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July 7, 2019

Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli.

As a highly valued keto-carotenoid, astaxanthin is widely used in nutritional supplements and pharmaceuticals. Therefore, the demand for biosynthetic astaxanthin and improved efficiency of astaxanthin biosynthesis has driven the investigation of metabolic engineering of native astaxanthin producers and heterologous hosts. However, microbial resources for astaxanthin are limited. In this study, we found that the a-Proteobacterium Sphingomonas sp. ATCC 55669 could produce astaxanthin naturally. We used whole-genome sequencing to identify the astaxanthin biosynthetic pathway using a combined PacBio-Illumina approach. The putative astaxanthin biosynthetic pathway in Sphingomonas sp. ATCC 55669 was predicted. For further confirmation, a high-efficiency targeted engineering carotenoid synthesis platform was constructed in E. coli for identifying the functional roles of candidate genes. All genes involved in astaxanthin biosynthesis showed discrete distributions on the chromosome. Moreover, the overexpression of exogenous E. coli idi in Sphingomonas sp. ATCC 55669 increased astaxanthin production by 5.4-fold. This study described a new astaxanthin producer and provided more biosynthesis components for bioengineering of astaxanthin in the future. © 2015 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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July 7, 2019

Assembly and characterization of the MHC class I region of the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis).

The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis; YFP) is the sole freshwater subspecies of N. asiaeorientalis and is now critically endangered. Major histocompatibility complex (MHC) is a family of highly polymorphic genes that play an important immunological role in antigen presentation in the vertebrates. Currently, however, little is known about MHC region in the genome of the YFP, which hampers conservation genetics and evolutionary ecology study using MHC genes. In this work, a nucleotide sequence of 774,811 bp covering the YFP MHC class I region was obtained by screening a YFP bacterial artificial chromosome (BAC) library, followed by sequencing and assembly of positive BAC clones. A total of 45 genes were successfully annotated, of which four were MHC class I genes. There are high similarities among the four YFP MHC class I genes (>94 %). Divergence in the coding region of the four YFP MHC class I genes is mainly localized to exons 2 and 3, which encode the antigen-binding sites of MHC class I genes. Additionally, comparison of the MHC structure in YFP to those of cattle, sheep, and pig showed that MHC class I genes are located in genome regions with regard to the conserved genes, and the YFP contains the fewest MHC class I genes among these species. This is the first report characterizing a cetacean MHC class I region and describing its organization, which would be valuable for further investigation of adaptation in natural populations of the YFP and other cetaceans.

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July 7, 2019

Novel FANCI mutations in Fanconi anemia with VACTERL association.

Fanconi anemia (FA) is an inherited bone marrow failure syndrome caused by mutations in DNA repair genes; some of these patients may have features of the VACTERL association. Autosomal recessive mutations in FANCI are a rare cause of FA. We identified FANCI mutations by next generation sequencing in three patients in our FA cohort among several whose mutated gene was unknown. Four of the six mutations are novel and all mutations are likely deleterious to protein function. There are now 16 reported cases of FA due to FANCI of whom 7 have at least 3 features of the VACTERL association (44%). This suggests that the VACTERL association in patients with FA may be seen in patients with FANCI mutations more often than previously recognized. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

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July 7, 2019

Timing, rates and spectra of human germline mutation.

Germline mutations are a driving force behind genome evolution and genetic disease. We investigated genome-wide mutation rates and spectra in multi-sibling families. The mutation rate increased with paternal age in all families, but the number of additional mutations per year differed by more than twofold between families. Meta-analysis of 6,570 mutations showed that germline methylation influences mutation rates. In contrast to somatic mutations, we found remarkable consistency in germline mutation spectra between the sexes and at different paternal ages. In parental germ line, 3.8% of mutations were mosaic, resulting in 1.3% of mutations being shared by siblings. The number of these shared mutations varied significantly between families. Our data suggest that the mutation rate per cell division is higher during both early embryogenesis and differentiation of primordial germ cells but is reduced substantially during post-pubertal spermatogenesis. These findings have important consequences for the recurrence risks of disorders caused by de novo mutations.

