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September 22, 2019

FRI-4 carbapenemase-producing Enterobacter cloacae complex isolated in Tokyo, Japan.

A carbapenem-resistant Enterobacter cloacae complex isolated in Tokyo, Japan, produced a carbapenemase that was detected by a Carba NP test and a modified carbapenem inactivation method, but none of the ‘Big Five’ carbapenemase genes was detected by PCR. This study aimed to identify the carbapenemase.Carbapenemase genes were screened by WGS. Next, we generated a recombinant plasmid in which the carbapenemase gene was inserted. We also extracted the carbapenemase gene-carrying plasmid from the E. cloacae complex. The effects of both plasmids on the antibiotic susceptibility of Escherichia coli were then tested. The carbapenemase gene-carrying plasmid in the E. cloacae complex was completely sequenced.A novel carbapenemase gene, blaFRI-4, encoded an amino acid sequence that was 93.2% identical to French imipenemase (FRI-1). E. coli transformed with blaFRI-4 showed reduced carbapenem susceptibility. A complete sequence of the blaFRI-4-carrying 98?508?bp IncFII/IncR plasmid (pTMTA61661) showed that blaFRI-4 and the surrounding region (18.7?kb) were duplicated.The FRI-4-producing E. cloacae complex was isolated in Japan, whereas all other FRI variants have been found in Europe, suggesting that the spread of FRI carbapenemases is global.

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September 22, 2019

Plasmid and chromosomal integration of four novel blaIMP-carrying transposons from Pseudomonas aeruginosa, Klebsiella pneumoniae and an Enterobacter sp.

To provide detailed genetic characterization of four novel blaIMP-carrying transposons from Pseudomonas aeruginosa, Klebsiella pneumoniae and an Enterobacter sp.P. aeruginosa 60512, K. pneumoniae 447, P. aeruginosa 12939 and Enterobacter sp. A1137 were subjected to genome sequencing. The complete nucleotide sequences of two plasmids (p60512-IMP from the 60512 isolate and p447-IMP from the 447 isolate) and two chromosomes (the 12939 and A1137 isolates) were determined, then a genomic comparison of p60512-IMP, p447-IMP and four novel blaIMP-carrying transposons (Tn6394, Tn6375, Tn6411 and Tn6397) with related sequences was performed. Transferability of the blaIMP gene and bacterial antimicrobial susceptibility were tested.Tn6394 and Tn6375 were located in p60512-IMP and p447-IMP, respectively, while Tn6411 and Tn6397 were integrated into the 12939 and A1137 chromosomes, respectively. Tn6394 was an ISPa17-based transposition unit that harboured the integron In992 (carrying blaIMP-1). In73 (carrying blaIMP-8), In73 and In992, together with the ISEcp1:IS1R-blaCTX-M-14-IS903D unit, the macAB-tolC region and the truncated aacC2-tmrB region, respectively, were integrated into the prototype transposons Tn1722, Tn1696 and Tn7, respectively, generating the Tn3-family unit transposons, Tn6375 and Tn6378, and the Tn7-family unit transposon Tn6411, respectively. Tn6397 was a large integrative and conjugative element carrying Tn6378.Complex events of transposition and homologous recombination have occurred during the original formation and further plasmid and chromosomal integration of these four transposons, promoting accumulation and spread of antimicrobial resistance genes.

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September 22, 2019

Full-length extension of HLA allele sequences by HLA allele-specific hemizygous Sanger sequencing (SSBT).

The gold standard for typing at the allele level of the highly polymorphic Human Leucocyte Antigen (HLA) gene system is sequence based typing. Since sequencing strategies have mainly focused on identification of the peptide binding groove, full-length sequence information is lacking for >90% of the HLA alleles. One of the goals of the 17th IHIWS workshop is to establish full-length sequences for as many HLA alleles as possible. In our component “Extension of HLA sequences by full-length HLA allele-specific hemizygous Sanger sequencing” we have used full-length hemizygous Sanger Sequence Based Typing to achieve this goal. We selected samples of which full length sequences were not available in the IPD-IMGT/HLA database. In total we have generated the full-length sequences of 48 HLA-A, 45 -B and 31 -C alleles. For HLA-A extended alleles, 39/48 showed no intron differences compared to the first allele of the corresponding allele group, for HLA-B this was 26/45 and for HLA-C 20/31. Comparing the intron sequences to other alleles of the same allele group revealed that in 5/48 HLA-A, 16/45 HLA-B and 8/31 HLA-C alleles the intron sequence was identical to another allele of the same allele group. In the remaining 10 cases, the sequence either showed polymorphism at a conserved nucleotide or was the result of a gene conversion event. Elucidation of the full-length sequence gives insight in the polymorphic content of the alleles and facilitates the identification of its evolutionary origin. Copyright © 2018 American Society for Histocompatibility and Immunogenetics. All rights reserved.

