PacBio customers and thought leaders discuss the role SMRT sequencing is playing in comprehensive genomics: past, present, and future. Featuring J. Craig Venter, Gene Myers, Deanna Church, Jeong-Sun Seo and…
To track stepwise changes in genetic diversity and antimicrobial resistance in rapidly evolving OXA-232-producing Klebsiella pneumoniae ST14, an emerging carbapenem-resistant high-risk clone, in clinical settings.Twenty-six K. pneumoniae ST14 isolates were collected by the Korean Nationwide Surveillance of Antimicrobial Resistance system over the course of 1 year. Isolates were subjected to whole-genome sequencing and MIC determinations using 33 antibiotics from 14 classes.Single-nucleotide polymorphism (SNP) typing identified 72 unique SNP sites spanning the chromosomes of the isolates, dividing them into three clusters (I, II and III). The initial isolate possessed two plasmids with 18 antibiotic-resistance genes, including blaOXA-232, and exhibited resistance to 11 antibiotic classes. Four other plasmids containing 12 different resistance genes, including blaCTX-M-15 and strA/B, were introduced over time, providing additional resistance to aztreonam and streptomycin. Moreover, chromosomal integration of insertion sequence Ecp1-blaCTX-M-15 mediated the inactivation of mgrB responsible for colistin resistance in four isolates from cluster III. To the best of our knowledge, this is the first description of K. pneumoniae ST14 resistant to both carbapenem and colistin in South Korea. Furthermore, although some acquired genes were lost over time, the retention of 12 resistance genes and inactivation of mgrB provided resistance to 13 classes of antibiotics.We describe stepwise changes in OXA-232-producing K. pneumoniae ST14 in vivo over time in terms of antimicrobial resistance. Our findings contribute to our understanding of the evolution of emerging high-risk K. pneumoniae clones and provide reference data for future outbreaks.Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Although many ß-agarases that hydrolyze the ß-1,4 linkages of agarose have been biochemically characterized, only three a-agarases that hydrolyze the a-1,3 linkages are reported to date. In this study, a new a-agarase, AgaWS5, from Catenovulum sediminis WS1-A, a new agar-degrading marine bacterium, was biochemically characterized. AgaWS5 belongs to the glycoside hydrolase (GH) 96 family. AgaWS5 consists of 1295 amino acids (140 kDa) and has the 65% identity to an a-agarase, AgaA33, obtained from an agar-degrading bacterium Thalassomonas agarivorans JAMB-A33. AgaWS5 showed the maximum activity at a pH and temperature of 8 and 40 °C, respectively. AgaWS5 showed a cold-tolerance, and it retained more than 40% of its maximum enzymatic activity at 10 °C. AgaWS5 is predicted to have several calcium-binding sites. Thus, its activity was slightly enhanced in the presence of Ca2+, and was strongly inhibited by EDTA. The Km and Vmax of AgaWS5 for agarose were 10.6 mg/mL and 714.3 U/mg, respectively. Agarose-liquefication, thin layer chromatography, and mass and NMR spectroscopic analyses demonstrated that AgaWS5 is an endo-type a-agarase that degrades agarose and mainly produces agarotetraose. Thus, in this study, a novel cold-adapted GH96 agarotetraose-producing a-agarase was identified.
Horizontal transfer of plasmids encoding antimicrobial-resistance and virulence determinants has been instrumental in Staphylococcus aureus evolution, including the emergence of community-associated methicillin-resistant S. aureus (CA-MRSA). In the early 1990s the first CA-MRSA isolated in Western Australia (WA), WA-5, encoded cadmium, tetracycline and penicillin-resistance genes on plasmid pWBG753 (~30 kb). WA-5 and pWBG753 appeared only briefly in WA, however, fusidic-acid-resistance plasmids related to pWBG753 were also present in the first European CA-MRSA at the time. Here we characterized a 72-kb conjugative plasmid pWBG731 present in multiresistant WA-5-like clones from the same period. pWBG731 was a cointegrant formed from pWBG753 and a pWBG749-family conjugative plasmid. pWBG731 carried mupirocin, trimethoprim, cadmium and penicillin-resistance genes. The stepwise evolution of pWBG731 likely occurred through the combined actions of IS257, IS257-dependent miniature inverted-repeat transposable elements (MITEs) and the BinL resolution system of the ß-lactamase transposon Tn552 An evolutionary intermediate ~42-kb non-conjugative plasmid pWBG715, possessed the same resistance genes as pWBG731 but retained an integrated copy of the small tetracycline-resistance plasmid pT181. IS257 likely facilitated replacement of pT181 with conjugation genes on pWBG731, thus enabling autonomous transfer. Like conjugative plasmid pWBG749, pWBG731 also mobilized non-conjugative plasmids carrying oriT mimics. It seems likely that pWBG731 represents the product of multiple recombination events between the WA-5 pWBG753 plasmid and other mobile genetic elements present in indigenous CA-MSSA. The molecular evolution of pWBG731 saliently illustrates how diverse mobile genetic elements can together facilitate rapid accrual and horizontal dissemination of multiresistance in S. aureus CA-MRSA.Copyright © 2019 American Society for Microbiology.