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July 7, 2019

Long read and single molecule DNA sequencing simplifies genome assembly and TAL effector gene analysis of Xanthomonas translucens.

The species Xanthomonas translucens encompasses a complex of bacterial strains that cause diseases and yield loss on grass species including important cereal crops. Three pathovars, X. translucens pv. undulosa, X. translucens pv. translucens and X. translucens pv.cerealis, have been described as pathogens of wheat, barley, and oats. However, no complete genome sequence for a strain of this complex is currently available.A complete genome sequence of X. translucens pv. undulosa strain XT4699 was obtained by using PacBio long read, single molecule, real time (SMRT) DNA sequences and Illumina sequences. Draft genome sequences of nineteen additional X. translucens strains, which were collected from wheat or barley in different regions and at different times, were generated by Illumina sequencing. Phylogenetic relationships among different Xanthomonas strains indicates that X. translucens are members of a distinct clade from so-called group 2 xanthomonads and three pathovars of this species, undulosa, translucens and cerealis, represent distinct subclades in the group 1 clade. Knockout mutation of type III secretion system of XT4699 eliminated the ability to cause water-soaking symptoms on wheat and barley and resulted in a reduction in populations on wheat in comparison to the wild type strain. Sequence comparison of X. translucens strains revealed the genetic variation on type III effector repertories among different pathovars or within one pathovar. The full genome sequence of XT4699 reveals the presence of eight members of the Transcription-Activator Like (TAL) effector genes, which are phylogenetically distant from previous known TAL effector genes of group 2 xanthomonads. Microarray and qRT-PCR analyses revealed TAL effector-specific wheat gene expression modulation.PacBio long read sequencing facilitates the assembly of Xanthomonas genomes and the multiple TAL effector genes, which are difficult to assemble from short read platforms. The complete genome sequence of X. translucens pv. undulosa strain XT4699 and draft genome sequences of nineteen additional X. translucens strains provides a resource for further genetic analyses of pathogenic diversity and host range of the X. translucens species complex. TAL effectors of XT4699 strain play roles in modulating wheat host gene expressions.

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July 7, 2019

Oral phage therapy of acute bacterial diarrhea with two coliphage preparations: A randomized trial in children from Bangladesh

Background Antibiotic resistance is rising in important bacterial pathogens. Phage therapy (PT), the use of bacterial viruses infecting the pathogen in a species-specific way, is a potential alternative. Method T4-like coliphages or a commercial Russian coliphage product or placebo was orally given over 4 days to Bangladeshi children hospitalized with acute bacterial diarrhea. Safety of oral phage was assessed clinically and by functional tests; coliphage and Escherichia coli titers and enteropathogens were determined in stool and quantitative diarrhea parameters (stool output, stool frequency) were measured. Stool microbiota was studied by 16S rRNA gene sequencing; the genomes of four fecal Streptococcus isolates were sequenced. Findings No adverse events attributable to oral phage application were observed (primary safety outcome). Fecal coliphage was increased in treated over control children, but the titers did not show substantial intestinal phage replication (secondary microbiology outcome). 60% of the children suffered from a microbiologically proven E. coli diarrhea; the most frequent diagnosis was ETEC infections. Bacterial co-pathogens were also detected. Half of the patients contained phage-susceptible E. coli colonies in the stool. E. coli represented less than 5% of fecal bacteria. Stool ETEC titers showed only a short-lived peak and were otherwise close to the replication threshold determined for T4 phage in vitro. An interim analysis after the enrollment of 120 patients showed no amelioration in quantitative diarrhea parameter by PT over standard care (tertiary clinical outcome). Stool microbiota was characterized by an overgrowth with Streptococcus belonging to the Streptococcus gallolyticus and Streptococcus salivarius species groups, their abundance correlated with quantitative diarrhea outcome, but genome sequencing did not identify virulence genes. Interpretation Oral coliphages showed a safe gut transit in children, but failed to achieve intestinal amplification and to improve diarrhea outcome, possibly due to insufficient phage coverage and too low E. coli pathogen titers requiring higher oral phage doses. More knowledge is needed on in vivo phage–bacterium interaction and the role of E. coli in childhood diarrhea for successful PT. Funding The study was supported by a grant from Nestlé Nutrition and Nestlé Health Science. The trial was registered with Identifier NCT00937274 at ClinicalTrials.gov.