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September 22, 2019

The genomic architecture and molecular evolution of ant odorant receptors.

The massive expansions of odorant receptor (OR) genes in ant genomes are notable examples of rapid genome evolution and adaptive gene duplication. However, the molecular mechanisms leading to gene family expansion remain poorly understood, partly because available ant genomes are fragmentary. Here, we present a highly contiguous, chromosome-level assembly of the clonal raider ant genome, revealing the largest known OR repertoire in an insect. While most ant ORs originate via local tandem duplication, we also observe several cases of dispersed duplication followed by tandem duplication in the most rapidly evolving OR clades. We found that areas of unusually high transposable element density (TE islands) were depauperate in ORs in the clonal raider ant, and found no evidence for retrotransposition of ORs. However, OR loci were enriched for transposons relative to the genome as a whole, potentially facilitating tandem duplication by unequal crossing over. We also found that ant OR genes are highly AT-rich compared to other genes. In contrast, in flies, OR genes are dispersed and largely isolated within the genome, and we find that fly ORs are not AT-rich. The genomic architecture and composition of ant ORs thus show convergence with the unrelated vertebrate ORs rather than the related fly ORs. This might be related to the greater gene numbers and/or potential similarities in gene regulation between ants and vertebrates as compared to flies.© 2018 McKenzie and Kronauer; Published by Cold Spring Harbor Laboratory Press.

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September 22, 2019

A metabolic and genomic assessment of sugar fermentation profiles of the thermophilic Thermotogales, Fervidobacterium pennivorans.

A metabolic, genomic and proteomic assessment of Fervidobacterium pennivorans strains was undertaken to clarify the metabolic and genetic capabilities of this Thermotogales species. The type strain Ven5 originally isolated from a hot mud spa in Italy, and a newly isolated strain (DYC) from a hot spring at Ngatamariki, New Zealand, were compared for metabolic and genomic differences. The fermentation profiles of both strains on cellobiose generated similar major end products (acetate, alanine, glutamate, H2, and CO2). The vast majority of end products produced were redox neutral, and carbon balances were in the range of 95-115%. Each strain showed distinct fermentation profiles on sugar substrates. The genome of strain DYC was sequenced and shown to have high sequence similarity and synteny with F. pennivorans Ven5 genome, suggesting they are the same species. The unique genome regions in Ven5, corresponded to genes involved in the Entner-Doudoroff pathway confirming our observation of DYC’s inability to utilize gluconate. Genome analysis was able to elucidate pathways involved in production of the observed end-products with the exception of alanine and glutamate synthesis which were resolved with less clarity due to poor sequence identity and missing critical enzymes within the pathway, respectively.

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September 22, 2019

Thermus sediminis sp. nov., a thiosulfate-oxidizing and arsenate-reducing organism isolated from Little Hot Creek in the Long Valley Caldera, California.

Thermus species are widespread in natural and artificial thermal environments. Two new yellow-pigmented strains, L198T and L423, isolated from Little Hot Creek, a geothermal spring in eastern California, were identified as novel organisms belonging to the genus Thermus. Cells are Gram-negative, rod-shaped, and non-motile. Growth was observed at temperatures from 45 to 75 °C and at salinities of 0-2.0% added NaCl. Both strains grow heterotrophically or chemolithotrophically by oxidation of thiosulfate to sulfate. L198T and L423 grow by aerobic respiration or anaerobic respiration with arsenate as the terminal electron acceptor. Values for 16S rRNA gene identity (=?97.01%), digital DNA-DNA hybridization (=?32.7%), OrthoANI (=?87.5%), and genome-to-genome distance (0.13) values to all Thermus genomes were less than established criteria for microbial species. The predominant respiratory quinone was menaquinone-8 and the major cellular fatty acids were iso-C15:0, iso-C17:0 and anteiso-C15:0. One unidentified phospholipid (PL1) and one unidentified glycolipid (GL1) dominated the polar lipid pattern. The new strains could be differentiated from related taxa by ß-galactosidase and ß-glucosidase activity and the presence of hydroxy fatty acids. Based on phylogenetic, genomic, phenotypic, and chemotaxonomic evidence, the novel species Thermus sediminis sp. nov. is proposed, with the type strain L198T (=?CGMCC 1.13590T?=?KCTC XXX).

