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July 7, 2019

Short communication: Single molecule, real-time sequencing technology revealed species- and strain-specific methylation patterns of 2 Lactobacillus strains.

Pacific Biosciences’ (Menlo Park, CA) single molecule, real-time sequencing technology was reported to have some advantages in generating finished genomes and characterizing the epigenome of bacteria. In the present study, this technology was used to sequence 2 Lactobacillus strains, Lactobacillus casei Zhang and Lactobacillus plantarum P-8. Previously, the former bacterium was sequenced by an Applied Biosystems 3730 DNA analyzer (Grand Island, NY), whereas the latter one was analyzed with Roche 454 (Indianapolis, IN) and Illumina sequencing technologies (San Diego, CA). The results showed that single molecule, real-time sequencing resulted in high-quality, finished genomes for both strains. Interestingly, epigenome analysis indicates the presence of 1 active N(6)-methyladenine methyltransferase in L. casei Zhang, but none in L. plantarum P-8. Our study revealed for the first time a completely different methylation pattern in 2 Lactobacillus strains. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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July 7, 2019

Draft genome sequences of gammaproteobacterial methanotrophs isolated from lake washington sediment.

The genomes of Methylosarcina lacus LW14(T) (=ATCC BAA-1047(T) = JCM 13284(T)), Methylobacter sp. strain 21/22, Methylobacter sp. strain 31/32, Methylomonas sp. strain LW13, Methylomonas sp. strain MK1, and Methylomonas sp. strain 11b were sequenced and are reported here. All the strains are obligately methanotrophic bacteria isolated from the sediment of Lake Washington. Copyright © 2015 Kalyuzhnaya et al.

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July 7, 2019

The genome of Dendrobium officinale illuminates the biology of the important traditional Chinese orchid herb.

Dendrobium officinale Kimura et Migo is a traditional Chinese orchid herb that has both ornamental value and a broad range of therapeutic effects. Here, we report the first de novo assembled 1.35 Gb genome sequences for D. officinale by combining the second-generation Illumina Hiseq 2000 and third-generation PacBio sequencing technologies. We found that orchids have a complete inflorescence gene set and have some specific inflorescence genes. We observed gene expansion in gene families related to fungus symbiosis and drought resistance. We analyzed biosynthesis pathways of medicinal components of D. officinale and found extensive duplication of SPS and SuSy genes, which are related to polysaccharide generation, and that the pathway of D. officinale alkaloid synthesis could be extended to generate 16-epivellosimine. The D. officinale genome assembly demonstrates a new approach to deciphering large complex genomes and, as an important orchid species and a traditional Chinese medicine, the D. officinale genome will facilitate future research on the evolution of orchid plants, as well as the study of medicinal components and potential genetic breeding of the dendrobe. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

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July 7, 2019

GAML: genome assembly by maximum likelihood.

Resolution of repeats and scaffolding of shorter contigs are critical parts of genome assembly. Modern assemblers usually perform such steps by heuristics, often tailored to a particular technology for producing paired or long reads.We propose a new framework that allows systematic combination of diverse sequencing datasets into a single assembly. We achieve this by searching for an assembly with the maximum likelihood in a probabilistic model capturing error rate, insert lengths, and other characteristics of the sequencing technology used to produce each dataset. We have implemented a prototype genome assembler GAML that can use any combination of insert sizes with Illumina or 454 reads, as well as PacBio reads. Our experiments show that we can assemble short genomes with N50 sizes and error rates comparable to ALLPATHS-LG or Cerulean. While ALLPATHS-LG and Cerulean require each a specific combination of datasets, GAML works on any combination.We have introduced a new probabilistic approach to genome assembly and demonstrated that this approach can lead to superior results when used to combine diverse set of datasets from different sequencing technologies. Data and software is available at http://compbio.fmph.uniba.sk/gaml.

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July 7, 2019

BreakSeek: a breakpoint-based algorithm for full spectral range INDEL detection.

Although recent developed algorithms have integrated multiple signals to improve sensitivity for insertion and deletion (INDEL) detection, they are far from being perfect and still have great limitations in detecting a full size range of INDELs. Here we present BreakSeek, a novel breakpoint-based algorithm, which can unbiasedly and efficiently detect both homozygous and heterozygous INDELs, ranging from several base pairs to over thousands of base pairs, with accurate breakpoint and heterozygosity rate estimations. Comprehensive evaluations on both simulated and real datasets revealed that BreakSeek outperformed other existing methods on both sensitivity and specificity in detecting both small and large INDELs, and uncovered a significant amount of novel INDELs that were missed before. In addition, by incorporating sophisticated statistic models, we for the first time investigated and demonstrated the importance of handling false and conflicting signals for multi-signal integrated methods.© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 7, 2019

The Streptomyces leeuwenhoekii genome: de novo sequencing and assembly in single contigs of the chromosome, circular plasmid pSLE1 and linear plasmid pSLE2.