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July 7, 2019

Complete sequences of multidrug resistance plasmids bearing rmtD1 and rmtD2 16S ribosomal RNA methyltransferase genes.

Complete nucleotide sequences were determined for two plasmids bearing rmtD group 16S rRNA methyltransferase genes. pKp64/11 was 78 kb in size, belonged to the IncL/M group, and harbored blaTEM-1b, sul1, qacE?1, dfrA22, and rmtD1 across two multidrug resistance regions (MRRs). pKp368/10 was 170 kb in size, belonged to the IncA/C group, and harbored acrB, sul1, qacE?1, ant(3?)-Ia, aac(6′)-Ib, cat, rmtD2, and blaCTX-M-8 across three MRRs. The rmtD-containing regions shared a conserved motif, suggesting a common origin for the two rmtD alleles. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

In vitro selection of miltefosine resistance in promastigotes of Leishmania donovani from Nepal: genomic and metabolomic characterization.

In this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb-S; and antimony resistant Sb-R). MIL-R was easily induced in both strains using the promastigote-stage, but a significant increase in MIL-R in the intracellular amastigote compared to the corresponding wild-type did not occur until promastigotes had adapted to 12.2 µM MIL. A variety of common and strain-specific genetic changes were discovered in MIL-adapted parasites, including deletions at the LdMT transporter gene, single-base mutations and changes in somy. The most obvious lipid changes in MIL-R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL-R parasites, with more genetic changes occurring in Sb-R compared with Sb-S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb-R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite’s biochemical pathways and how they are genetically regulated to be verified fully. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

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July 7, 2019

Refinement of the canine CD1 locus topology and investigation of antibody binding to recombinant canine CD1 isoforms.

CD1 molecules are antigen-presenting glycoproteins primarily found on dendritic cells (DCs) responsible for lipid antigen presentation to CD1-restricted T cells. Despite their pivotal role in immunity, little is known about CD1 protein expression in dogs, notably due to lack of isoform-specific antibodies. The canine (Canis familiaris) CD1 locus was previously found to contain three functional CD1A genes: canCD1A2, canCD1A6, and canCD1A8, where two variants of canCD1A8, canCD1A8.1 and canCD1A8.2, were assumed to be allelic variants. However, we hypothesized that these rather represented two separate genes. Sequencing of three overlapping bacterial artificial chromosomes (BACs) spanning the entire canine CD1 locus revealed canCD1A8.2 and canCD1A8.1 to be located in tandem between canCD1A7 and canCD1C, and canCD1A8.1 was consequently renamed canCD1A9. Green fluorescent protein (GFP)-fused canine CD1 transcripts were recombinantly expressed in 293T cells. All proteins showed a highly positive GFP expression except for canine CD1d and a splice variant of canine CD1a8 lacking exon 3. Probing with a panel of anti-CD1 monoclonal antibodies (mAbs) showed that Ca13.9H11 and Ca9.AG5 only recognized canine CD1a8 and CD1a9 isoforms, and Fe1.5F4 mAb solely recognized canine CD1a6. Anti-CD1b mAbs recognized the canine CD1b protein, but also bound CD1a2, CD1a8, and CD1a9. Interestingly, Ca9.AG5 showed allele specificity based on a single nucleotide polymorphism (SNP) located at position 321. Our findings have refined the structure of the canine CD1 locus and available antibody specificity against canine CD1 proteins. These are important fundamentals for future investigation of the role of canine CD1 in lipid immunity.