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September 22, 2019

Phenazines in plant-beneficial Pseudomonas spp.: biosynthesis, regulation, function and genomics.

Plant-beneficial phenazine-producing Pseudomonas spp. are proficient biocontrol agents of soil-dwelling plant pathogens. Phenazines are redox-active molecules that display broad-spectrum antibiotic activity toward many fungal, bacterial and oomycete plant pathogens. Phenazine compounds also play a role in the persistence and survival of Pseudomonas spp. in the rhizosphere. This mini-review focuses on plant-beneficial phenazine-producing Pseudomonas spp. from the P. fluorescens species complex, which includes numerous well-known phenazine-producing strains of biocontrol interest. In this review the current knowledge on phenazine biosynthesis and regulation, the role played by phenazines in biocontrol and rhizosphere colonization, as well as exciting new advances in the genomics of plant-beneficial phenazine-producing Pseudomonas spp. will be discussed.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

Genome mining of Streptomyces xinghaiensis NRRL B-24674T for the discovery of the gene cluster involved in anticomplement activities and detection of novel xiamycin analogs.

Marine actinobacterium Streptomyces xinghaiensis NRRL B-24674T has been characterized as a novel species, but thus far, its biosynthetic potential remains unexplored. In this study, the high-quality genome sequence of S. xinghaiensis NRRL B-24674T was obtained, and the production of anticomplement agents, xiamycin analogs, and siderophores was investigated by genome mining. Anticomplement compounds are valuable for combating numerous diseases caused by the abnormal activation of the human complement system. The biosynthetic gene cluster (BGC) nrps1 resembles that of complestatins, which are potent microbial-derived anticomplement agents. The identification of the nrps1 BGC revealed a core peptide that differed from that in complestatin; thus, we studied the anticomplement activity of this strain. The culture broth of S. xinghaiensis NRRL B-24674T displayed good anticomplement activity. Subsequently, the disruption of the genes in the nrps1 BGC resulted in the loss of anticomplement activity, confirming the involvement of this BGC in the biosynthesis of anticomplement agents. In addition, the mining of the BGC tep5, which resembles that of the antiviral pentacyclic indolosesquiterpene xiamycin, resulted in the discovery of nine xiamycin analogs, including three novel compounds. In addition to the BGCs responsible for desferrioxamine B, neomycin, ectoine, and carotenoid, 18 BGCs present in the genome are predicted to be novel. The results of this study unveil the potential of S. xinghaiensis as a producer of novel anticomplement agents and provide a basis for further exploration of the biosynthetic potential of S. xinghaiensis NRRL B-24674T for the discovery of novel bioactive compounds by genome mining.

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September 22, 2019

Alpha- and beta-mannan utilization by marine Bacteroidetes.

Marine microscopic algae carry out about half of the global carbon dioxide fixation into organic matter. They provide organic substrates for marine microbes such as members of the Bacteroidetes that degrade algal polysaccharides using carbohydrate-active enzymes (CAZymes). In Bacteroidetes genomes CAZyme encoding genes are mostly grouped in distinct regions termed polysaccharide utilization loci (PULs). While some studies have shown involvement of PULs in the degradation of algal polysaccharides, the specific substrates are for the most part still unknown. We investigated four marine Bacteroidetes isolated from the southern North Sea that harbour putative mannan-specific PULs. These PULs are similarly organized as PULs in human gut Bacteroides that digest a- and ß-mannans from yeasts and plants respectively. Using proteomics and defined growth experiments with polysaccharides as sole carbon sources we could show that the investigated marine Bacteroidetes express the predicted functional proteins required for a- and ß-mannan degradation. Our data suggest that algal mannans play an as yet unknown important role in the marine carbon cycle, and that biochemical principles established for gut or terrestrial microbes also apply to marine bacteria, even though their PULs are evolutionarily distant.© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

Genomic analysis of Picochlorum species reveals how microalgae may adapt to variable environments.

