus subtilis 7PJ-16 Strain, a Potential Biocontrol Agent of Mulberry Fruit Sclerotiniose.

Bacillus sp. 7PJ-16, an endophytic bacterium isolated from a healthy mulberry stem and previously identified as Bacillus tequilensis 7PJ-16, exhibits strong antifungal activity and has the capacity to promote plant growth. This strain was studied for its effectiveness as a biocontrol agent to reduce mulberry fruit sclerotiniose in the field and as a growth-promoting agent for mulberry in the greenhouse. In field studies, the cell suspension and supernatant of strain 7PJ-16 exhibited biocontrol efficacy and the lowest disease incidence was reduced down to only 0.80%. In greenhouse experiments, the cell suspension (1.0?×?106 and 1.0?×?105 CFU/mL) and the cell-free supernatant (100-fold and 1000-fold dilution) stimulated mulberry seed germination and promoted mulberry seedling growth. In addition, to accurately identify the 7PJ-16 strain and further explore the mechanisms of its antifungal and growth-promoting properties, the complete genome of this strain was sequenced and annotated. The 7PJ-16 genome is comprised of two circular plasmids and a 4,209,045-bp circular chromosome, containing 4492 protein-coding genes and 116 RNA genes. This strain was ultimately designed as Bacillus subtilis based on core genome sequence analyses using a phylogenomic approach. In this genome, we identified a series of gene clusters that function in the synthesis of non-ribosomal peptides (surfactin, fengycin, bacillibactin, and bacilysin) as well as the ribosome-dependent synthesis of tasA and bacteriocins (subtilin, subtilosin A), which are responsible for the biosynthesis of numerous antimicrobial metabolites. Additionally, several genes with function that promote plant growth, such as indole-3-acetic acid biosynthesis, the production of volatile substances, and siderophores synthesis, were also identified. The information described in this study has established a good foundation for understanding the beneficial interactions between endophytes and host plants, and facilitates the further application of B. subtilis 7PJ-16 as an agricultural biofertilizer and biocontrol agent.

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