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July 7, 2019

Unlocking the biological potential of Euglena gracilis: evolution, cell biology and significance to parasitism

Photosynthetic euglenids are major components of aquatic ecosystems and relatives of trypanosomes. Euglena gracilis has considerable biotechnological potential and great adaptability, but exploitation remains hampered by the absence of a comprehensive gene catalogue. We address this by genome, RNA and protein sequencing: the E. gracilis genome is >2Gb, with 36,526 predicted proteins. Large lineage-specific paralog families are present, with evidence for flexibility in environmental monitoring, divergent mechanisms for metabolic control, and novel solutions for adaptation to extreme environments. Contributions from photosynthetic eukaryotes to the nuclear genome, consistent with the shopping bag model are found, together with transitions between kinetoplastid and canonical systems. Control of protein expression is almost exclusively post-transcriptional. These data are a major advance in understanding the nuclear genomes of euglenids and provide a platform for investigating the contributions of E. gracilis and its relatives to the biosphere.

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July 7, 2019

Linear peptides are the major products of a biosynthetic pathway that encodes for cyclic depsipeptides.

Three new dentigerumycin analogues are produced by Streptomyces sp. M41, a bacterium isolated from a South African termite, Macrotermes natalensis. The structures of the complex nonribosomal peptide synthetase-polyketide synthase (NRPS/PKS) hybrid compounds were determined by 1D- and 2D-NMR spectroscopy, high-resolution mass spectrometry, and circular dichroism (CD) spectroscopy. Both cyclic and linear peptides are reported, and the genetic organization of the NRPS modules within the biosynthetic gene cluster accounts for the observed structural diversity.

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July 7, 2019

Genetic and functional characterization of an extracellular modular GH6 endo-ß-1,4-glucanase from an earthworm symbiont, Cellulosimicrobium funkei HY-13.

The gene (1608-bp) encoding a GH6 endo-ß-1,4-glucanase (CelL) from the earthworm-symbiotic bacterium Cellulosimicrobium funkei HY-13 was cloned from its whole genome sequence, expressed recombinantly, and biochemically characterized. CelL (56.0 kDa) is a modular enzyme consisting of an N-terminal catalytic GH6 domain (from Val57 to Pro396), which is 71 % identical to a GH6 protein (accession no.: WP_034662937) from Cellulomonas sp. KRMCY2, together with a C-terminal CBM 2 domain (from Cys429 to Cys532). The highest catalytic activity of CelL toward carboxymethylcellulose (CMC) was observed at 50 °C and pH 5.0, and was relatively stable at a broad pH range of 4.0-10.0. The enzyme was capable of efficiently hydrolyzing the cellulosic polymers in the order of barley ß-1,3-1,4-D-glucan > CMC > lichenan > Avicel > konjac glucomannan. However, cellobiose, cellotriose, p-nitrophenyl derivatives of mono- and disaccharides, or structurally unrelated carbohydrate polymers including ß-1,3-D-glucan, ß-1,4-D-galactomannan, and ß-1,4-D-xylan were not susceptible to CelL. The enzymatic hydrolysis of cellopentaose resulted in the production of a mixture of 68.6 % cellobiose and 31.4 % cellotriose but barley ß-1,3-1,4-D-glucan was 100 % degraded to cellotriose by CelL. The enzyme strongly bound to Avicel, ivory nut mannan, and chitin but showed relatively weak binding affinity to lichenan, lignin, or poly(3-hydroxybutyrate) granules.

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July 7, 2019

hybridSPAdes: an algorithm for hybrid assembly of short and long reads.

Recent advances in single molecule real-time (SMRT) and nanopore sequencing technologies have enabled high-quality assemblies from long and inaccurate reads. However, these approaches require high coverage by long reads and remain expensive. On the other hand, the inexpensive short reads technologies produce accurate but fragmented assemblies. Thus, a hybrid approach that assembles long reads (with low coverage) and short reads has a potential to generate high-quality assemblies at reduced cost.We describe hybridSPAdes algorithm for assembling short and long reads and benchmark it on a variety of bacterial assembly projects. Our results demonstrate that hybridSPAdes generates accurate assemblies (even in projects with relatively low coverage by long reads) thus reducing the overall cost of genome sequencing. We further present the first complete assembly of a genome from single cells using SMRT reads.hybridSPAdes is implemented in C++?as a part of SPAdes genome assembler and is publicly available at http://bioinf.spbau.ru/en/spades CONTACT: d.antipov@spbu.ruSupplementary information: supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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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

Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation.

Butanol is currently one of the most discussed biofuels. Its use provides many benefits in comparison to bio-ethanol, but the price of its fermentative production is still high. Genetic improvements could help solve many problems associated with butanol production during ABE fermentation, such as its toxicity, low concentration achievable in the cultivation medium, the need for a relatively expensive substrate, and many more. Clostridium pasteurianum NRRL B-598 is non-type strain producing butanol, acetone, and a negligible amount of ethanol. Its main benefits are high oxygen tolerance, utilization of a wide range of carbon and nitrogen sources, and the availability of its whole genome sequence. However, there is no established method for the transfer of foreign DNA into this strain; this is the next step necessary for progress in its use for butanol production.We have described functional protocols for conjugation and transformation of the bio-butanol producer C. pasteurianum NRRL B-598 by foreign plasmid DNA. We show that the use of unmethylated plasmid DNA is necessary for efficient transformation or successful conjugation. Genes encoding DNA methylation and those for restriction-modification systems and antibiotic resistance were searched for in the whole genome sequence and their homologies with other clostridial bacteria were determined. Furthermore, activity of described novel type I restriction system was proved experimentally. The described electrotransformation protocol achieved an efficiency 1.2 × 10(2) cfu/µg DNA after step-by-step optimization and an efficiency of 1.6 × 10(2) cfu/µg DNA was achieved by the sonoporation technique using a standard laboratory ultrasound bath. The highest transformation efficiency was achieved using a combination of these approaches; sono/electroporation led to an increase in transformation efficiency, to 5.3 × 10(2) cfu/µg DNA.Both Dam and Dcm methylations are detrimental for transformation of C. pasteurianum NRRL B-598. Methods for conjugation, electroporation, sonoporation, and a combined method for sono/electroporation were established for this strain. The methods described could be used for genetic improvement of this strain, which is suitable for bio-butanol production.

