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July 7, 2019

Comprehensive molecular, genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40.

Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type.We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro).Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

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July 7, 2019

Whole-genome sequence of a Bordetella pertussis Brazilian vaccine strain.

Despite the reduction in incidence after vaccination, pertussis disease is still considered a public health problem worldwide, mainly due to recent and potential new outbreaks. We report here the complete genome of the Bordetella pertussis Butantan strain used in the Brazilian National Immunization Program as a whole-cell pertussis antigen to compose vaccines such as DTwP (diphtheria, tetanus, and whole-cell pertussis).

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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

Biochemical characterization of a Naegleria TET-like oxygenase and its application in single molecule sequencing of 5-methylcytosine.

Modified DNA bases in mammalian genomes, such as 5-methylcytosine ((5m)C) and its oxidized forms, are implicated in important epigenetic regulation processes. In human or mouse, successive enzymatic conversion of (5m)C to its oxidized forms is carried out by the ten-eleven translocation (TET) proteins. Previously we reported the structure of a TET-like (5m)C oxygenase (NgTET1) from Naegleria gruberi, a single-celled protist evolutionarily distant from vertebrates. Here we show that NgTET1 is a 5-methylpyrimidine oxygenase, with activity on both (5m)C (major activity) and thymidine (T) (minor activity) in all DNA forms tested, and provide unprecedented evidence for the formation of 5-formyluridine ((5f)U) and 5-carboxyuridine ((5ca)U) in vitro. Mutagenesis studies reveal a delicate balance between choice of (5m)C or T as the preferred substrate. Furthermore, our results suggest substrate preference by NgTET1 to (5m)CpG and TpG dinucleotide sites in DNA. Intriguingly, NgTET1 displays higher T-oxidation activity in vitro than mammalian TET1, supporting a closer evolutionary relationship between NgTET1 and the base J-binding proteins from trypanosomes. Finally, we demonstrate that NgTET1 can be readily used as a tool in (5m)C sequencing technologies such as single molecule, real-time sequencing to map (5m)C in bacterial genomes at base resolution.

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July 7, 2019

Insights on the emergence of Mycobacterium tuberculosis from the analysis of Mycobacterium kansasii.

By phylogenetic analysis, Mycobacterium kansasii is closely related to Mycobacterium tuberculosis. Yet, although both organisms cause pulmonary disease, M. tuberculosis is a global health menace, whereas M. kansasii is an opportunistic pathogen. To illuminate the differences between these organisms, we have sequenced the genome of M. kansasii ATCC 12478 and its plasmid (pMK12478) and conducted side-by-side in vitro and in vivo investigations of these two organisms. The M. kansasii genome is 6,432,277 bp, more than 2 Mb longer than that of M. tuberculosis H37Rv, and the plasmid contains 144,951 bp. Pairwise comparisons reveal conserved and discordant genes and genomic regions. A notable example of genomic conservation is the virulence locus ESX-1, which is intact and functional in the low-virulence M. kansasii, potentially mediating phagosomal disruption. Differences between these organisms include a decreased predicted metabolic capacity, an increased proportion of toxin-antitoxin genes, and the acquisition of M. tuberculosis-specific genes in the pathogen since their common ancestor. Consistent with their distinct epidemiologic profiles, following infection of C57BL/6 mice, M. kansasii counts increased by less than 10-fold over 6 weeks, whereas M. tuberculosis counts increased by over 10,000-fold in just 3 weeks. Together, these data suggest that M. kansasii can serve as an image of the environmental ancestor of M. tuberculosis before its emergence as a professional pathogen, and can be used as a model organism to study the switch from an environmental opportunistic pathogen to a professional host-restricted pathogen. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

Draft genome sequence of Kitasatospora griseola strain MF730-N6, a bafilomycin, terpentecin, and satosporin producer.

We report here the draft genome sequence of Kitasatospora griseola strain MF730-N6, a known producer of bafilomycin, terpentecin, and satosporins. The current assembly comprises 8 contigs covering 7.97 Mb. Genome annotation revealed 7,225 protein coding sequences, 100 tRNAs, 40 rRNA genes, and 23 secondary metabolite biosynthetic gene clusters. Copyright © 2015 Arens et al.

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July 7, 2019

Best practices in insect genome sequencing: What works and what doesn’t.

The last decade of decreasing DNA sequencing costs and proliferating sequencing services in core labs and companies has brought the de-novo genome sequencing and assembly of insect species within reach for many entomologists. However, sequence production alone is not enough to generate a high quality reference genome, and in many cases, poor planning can lead to extremely fragmented genome assemblies preventing high quality gene annotation and other desired analyses. Insect genomes can be problematic to assemble, due to combinations of high polymorphism, inability to breed for genome homozygocity, and small physical sizes limiting the quantity of DNA able to be isolated from a single individual. Recent advances in sequencing technology and assembly strategies are enabling a revolution for insect genome reference sequencing and assembly. Here we review historical and new genome sequencing and assembly strategies, with a particular focus on their application to arthropod genomes. We highlight both the need to design sequencing strategies for the requirements of the assembly software, and new long-read technologies that are enabling a return to traditional assembly approaches. Finally, we compare and contrast very cost effective short read draft genome strategies with the long read approaches that although entailing additional cost, bring a higher likelihood of success and the possibility of archival assembly qualities approaching that of finished genomes.

