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July 7, 2019

Genomic insights into the taxonomic status of the three subspecies of Bacillus subtilis.

Bacillus subtilis contains three subspecies, i.e., subspecies subtilis, spizizenii, and inaquosorum. As these subspecies are phenotypically indistinguishable, their differentiation has relied on phylogenetic analysis of multiple protein-coding gene sequences. B. subtilis subsp. inaquosorum is a recently proposed taxon that encompasses strain KCTC 13429(T) and related strains, which were previously classified as members of subspecies spizizenii. However, DNA-DNA hybridization (DDH) values among the three subspecies raised a question as to their independence. Thus, we evaluated the taxonomic status of subspecies inaquosorum using genome-based comparative analysis. In contrast to the previous experimental values of DDH, the inter-genomic relatedness inferred by average nucleotide identity (ANI) values indicated that subspecies inaquosorum and spizizenii were sufficiently different from subspecies subtilis and hence raised the possibility that the former two could be classified as separate species from B. subtilis. The genome-based tree also supported the separation of the two subspecies from B. subtilis. The exclusive presence of a subtilin synthesis system in subspecies spizizenii was a remarkable genetic characteristic that could even distinguish subspecies spizizenii from subspecies inaquosorum in addition to the low ANI values (<95%). Conclusively, the genome-based data obtained in this study demonstrated that subspecies inaquosorum and spizizenii are clearly distinguished from subspecies subtilis, and raises the possibility that these two subspecies could be classified as separate species from B. subtilis. In addition, the low ANI values between subspecies inaquosorum and spizizenii and the exclusive presence of subtilin synthesis genes in subspecies spizizenii also suggest circumscription of these two subspecies at the species level. Copyright © 2013 Elsevier GmbH. All rights reserved.

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July 7, 2019

The effects of read length, quality and quantity on microsatellite discovery and primer development: from Illumina to PacBio.

The advent of next-generation sequencing (NGS) technologies has transformed the way microsatellites are isolated for ecological and evolutionary investigations. Recent attempts to employ NGS for microsatellite discovery have used the 454, Illumina, and Ion Torrent platforms, but other methods including single-molecule real-time DNA sequencing (Pacific Biosciences or PacBio) remain viable alternatives. We outline a workflow from sequence quality control to microsatellite marker validation in three plant species using PacBio circular consensus sequencing (CCS). We then evaluate the performance of PacBio CCS in comparison with other NGS platforms for microsatellite isolation, through simulations that focus on variations in read length, read quantity and sequencing error rate. Although quality control of CCS reads reduced microsatellite yield by around 50%, hundreds of microsatellite loci that are expected to have improved conversion efficiency to functional markers were retrieved for each species. The simulations quantitatively validate the advantages of long reads and emphasize the detrimental effects of sequencing errors on NGS-enabled microsatellite development. In view of the continuing improvement in read length on NGS platforms, sequence quality and the corresponding strategies of quality control will become the primary factors to consider for effective microsatellite isolation. Among current options, PacBio CCS may be optimal for rapid, small-scale microsatellite development due to its flexibility in scaling sequencing effort, while platforms such as Illumina MiSeq will provide cost-efficient solutions for multispecies microsatellite projects. © 2014 John Wiley & Sons Ltd.

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July 7, 2019

Draft genome sequence of Kluyveromyces marxianus strain DMB1, isolated from sugarcane bagasse hydrolysate.

We determined the genome sequence of a thermotolerant yeast, Kluyveromyces marxianus strain DMB1, isolated from sugarcane bagasse hydrolysate, and the sequence provides further insights into the genomic differences between this strain and other reported K. marxianus strains. The genome described here is composed of 11,165,408 bases and has 4,943 protein-coding genes. Copyright © 2014 Suzuki et al.

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July 7, 2019

The dynamics of SecM-induced translational stalling.

SecM is an E. coli secretion monitor capable of stalling translation on the prokaryotic ribosome without cofactors. Biochemical and structural studies have demonstrated that the SecM nascent chain interacts with the 50S subunit exit tunnel to inhibit peptide bond formation. However, the timescales and pathways of stalling on an mRNA remain undefined. To provide a dynamic mechanism for stalling, we directly tracked the dynamics of elongation on ribosomes translating the SecM stall sequence (FSTPVWISQAQGIRAGP) using single-molecule fluorescence techniques. Within 1 min, three peptide-ribosome interactions work cooperatively over the last five codons of the SecM sequence, leading to severely impaired elongation rates beginning from the terminal proline and lasting four codons. Our results suggest that stalling is tightly linked to the dynamics of elongation and underscore the roles that the exit tunnel and nascent chain play in controlling fundamental steps in translation. opyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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July 7, 2019

A fault-tolerant method for HLA typing with PacBio data.