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July 7, 2019

Deciphering the streamlined genome of Streptomyces xiamenensis 318 as the producer of the anti-fibrotic drug candidate xiamenmycin.

Streptomyces xiamenensis 318, a moderate halophile isolated from a mangrove sediment, produces the anti-fibrotic compound xiamenmycin. The whole genome sequence of strain 318 was obtained through long-read single-molecule real-time (SMRT) sequencing, high-throughput Illumina HiSeq and 454 pyrosequencing technologies. The assembled genome comprises a linear chromosome as a single contig of 5,961,401-bp, which is considerably smaller than other reported complete genomes of the genus Streptomyces. Based on the antiSMASH pipeline, a total of 21?gene clusters were predicted to be involved in secondary metabolism. The gene cluster responsible for the biosynthesis of xiamenmycin resides in a strain-specific 61,387-bp genomic island belonging to the left-arm region. A core metabolic network consisting of 104 reactions that supports xiamenmycin biosynthesis was constructed to illustrate the necessary precursors derived from the central metabolic pathway. In accordance with the finding of a putative ikarugamycin gene cluster in the genome, the targeted chemical profiling of polycyclic tetramate macrolactams (PTMs) resulted in the identification of ikarugamycin. A successful genome mining for bioactive molecules with different skeletons suggests that the naturally minimized genome of S. xiamenensis 318 could be used as a blueprint for constructing a chassis cell with versatile biosynthetic capabilities for the production of secondary metabolites.

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July 7, 2019

Streptomyces thermoautotrophicus does not fix nitrogen.

Streptomyces thermoautotrophicus UBT1 has been described as a moderately thermophilic chemolithoautotroph with a novel nitrogenase enzyme that is oxygen-insensitive. We have cultured the UBT1 strain, and have isolated two new strains (H1 and P1-2) of very similar phenotypic and genetic characters. These strains show minimal growth on ammonium-free media, and fail to incorporate isotopically labeled N2 gas into biomass in multiple independent assays. The sdn genes previously published as the putative nitrogenase of S. thermoautotrophicus have little similarity to anything found in draft genome sequences, published here, for strains H1 and UBT1, but share >99% nucleotide identity with genes from Hydrogenibacillus schlegelii, a draft genome for which is also presented here. H. schlegelii similarly lacks nitrogenase genes and is a non-diazotroph. We propose reclassification of the species containing strains UBT1, H1, and P1-2 as a non-Streptomycete, non-diazotrophic, facultative chemolithoautotroph and conclude that the existence of the previously proposed oxygen-tolerant nitrogenase is extremely unlikely.

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July 7, 2019

Genomic and metagenomic analysis of microbes in a soil environment affected by the 2011 Great East Japan Earthquake tsunami.

The Great East Japan Earthquake of 2011 triggered large tsunami waves, which flooded broad areas of land along the Pacific coast of eastern Japan and changed the soil environment drastically. However, the microbial characteristics of tsunami-affected soil at the genomic level remain largely unknown. In this study, we isolated microbes from a soil sample using general low-nutrient and seawater-based media to investigate microbial characteristics in tsunami-affected soil.As expected, a greater proportion of strains isolated from the tsunami-affected soil than the unaffected soil grew in the seawater-based medium. Cultivable strains in both the general low-nutrient and seawater-based media were distributed in the genus Arthrobacter. Most importantly, whole-genome sequencing of four of the isolated Arthrobacter strains revealed independent losses of siderophore-synthesis genes from their genomes. Siderophores are low-molecular-weight, iron-chelating compounds that are secreted for iron uptake; thus, the loss of siderophore-synthesis genes indicates that these strains have adapted to environments with high-iron concentrations. Indeed, chemical analysis confirmed the investigated soil samples to be rich in iron, and culture experiments confirmed weak cultivability of some of these strains in iron-limited media. Furthermore, metagenomic analyses demonstrated over-representation of denitrification-related genes in the tsunami-affected soil sample, as well as the presence of pathogenic and marine-living genera and genes related to salt-tolerance.Collectively, the present results would provide an example of microbial characteristics of soil disturbed by the tsunami, which may give an insight into microbial adaptation to drastic environmental changes. Further analyses on microbial ecology after a tsunami are envisioned to develop a deeper understanding of the recovery processes of terrestrial microbial ecosystems.