Understanding how microalgae adapt to rapidly changing environments is not only important to science but can help clarify the potential impact of climate change on the biology of primary producers. We sequenced and analyzed the nuclear genome of multiple Picochlorum isolates (Chlorophyta) to elucidate strategies of environmental adaptation. It was previously found that coordinated gene regulation is involved in adaptation to salinity stress, and here we show that gene gain and loss also play key roles in adaptation. We determined the extent of horizontal gene transfer (HGT) from prokaryotes and their role in the origin of novel functions in the Picochlorum clade. HGT is an ongoing and dynamic process in this algal clade with adaptation being driven by transfer, divergence, and loss. One HGT candidate that is differentially expressed under salinity stress is indolepyruvate decarboxylase that is involved in the production of a plant auxin that mediates bacteria-diatom symbiotic interactions. Large differences in levels of heterozygosity were found in diploid haplotypes among Picochlorum isolates. Biallelic divergence was pronounced in P. oklahomensis (salt plains environment) when compared with its closely related sister taxon Picochlorum SENEW3 (brackish water environment), suggesting a role of diverged alleles in response to environmental stress. Our results elucidate how microbial eukaryotes with limited gene inventories expand habitat range from mesophilic to halophilic through allelic diversity, and with minor but important contributions made by HGT. We also explore how the nature and quality of genome data may impact inference of nuclear ploidy.

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September 22, 2019

Genomic discovery of the hypsin gene and biosynthetic pathways for terpenoids in Hypsizygus marmoreus.

Hypsizygus marmoreus (Beech mushroom) is a popular ingredient in Asian cuisine. The medicinal effects of its bioactive compounds such as hypsin and hypsiziprenol have been reported, but the genetic basis or biosynthesis of these components is unknown.In this study, we sequenced a reference strain of H. marmoreus (Haemi 51,987-8). We evaluated various assembly strategies, and as a result the Allpaths and PBJelly produced the best assembly. The resulting genome was 42.7 Mbp in length and annotated with 16,627 gene models. A putative gene (Hypma_04324) encoding the antifungal and antiproliferative hypsin protein with 75% sequence identity with the previously known N-terminal sequence was identified. Carbohydrate active enzyme analysis displayed the typical feature of white-rot fungi where auxiliary activity and carbohydrate-binding modules were enriched. The genome annotation revealed four terpene synthase genes responsible for terpenoid biosynthesis. From the gene tree analysis, we identified that terpene synthase genes can be classified into six clades. Four terpene synthase genes of H. marmoreus belonged to four different groups that implies they may be involved in the synthesis of different structures of terpenes. A terpene synthase gene cluster was well-conserved in Agaricomycetes genomes, which contained known biosynthesis and regulatory genes.Genome sequence analysis of this mushroom led to the discovery of the hypsin gene. Comparative genome analysis revealed the conserved gene cluster for terpenoid biosynthesis in the genome. These discoveries will further our understanding of the biosynthesis of medicinal bioactive molecules in this edible mushroom.

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September 22, 2019

Comparative genomic and methylome analysis of non-virulent D74 and virulent Nagasaki Haemophilus parasuis isolates.

Haemophilus parasuis is a respiratory pathogen of swine and the etiological agent of Glässer’s disease. H. parasuis isolates can exhibit different virulence capabilities ranging from lethal systemic disease to subclinical carriage. To identify genomic differences between phenotypically distinct strains, we obtained the closed whole-genome sequence annotation and genome-wide methylation patterns for the highly virulent Nagasaki strain and for the non-virulent D74 strain. Evaluation of the virulence-associated genes contained within the genomes of D74 and Nagasaki led to the discovery of a large number of toxin-antitoxin (TA) systems within both genomes. Five predicted hemolysins were identified as unique to Nagasaki and seven putative contact-dependent growth inhibition toxin proteins were identified only in strain D74. Assessment of all potential vtaA genes revealed thirteen present in the Nagasaki genome and three in the D74 genome. Subsequent evaluation of the predicted protein structure revealed that none of the D74 VtaA proteins contain a collagen triple helix repeat domain. Additionally, the predicted protein sequence for two D74 VtaA proteins is substantially longer than any predicted Nagasaki VtaA proteins. Fifteen methylation sequence motifs were identified in D74 and fourteen methylation sequence motifs were identified in Nagasaki using SMRT sequencing analysis. Only one of the methylation sequence motifs was observed in both strains indicative of the diversity between D74 and Nagasaki. Subsequent analysis also revealed diversity in the restriction-modification systems harbored by D74 and Nagasaki. The collective information reported in this study will aid in the development of vaccines and intervention strategies to decrease the prevalence and disease burden caused by H. parasuis.