Human leukocyte antigen (HLA) genes are critical genes involved in important biomedical aspects, including organ transplantation, autoimmune diseases and infectious diseases. The gene family contains the most polymorphic genes in humans and the difference between two alleles is only a single base pair substitution in many cases. The next generation sequencing (NGS) technologies could be used for high throughput HLA typing but in silico methods are still needed to correctly assign the alleles of a sample. Computer scientists have developed such methods for various NGS platforms, such as Illumina, Roche 454 and Ion Torrent, based on the characteristics of the reads they generate. However, the method for PacBio reads was less addressed, probably owing to its high error rates. The PacBio system has the longest read length among available NGS platforms, and therefore is the only platform capable of having exon 2 and exon 3 of HLA genes on the same read to unequivocally solve the ambiguity problem caused by the “phasing” issue.We proposed a new method BayesTyping1 to assign HLA alleles for PacBio circular consensus sequencing reads using Bayes’ theorem. The method was applied to simulated data of the three loci HLA-A, HLA-B and HLA-DRB1. The experimental results showed its capability to tolerate the disturbance of sequencing errors and external noise reads.The BayesTyping1 method could overcome the problems of HLA typing using PacBio reads, which mostly arise from sequencing errors of PacBio reads and the divergence of HLA genes, to some extent.

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July 7, 2019

Complete closed genome sequences of three Bibersteinia trehalosi nasopharyngeal isolates from cattle with shipping fever.

Bibersteinia trehalosi is a respiratory pathogen affecting cattle and related ruminants worldwide. B. trehalosi is closely related to Mannheimia haemolytica and is often associated with bovine respiratory disease complex (BRDC), a polymicrobial multifactorial disease. We present three complete closed genome sequences of this species generated using an automated assembly pipeline.

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July 7, 2019

Draft genome sequence of Kitasatospora cheerisanensis KCTC 2395, which produces plecomacrolide against phytopathogenic fungi.

Kitasatospora cheerisanensis KCTC 2395, which produces antifungal metabolites with bafilomycin derivatives, including bafilomycin C1-amide, was isolated from a soil sample at Mt. Jiri, South Korea. Here, we report its draft genome sequence, which contains 8.04 Mb with 73.6% G+C content and 7,810 protein-coding genes. Copyright © 2014 Hwang et al.

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July 7, 2019

Dubowitz syndrome is a complex comprised of multiple, genetically distinct and phenotypically overlapping disorders.

Dubowitz syndrome is a rare disorder characterized by multiple congenital anomalies, cognitive delay, growth failure, an immune defect, and an increased risk of blood dyscrasia and malignancy. There is considerable phenotypic variability, suggesting genetic heterogeneity. We clinically characterized and performed exome sequencing and high-density array SNP genotyping on three individuals with Dubowitz syndrome, including a pair of previously-described siblings (Patients 1 and 2, brother and sister) and an unpublished patient (Patient 3). Given the siblings’ history of bone marrow abnormalities, we also evaluated telomere length and performed radiosensitivity assays. In the siblings, exome sequencing identified compound heterozygosity for a known rare nonsense substitution in the nuclear ligase gene LIG4 (rs104894419, NM_002312.3:c.2440C>T) that predicts p.Arg814X (MAF:0.0002) and an NM_002312.3:c.613delT variant that predicts a p.Ser205Leufs*29 frameshift. The frameshift mutation has not been reported in 1000 Genomes, ESP, or ClinSeq. These LIG4 mutations were previously reported in the sibling sister; her brother had not been previously tested. Western blotting showed an absence of a ligase IV band in both siblings. In the third patient, array SNP genotyping revealed a de novo ~ 3.89 Mb interstitial deletion at chromosome 17q24.2 (chr 17:62,068,463-65,963,102, hg18), which spanned the known Carney complex gene PRKAR1A. In all three patients, a median lymphocyte telomere length of = 1st centile was observed and radiosensitivity assays showed increased sensitivity to ionizing radiation. Our work suggests that, in addition to dyskeratosis congenita, LIG4 and 17q24.2 syndromes also feature shortened telomeres; to confirm this, telomere length testing should be considered in both disorders. Taken together, our work and other reports on Dubowitz syndrome, as currently recognized, suggest that it is not a unitary entity but instead a collection of phenotypically similar disorders. As a clinical entity, Dubowitz syndrome will need continual re-evaluation and re-definition as its constituent phenotypes are determined.

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July 7, 2019

Strain Kaplan of pseudorabies virus genome sequenced by PacBio single-molecule real-time sequencing technology.

Pseudorabies virus (PRV) is a neurotropic herpesvirus that causes Aujeszky’s disease in pigs. PRV strains are widely used as transsynaptic tracers for mapping neural circuits. We present here the complete and fully annotated genome sequence of strain Kaplan of PRV, determined by Pacific Biosciences RSII long-read sequencing technology. Copyright © 2014 Tombácz et al.

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July 7, 2019

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence.

Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.

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July 7, 2019

The oxygen-independent metabolism of cyclic monoterpenes in Castellaniella defragrans 65Phen.

The facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic ß-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway.Castellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95 Mb contained a 70 kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58EuT, than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate.The genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen.

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July 7, 2019

Improved draft genome sequence of Clostridium pasteurianum strain ATCC 6013 (DSM 525) using a hybrid next-generation sequencing approach.

We present an improved draft genome sequence for Clostridium pasteurianum strain ATCC 6013 (DSM 525), the type strain of the species and an important solventogenic bacterium with industrial potential. Availability of a near-complete genome sequence will enable strain engineering of this promising bacterium. Copyright © 2014 Pyne et al.

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