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July 7, 2019

Genomes and virulence difference between two physiological races of Phytophthora nicotianae.

Black shank is a severe plant disease caused by the soil-borne pathogen Phytophthora nicotianae. Two physiological races of P. nicotianae, races 0 and 1, are predominantly observed in cultivated tobacco fields around the world. Race 0 has been reported to be more aggressive, having a shorter incubation period, and causing worse root rot symptoms, while race 1 causes more severe necrosis. The molecular mechanisms underlying the difference in virulence between race 0 and 1 remain elusive.We assembled and annotated the genomes of P. nicotianae races 0 and 1, which were obtained by a combination of PacBio single-molecular real-time sequencing and second-generation sequencing (both HiSeq and MiSeq platforms). Gene family analysis revealed a highly expanded ATP-binding cassette transporter gene family in P. nicotianae. Specifically, more RxLR effector genes were found in the genome of race 0 than in that of race 1. In addition, RxLR effector genes were found to be mainly distributed in gene-sparse, repeat-rich regions of the P. nicotianae genome.These results provide not only high quality reference genomes of P. nicotianae, but also insights into the infection mechanisms of P. nicotianae and its co-evolution with the host plant. They also reveal insights into the difference in virulence between the two physiological races.

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July 7, 2019

Transcriptional profiling the 150 kb linear megaplasmid of Borrelia turicatae suggests a role in vector colonization and initiating mammalian infection.

Adaptation is key for survival as vector-borne pathogens transmit between the arthropod and vertebrate, and temperature change is an environmental signal inducing alterations in gene expression of tick-borne spirochetes. While plasmids are often associated with adaptation, complex genomes of relapsing fever spirochetes have hindered progress in understanding the mechanisms of vector colonization and transmission. We utilized recent advances in genome sequencing to generate the most complete version of the Borrelia turicatae 150 kb linear megaplasmid (lp150). Additionally, a transcriptional analysis of open reading frames (ORFs) in lp150 was conducted and identified regions that were up-regulated during in vitro cultivation at tick-like growth temperatures (22°C), relative to bacteria grown at 35°C and infected murine blood. Evaluation of the 3′ end of lp150 identified a cluster of ORFs that code for putative surface lipoproteins. With a microbe’s surface proteome serving important roles in pathogenesis, we confirmed the ORFs expression in vitro and in the tick compared to spirochetes infecting murine blood. Transcriptional evaluation of lp150 indicates the plasmid likely has essential roles in vector colonization and/or initiating mammalian infection. These results also provide a much needed transcriptional framework to delineate the molecular mechanisms utilized by relapsing fever spirochetes during their enzootic cycle.

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July 7, 2019

Resolving complex structural genomic rearrangements using a randomized approach.

Complex chromosomal rearrangements are structural genomic alterations involving multiple instances of deletions, duplications, inversions, or translocations that co-occur either on the same chromosome or represent different overlapping events on homologous chromosomes. We present SVelter, an algorithm that identifies regions of the genome suspected to harbor a complex event and then resolves the structure by iteratively rearranging the local genome structure, in a randomized fashion, with each structure scored against characteristics of the observed sequencing data. SVelter is able to accurately reconstruct complex chromosomal rearrangements when compared to well-characterized genomes that have been deeply sequenced with both short and long reads.

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