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September 22, 2019

A complete Leishmania donovani reference genome identifies novel genetic variations associated with virulence.

Leishmania donovani is responsible for visceral leishmaniasis, a neglected and lethal parasitic disease with limited treatment options and no vaccine. The study of L. donovani has been hindered by the lack of a high-quality reference genome and this can impact experimental outcomes including the identification of virulence genes, drug targets and vaccine development. We therefore generated a complete genome assembly by deep sequencing using a combination of second generation (Illumina) and third generation (PacBio) sequencing technologies. Compared to the current L. donovani assembly, the genome assembly reported within resulted in the closure over 2,000 gaps, the extension of several chromosomes up to telomeric repeats and the re-annotation of close to 15% of protein coding genes and the annotation of hundreds of non-coding RNA genes. It was possible to correctly assemble the highly repetitive A2 and Amastin virulence gene clusters. A comparative sequence analysis using the improved reference genome confirmed 70 published and identified 15 novel genomic differences between closely related visceral and atypical cutaneous disease-causing L. donovani strains providing a more complete map of genes associated with virulence and visceral organ tropism. Bioinformatic tools including protein variation effect analyzer and basic local alignment search tool were used to prioritize a list of potential virulence genes based on mutation severity, gene conservation and function. This complete genome assembly and novel information on virulence factors will support the identification of new drug targets and the development of a vaccine for L. donovani.

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September 22, 2019

Streptococcus suis contains multiple phase-variable methyltransferases that show a discrete lineage distribution.

Streptococcus suis is a major pathogen of swine, responsible for a number of chronic and acute infections, and is also emerging as a major zoonotic pathogen, particularly in South-East Asia. Our study of a diverse population of S. suis shows that this organism contains both Type I and Type III phase-variable methyltransferases. In all previous examples, phase-variation of methyltransferases results in genome wide methylation differences, and results in differential regulation of multiple genes, a system known as the phasevarion (phase-variable regulon). We hypothesized that each variant in the Type I and Type III systems encoded a methyltransferase with a unique specificity, and could therefore control a distinct phasevarion, either by recombination-driven shuffling between different specificities (Type I) or by biphasic on-off switching via simple sequence repeats (Type III). Here, we present the identification of the target specificities for each Type III allelic variant from S. suis using single-molecule, real-time methylome analysis. We demonstrate phase-variation is occurring in both Type I and Type III methyltransferases, and show a distinct association between methyltransferase type and presence, and population clades. In addition, we show that the phase-variable Type I methyltransferase was likely acquired at the origin of a highly virulent zoonotic sub-population.

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September 22, 2019

Excision-reintegration at a pneumococcal phase-variable restriction-modification locus drives within- and between-strain epigenetic differentiation and inhibits gene acquisition.

Phase-variation of Type I restriction-modification systems can rapidly alter the sequence motifs they target, diversifying both the epigenetic patterns and endonuclease activity within clonally descended populations. Here, we characterize the Streptococcus pneumoniae SpnIV phase-variable Type I RMS, encoded by the translocating variable restriction (tvr) locus, to identify its target motifs, mechanism and regulation of phase variation, and effects on exchange of sequence through transformation. The specificity-determining hsdS genes were shuffled through a recombinase-mediated excision-reintegration mechanism involving circular intermediate molecules, guided by two types of direct repeat. The rate of rearrangements was limited by an attenuator and toxin-antitoxin system homologs that inhibited recombinase gene transcription. Target motifs for both the SpnIV, and multiple Type II, MTases were identified through methylation-sensitive sequencing of a panel of recombinase-null mutants. This demonstrated the species-wide diversity observed at the tvr locus can likely specify nine different methylation patterns. This will reduce sequence exchange in this diverse species, as the native form of the SpnIV RMS was demonstrated to inhibit the acquisition of genomic islands by transformation. Hence the tvr locus can drive variation in genome methylation both within and between strains, and limits the genomic plasticity of S. pneumoniae.

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