Next Generation DNA Sequencing (NGS) and genome mining of actinomycetes and other microorganisms is currently one of the most promising strategies for the discovery of novel bioactive natural products, potentially revealing novel chemistry and enzymology involved in their biosynthesis. This approach also allows rapid insights into the biosynthetic potential of microorganisms isolated from unexploited habitats and ecosystems, which in many cases may prove difficult to culture and manipulate in the laboratory. Streptomyces leeuwenhoekii (formerly Streptomyces sp. strain C34) was isolated from the hyper-arid high-altitude Atacama Desert in Chile and shown to produce novel polyketide antibiotics.Here we present the de novo sequencing of the S. leeuwenhoekii linear chromosome (8 Mb) and two extrachromosomal replicons, the circular pSLE1 (86 kb) and the linear pSLE2 (132 kb), all in single contigs, obtained by combining Pacific Biosciences SMRT (PacBio) and Illumina MiSeq technologies. We identified the biosynthetic gene clusters for chaxamycin, chaxalactin, hygromycin A and desferrioxamine E, metabolites all previously shown to be produced by this strain (J Nat Prod, 2011, 74:1965) and an additional 31 putative gene clusters for specialised metabolites. As well as gene clusters for polyketides and non-ribosomal peptides, we also identified three gene clusters encoding novel lasso-peptides.The S. leeuwenhoekii genome contains 35 gene clusters apparently encoding the biosynthesis of specialised metabolites, most of them completely novel and uncharacterised. This project has served to evaluate the current state of NGS for efficient and effective genome mining of high GC actinomycetes. The PacBio technology now permits the assembly of actinomycete replicons into single contigs with >99 % accuracy. The assembled Illumina sequence permitted not only the correction of omissions found in GC homopolymers in the PacBio assembly (exacerbated by the high GC content of actinomycete DNA) but it also allowed us to obtain the sequences of the termini of the chromosome and of a linear plasmid that were not assembled by PacBio. We propose an experimental pipeline that uses the Illumina assembled contigs, in addition to just the reads, to complement the current limitations of the PacBio sequencing technology and assembly software.

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July 7, 2019

Essential roles of methionine and S-adenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis.

Multidrug resistance, strong side effects, and compliance problems in TB chemotherapy mandate new ways to kill Mycobacterium tuberculosis (Mtb). Here we show that deletion of the gene encoding homoserine transacetylase (metA) inactivates methionine and S-adenosylmethionine (SAM) biosynthesis in Mtb and renders this pathogen exquisitely sensitive to killing in immunocompetent or immunocompromised mice, leading to rapid clearance from host tissues. Mtb ?metA is unable to proliferate in primary human macrophages, and in vitro starvation leads to extraordinarily rapid killing with no appearance of suppressor mutants. Cell death of Mtb ?metA is faster than that of other auxotrophic mutants (i.e., tryptophan, pantothenate, leucine, biotin), suggesting a particularly potent mechanism of killing. Time-course metabolomics showed complete depletion of intracellular methionine and SAM. SAM depletion was consistent with a significant decrease in methylation at the DNA level (measured by single-molecule real-time sequencing) and with the induction of several essential methyltransferases involved in biotin and menaquinone biosynthesis, both of which are vital biological processes and validated targets of antimycobacterial drugs. Mtb ?metA could be partially rescued by biotin supplementation, confirming a multitarget cell death mechanism. The work presented here uncovers a previously unidentified vulnerability of Mtb-the incapacity to scavenge intermediates of SAM and methionine biosynthesis from the host. This vulnerability unveils an entirely new drug target space with the promise of rapid killing of the tubercle bacillus by a new mechanism of action.

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July 7, 2019

A multidrug resistance plasmid contains the molecular switch for type VI secretion in Acinetobacter baumannii.

Infections with Acinetobacter baumannii, one of the most troublesome and least studied multidrug-resistant superbugs, are increasing at alarming rates. A. baumannii encodes a type VI secretion system (T6SS), an antibacterial apparatus of Gram-negative bacteria used to kill competitors. Expression of the T6SS varies among different strains of A. baumannii, for which the regulatory mechanisms are unknown. Here, we show that several multidrug-resistant strains of A. baumannii harbor a large, self-transmissible resistance plasmid that carries the negative regulators for T6SS. T6SS activity is silenced in plasmid-containing, antibiotic-resistant cells, while part of the population undergoes frequent plasmid loss and activation of the T6SS. This activation results in T6SS-mediated killing of competing bacteria but renders A. baumannii susceptible to antibiotics. Our data show that a plasmid that has evolved to harbor antibiotic resistance genes plays a role in the differentiation of cells specialized in the elimination of competing bacteria.

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July 7, 2019

Sequencing of plasmids pAMBL1 and pAMBL2 from Pseudomonas aeruginosa reveals a blaVIM-1 amplification causing high-level carbapenem resistance.

Carbapenemases are a major concern for the treatment of infectious diseases caused by Gram-negative bacteria. Although plasmids are responsible for the spread of resistance genes among these pathogens, there is limited information on the nature of the mobile genetic elements carrying carbapenemases in Pseudomonas aeruginosa.We combined data from two different next-generation sequencing platforms, Illumina HiSeq2000 and PacBio RSII, to obtain the complete nucleotide sequences of two blaVIM-1-carrying plasmids (pAMBL1 and pAMBL2) isolated from P. aeruginosa clinical isolates.Plasmid pAMBL1 has 26?440 bp and carries a RepA_C family replication protein. pAMBL1 is similar to plasmids pNOR-2000 and pKLC102 from P. aeruginosa and pAX22 from Achromobacter xylosoxidans, which also carry VIM-type carbapenemases. pAMBL2 is a 24?133 bp plasmid with a replication protein that belongs to the Rep_3 family. It shows a high degree of homology with a fragment of the blaVIM-1-bearing plasmid pPC9 from Pseudomonas putida. Plasmid pAMBL2 carries three copies of the blaVIM-1 cassette in an In70 class 1 integron conferring, unlike pAMBL1, high-level resistance to carbapenems.We present two new plasmids coding for VIM-1 carbapenemase from P. aeruginosa and report that the presence of three copies of blaVIM-1 in pAMBL2 produces high-level resistance to carbapenems.© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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