Genome-wide association studies (GWAS) have identified many genomic loci associated with risk for schizophrenia, but unambiguous identification of the relationship between disease-associated variants and specific genes, and in particular their effect on risk conferring transcripts, has proven difficult. To better understand the specific molecular mechanism(s) at the schizophrenia locus in 11q25, we undertook cis expression quantitative trait loci (cis-eQTL) mapping for this 2 megabase genomic region using postmortem human brain samples. To comprehensively assess the effects of genetic risk upon local expression, we evaluated multiple transcript features: genes, exons, and exon-exon junctions in multiple brain regions-dorsolateral prefrontal cortex (DLPFC), hippocampus, and caudate. Genetic risk variants strongly associated with expression of SNX19 transcript features that tag multiple rare classes of SNX19 transcripts, whereas they only weakly affected expression of an exon-exon junction that tags the majority of abundant transcripts. The most prominent class of SNX19 risk-associated transcripts is predicted to be overexpressed, defined by an exon-exon splice junction between exons 8 and 10 (junc8.10) and that is predicted to encode proteins that lack the characteristic nexin C terminal domain. Risk alleles were also associated with either increased or decreased expression of multiple additional classes of transcripts. With RACE, molecular cloning, and long read sequencing, we found a number of novel SNX19 transcripts that further define the set of potential etiological transcripts. We explored epigenetic regulation of SNX19 expression and found that DNA methylation at CpG sites near the primary transcription start site and within exon 2 partially mediate the effects of risk variants on risk-associated expression. ATAC sequencing revealed that some of the most strongly risk-associated SNPs are located within a region of open chromatin, suggesting a nearby regulatory element is involved. These findings indicate a potentially complex molecular etiology, in which risk alleles for schizophrenia generate epigenetic alterations and dysregulation of multiple classes of SNX19 transcripts.
Azospirillum sp. strains TSA2S and TSH100 are plant growth-promoting rhizobacteria with the capacity to mitigate N2O from agricultural soil. They were isolated from the rhizosphere of paddy soil in Tokyo, Japan. Here, we present the genome sequences of these two strains.Copyright © 2019 Gao et al.
The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in S. hawaiiensis NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4S,2R)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional clpP homologous gene (named clpPADEP) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpPADEP in three ADEP-sensitive Streptomyces species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant.IMPORTANCE Antibiotic acyldepsipeptides (ADEPs) represent a promising new class of potent antibiotics and, at the same time, are valuable tools to study the molecular functioning of their target, ClpP, the proteolytic core of the bacterial caseinolytic protease. Here, we present a straightforward purification procedure for ADEP1 that yields substantial amounts of the pure compound in a time- and cost-efficient manner, which is a prerequisite to conveniently study the antimicrobial effects of ADEP and the operating mode of bacterial ClpP machineries in diverse bacteria. Identification and characterization of the ADEP biosynthetic gene cluster in Streptomyces hawaiiensis NRRL 15010 enables future bioinformatics screenings for similar gene clusters and/or subclusters to find novel natural compounds with specific substructures. Most strikingly, we identified a cluster-associated clpP homolog (named clpPADEP) as an ADEP resistance gene. ClpPADEP constitutes a novel bacterial resistance factor that alone is necessary and sufficient to confer high-level ADEP resistance to Streptomyces across species.Copyright © 2019 American Society for Microbiology.
Here, we report the full-genome sequence of an NAD-hemin-independent Avibacterium paragallinarum serovar C-2 strain, FARPER-174, isolated from layer hens in Peru. This genome contained 12 potential genomic islands that include ribosomal protein-coding genes, a nadR gene, hemocin-coding genes, sequences of fagos, an rtx operon, and drug resistance genes. Copyright © 2019 Tataje-Lavanda et al.
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.
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.
A novel facultative anaerobic, Gram-stain-negative coccus, designated strain ChDC B345T, was isolated from human pericoronitis lesion and was characterized by polyphasic taxonomic analysis. The 16S ribosomal RNA gene (16S rDNA) sequence revealed that the strain belonged to the genus Streptococcus. The 16S rDNA sequence of strain ChDC B345T was most closely related to those of Streptococcus mitis NCTC 12261T (99.5%) and Streptococcus pseudopneumoniae ATCC BAA-960T (99.5%). Complete genome of strain ChDC B345T was 1,972,471 bp in length and the G?+?C content was 40.2 mol%. Average nucleotide identity values between strain ChDC B345T and S. pseudopneumoniae ATCC BAA-960T or S. mitis NCTC 12261T were 92.17% and 93.63%, respectively. Genome-to-genome distance values between strain ChDC B345T and S. pseudopneumoniae ATCC BAA-960T or S. mitis NCTC 12261T were 47.8% (45.2-50.4%) and 53.0% (51.0-56.4%), respectively. Based on these results, strain ChDC B345T (=?KCOM 1679T?=?JCM 33299T) should be classified as a novel species of genus Streptococcus, for which we propose the name Streptococcus gwangjuense sp. nov.