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July 7, 2019

Core genome and plasmidome of the quorum-quenching bacterium Rhodococcus erythropolis.

Rhodococcus erythropolis is a worldwide-distributed actinobacterium that exhibits a remarkable metabolic versatility illustrated by its ability to degrade complex compounds, such as quorum-sensing signals N-acylhomoserine lactones (NAHLs), phenols, sterols and fuel derivatives. Because of its catabolic properties, R. erythropolis strains are proposed as anti-biofouling agents against NAHL-dependent biofilms, biocontrol agents against NAHL-emitting plant pathogens, and bioremediation agents in contaminated waters and soils. Here, we used the PacBio technology to resolve the complete genome sequence of the biocontrol strain R. erythropolis R138. Its genome consisted in a circular chromosome (6,236,862 bp), a linear plasmid pLRE138 (477,915 bp) and a circular plasmid pCRE138 (91,729 bp). In addition, draft genomes of five R. erythropolis strains were determined by Illumina technology and compared with the other five R. erythropolis genomes that are available in public databases: 5,825 common CDSs were present in all of the eleven analyzed genomes and represented up to 87 % of those identified in R. erythropolis R138. This study highlighted the high proportion of core-genome genes in R. erythropolis, but a high variability of the plasmid content. Key-metabolic pathways which are involved in the degradation of complex molecules, such as NAHLs and phenol, catechol and sterol derivatives are coded by the R. erythropolis core-genome.

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July 7, 2019

Active site and laminarin binding in glycoside hydrolase family 55.

The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-ß-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 ß-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100-10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ~30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties.© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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July 7, 2019

Genomics of methylotrophy in gram-positive methylamine-utilizing bacteria

Gram-positive methylotrophic bacteria have been known for a long period of time, some serving as model organisms for characterizing the specific details of methylotrophy pathways/enzymes within this group. However, genome-based knowledge of methylotrophy within this group has been so far limited to a single species, Bacillus methanolicus (Firmicutes). The paucity of whole-genome data for Gram-positive methylotrophs limits our global understanding of methylotrophy within this group, including their roles in specific biogeochemical cycles, as well as their biotechnological potential. Here, we describe the isolation of seven novel strains of Gram-positive methylotrophs that include two strains of Bacillus and five representatives of Actinobacteria classified within two genera, Arthrobacter and Mycobacterium. We report whole-genome sequences for these isolates and present comparative analysis of the methylotrophy functional modules within these genomes. The genomic sequences of these seven novel organisms, all capable of growth on methylated amines, present an important reference dataset for understanding the genomic basis of methylotrophy in Gram-positive methylotrophic bacteria. This study is a major contribution to the field of methylotrophy, aimed at closing the gap in the genomic knowledge of methylotrophy within this diverse group of bacteria.

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July 7, 2019

Whole-genome sequencing for comparative genomics and de novo genome assembly.

Next-generation sequencing technologies for whole-genome sequencing of mycobacteria are rapidly becoming an attractive alternative to more traditional sequencing methods. In particular this technology is proving useful for genome-wide identification of mutations in mycobacteria (comparative genomics) as well as for de novo assembly of whole genomes. Next-generation sequencing however generates a vast quantity of data that can only be transformed into a usable and comprehensible form using bioinformatics. Here we describe the methodology one would use to prepare libraries for whole-genome sequencing, and the basic bioinformatics to identify mutations in a genome following Illumina HiSeq or MiSeq sequencing, as well as de novo genome assembly following sequencing using Pacific Biosciences (PacBio).

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July 7, 2019

Saccharina genomes provide novel insight into kelp biology.

Seaweeds are essential for marine ecosystems and have immense economic value. Here we present a comprehensive analysis of the draft genome of Saccharina japonica, one of the most economically important seaweeds. The 537-Mb assembled genomic sequence covered 98.5% of the estimated genome, and 18,733 protein-coding genes are predicted and annotated. Gene families related to cell wall synthesis, halogen concentration, development and defence systems were expanded. Functional diversification of the mannuronan C-5-epimerase and haloperoxidase gene families provides insight into the evolutionary adaptation of polysaccharide biosynthesis and iodine antioxidation. Additional sequencing of seven cultivars and nine wild individuals reveal that the genetic diversity within wild populations is greater than among cultivars. All of the cultivars are descendants of a wild S. japonica accession showing limited admixture with S. longissima. This study represents an important advance toward improving yields and economic traits in Saccharina and provides an invaluable resource for plant genome studies.

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July 7, 2019

Complete genome sequence of Mycoplasma yeatsii strain GM274B (ATCC 43094).

Mycoplasma yeatsii is a goat mycoplasma species that, although an obligate parasite, accommodates this lifestyle as an inapparent commensalist. High-frequency transformation has also been reported for this species. The complete 895,051-bp genome sequence of strain GM274B has been determined, enabling an analysis of the features of this potential cloning host. Copyright © 2015 Calcutt et al.

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