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July 7, 2019

Complete genome of Streptomyces hygroscopicus subsp. limoneus KCTC 1717 (=KCCM 11405), a soil bacterium producing validamycin and diverse secondary metabolites.

Streptomyces hygroscopicus subsp. limoneus is a Gram-positive, aerobic, aerial mycelial, spore-forming bacterium that was first isolated from a soil sample in Akashi City, Hyogo Prefecture, Japan. We here report the complete genome of S. hygroscopicus subsp. limoneus KCTC 1717 (=KCCM 11405=IFO 12704=ATCC 21432), which consists of 10,537,932bp (G+C content of 71.96%) with two linear chromosomes, 8983 protein-coding genes, 67 tRNAs and 6 rRNA operons. Genes related to biosynthesis of validamycin, valienamine and diverse secondary metabolites were detected in this genome. Genomic data is thus expected to considerably improve our understanding of how industrially important aminocyclitols are biosynthesized by microbial cells. Copyright © 2015 Elsevier B.V. All rights reserved.

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July 7, 2019

The genome analysis of Candidatus Burkholderia crenata reveals that secondary metabolism may be a key function of the Ardisia crenata leaf nodule symbiosis.

A majority of Ardisia species harbour Burkholderia sp. bacteria within specialized leaf nodules. The bacteria are transmitted hereditarily and have not yet been cultured outside of their host. Because the plants cannot develop beyond the seedling stage without their symbionts, the symbiosis is considered obligatory. We sequenced for the first time the genome of Candidatus Burkholderia crenata (Ca. B. crenata), the leaf nodule symbiont of Ardisia crenata. The genome of Ca. B. crenata is the smallest Burkholderia genome to date. It contains a large amount of insertion sequences and pseudogenes and displays features consistent with reductive genome evolution. The genome does not encode functions commonly associated with plant symbioses such as nitrogen fixation and plant hormone metabolism. However, we identified unique genes with a predicted role in secondary metabolism in the genome of Ca. B. crenata. Specifically, we provide evidence that the bacterial symbionts are responsible for the synthesis of compound FR900359, a cyclic depsipeptide with biomedical properties previously isolated from leaves of A.?crenata. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

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July 7, 2019

Genome sequence of Shimia str. SK013, a representative of the Roseobacter group isolated from marine sediment.

Shimia strain SK013 is an aerobic, Gram-negative, rod shaped alphaproteobacterium affiliated with the Roseobacter group within the family Rhodobacteraceae. The strain was isolated from surface sediment (0-1 cm) of the Skagerrak at 114 m below sea level. The 4,049,808 bp genome of Shimia str. SK013 comprises 3,981 protein-coding genes and 47 RNA genes. It contains one chromosome and no extrachromosomal elements. The genome analysis revealed the presence of genes for a dimethylsulfoniopropionate lyase, demethylase and the trimethylamine methyltransferase (mttB) as well as genes for nitrate, nitrite and dimethyl sulfoxide reduction. This indicates that Shimia str. SK013 is able to switch from aerobic to anaerobic metabolism and thus is capable of aerobic and anaerobic sulfur cycling at the seafloor. Among the ability to convert other sulfur compounds it has the genetic capacity to produce climatically active dimethyl sulfide. Growth on glutamate as a sole carbon source results in formation of cell-connecting filaments, a putative phenotypic adaptation of the surface-associated strain to the environmental conditions at the seafloor. Genome analysis revealed the presence of a flagellum (fla1) and a type IV pilus biogenesis, which is speculated to be a prerequisite for biofilm formation. This is also related to genes responsible for signalling such as N-acyl homoserine lactones, as well as quip-genes responsible for quorum quenching and antibiotic biosynthesis. Pairwise similarities of 16S rRNA genes (98.56 % sequence similarity to the next relative S. haliotis) and the in silico DNA-DNA hybridization (21.20 % sequence similarity to S. haliotis) indicated Shimia str. SK013 to be considered as a new species. The genome analysis of Shimia str. SK013 offered first insights into specific physiological and phenotypic adaptation mechanisms of Roseobacter-affiliated bacteria to the benthic environment.

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July 7, 2019

Metabolomics-guided analysis of isocoumarin production by Streptomyces species MBT76 and biotransformation of flavonoids and phenylpropanoids.

Actinomycetes produce the majority of the antibiotics currently in clinical use. The efficiency of antibiotic production is affected by multiple factors such as nutrients, pH, temperature and growth phase. Finding the optimal harvesting time is crucial for successful isolation of the desired bioactive metabolites from actinomycetes, but for this conventional chemical analysis has limitations due to the metabolic complexity. This study explores the utility of NMR-based metabolomics for (1) optimizing fermentation time for the production of known and/or unknown bioactive compounds produced by actinomycetes; (2) elucidating the biosynthetic pathway for microbial natural products; and (3) facilitating the biotransformation of nature-abundant chemicals.

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