The stingless bee Melipona bicolor is the only bee in which true polygyny occurs. Its mitochondrial genome was first sequenced in 2008, but it was incomplete and no information about its transcription was known. We combined short and long reads of M. bicolor DNA with RNASeq data to obtain insights about mitochondrial evolution and gene expression in bees. The complete genome has 15,001?bp, including a control region of 255?bp that contains all conserved structures described in honeybees with the highest AT content reported so far for bees (98.1%), displaying a compact but functional region. Gene expression control is similar to other insects however unusual patterns of expression may suggest the existence of different isoforms for the mitochondrially encoded 12S rRNA. Results reveal unique and shared features of the mitochondrial genome in terms of sequence evolution and gene expression making M. bicolor an interesting model to study mitochondrial genomic evolution. Copyright © 2019 Elsevier B.V. All rights reserved.
We report a family with progressive myoclonic epilepsy who underwent whole-exome sequencing but was negative for pathogenic variants. Similar clinical courses of a devastating neurodegenerative phenotype of two affected siblings were highly suggestive of a genetic etiology, which indicates that the survey of genetic variation by whole-exome sequencing was not comprehensive. To investigate the presence of a variant that remained unrecognized by standard genetic testing, PacBio long-read sequencing was performed. Structural variant (SV) detection using low-coverage (6×) whole-genome sequencing called 17,165 SVs (7,216 deletions and 9,949 insertions). Our SV selection narrowed down potential candidates to only five SVs (two deletions and three insertions) on the genes tagged with autosomal recessive phenotypes. Among them, a 12.4-kb deletion involving the CLN6 gene was the top candidate because its homozygous abnormalities cause neuronal ceroid lipofuscinosis. This deletion included the initiation codon and was found in a GC-rich region containing multiple repetitive elements. These results indicate the presence of a causal variant in a difficult-to-sequence region and suggest that such variants that remain enigmatic after the application of current whole-exome sequencing technology could be uncovered by unbiased application of long-read whole-genome sequencing.
The fungus Penicillium purpurogenum grows on a variety of natural carbon sources and secretes a large number of enzymes which degrade the polysaccharides present in lignocellulose. In this work, the gene coding for a novel endoxylanase has been identified in the genome of the fungus. This gene (xynd) possesses four introns. The cDNA has been expressed in Pichia pastoris and characterized. The enzyme, XynD, belongs to family 10 of the glycoside hydrolases. Mature XynD has a calculated molecular weight of 40,997. It consists of 387 amino acid residues with an N-terminal catalytic module, a linker rich in ser and thr residues, and a C-terminal family 1 carbohydrate-binding module. XynD shows the highest identity (97%) to a putative endoxylanase from Penicillium subrubescens but its highest identity to a biochemically characterized xylanase (XYND from Penicillium funiculosum) is only 68%. The enzyme has a temperature optimum of 60 °C, and it is highly stable in its pH optimum range of 6.5-8.5. XynD is the fourth biochemically characterized endoxylanase from P. purpurogenum, confirming the rich potential of this fungus for lignocellulose biodegradation. XynD, due to its wide pH optimum and stability, may be a useful enzyme in biotechnological procedures related to this biodegradation process.
Allelic differences in A and B mating-type loci are a prerequisite for the progression of mating in the genus Pleurotus eryngii; thus, the crossing is hampered by this biological barrier in inbreeding. Molecular markers linked to mating types of P. eryngii KNR2312 were investigated with randomly amplified polymorphic DNA to enhance crossing efficiency. An A4-linked sequence was identified and used to find the adjacent genomic region with the entire motif of the A locus from a contig sequenced by PacBio. The sequence-characterized amplified region marker 7-2299 distinguished A4 mating-type monokaryons from KNR2312 and other strains. A BLAST search of flanked sequences revealed that the A4 locus had a general feature consisting of the putative HD1 and HD2 genes. Both putative HD transcription factors contain a homeodomain sequence and a nuclear localization sequence; however, valid dimerization motifs were found only in the HD1 protein. The ACAAT motif, which was reported to have relevance to sex determination, was found in the intergenic region. The SCAR marker could be applicable in the classification of mating types in the P. eryngii breeding program, and the A4 locus could be the basis for a multi-allele detection marker.
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