Phytoplankton consortia as a blueprint for mutually beneficial eukaryote-bacteria ecosystems based on the biocoenosis of Botryococcus consortia.

Bacteria occupy all major ecosystems and maintain an intensive relationship to the eukaryotes, developing together into complex biomes (i.e., phycosphere and rhizosphere). Interactions between eukaryotes and bacteria range from cooperative to competitive, with the associated microorganisms affecting their host`s development, growth and health. Since the advent of non-culture dependent analytical techniques such as metagenome sequencing, consortia have been described at the phylogenetic level but rarely functionally. Multifaceted analysis of the microbial consortium of the ancient phytoplankton Botryococcus as an attractive model food web revealed that its all abundant bacterial members belong to a niche of biotin auxotrophs, essentially depending on the microalga. In addition, hydrocarbonoclastic bacteria without vitamin auxotrophies seem adversely to affect the algal cell morphology. Synthetic rearrangement of a minimal community consisting of an alga, a mutualistic and a parasitic bacteria underpins the model of a eukaryote that maintains its own mutualistic microbial community to control its surrounding biosphere. This model of coexistence, potentially useful for defense against invaders by a eukaryotic host could represent ecologically relevant interactions that cross species boundaries. Metabolic and system reconstruction is an opportunity to unravel the relationships within the consortia and provide a blueprint for the construction of mutually beneficial synthetic ecosystems.

Whole-Genome Sequence of Pseudoalteromonas sp. NC201, a Probiotic Strain for Litopenaeus stylirostris Hatcheries in New Caledonia.

The marine bacterium Pseudoalteromonas sp. strain NC201 has shown probiotic potential in Litopenaeus stylirostris rearing. In this study, the complete genome of NC201 was sequenced. This genome consists of a chromosome (4.13 Mb) and a chromid (1.24 Mb). The genome contains gene clusters coding for antibacterial peptides and secondary metabolites.

Biosynthetic reconstitution of deoxysugar phosphoramidate metalloprotease inhibitors using an N-P-bond-forming kinase.

Phosphoramidon is a potent metalloprotease inhibitor and a widespread tool in cell biology research. It contains a dipeptide backbone that is uniquely linked to a 6-deoxysugar via a phosphoramidate bridge. Herein, we report the identification of a gene cluster for the formation of phosphoramidon and its detailed characterization. In vitro reconstitution of the biosynthesis established TalE as a phosphoramidate-forming kinase and TalC as the glycosyltransferase which installs the l-rhamnose moiety by phosphoester linkage.

Fabry disease: Detection of Alu-mediated exon duplication by NGS.

Monogenetic diseases can be analyzed routinely by targeted DNA sequencing. If causative variants are not found, complementary methods like RNA sequencing or analysis of copy number variations by multiplex ligation-dependent probe amplification have to be considered. In the latter, especially exonic duplications or deletions can be detected, but the precise sites of mutations remain unclear. As we demonstrate in this casuistic report of Fabry disease, next-generation sequencing (NGS) of a long-range PCR product can identify the recombination site directly and illuminate the underlying molecular mechanism.

Bioinformatics in Germany: toward a national-level infrastructure.

The German Network for Bioinformatics Infrastructure (de.NBI) is a national initiative funded by the German Federal Ministry of Education and Research (BMBF). The mission of de.NBI is (i) to provide high-quality bioinformatics services to users in basic and applied life sciences research from academia, industry and biomedicine; (ii) to offer bioinformatics training to users in Germany and Europe through a wide range of workshops and courses; and (iii) to foster the cooperation of the German bioinformatics community with international network structures such as the European life-sciences Infrastructure for biological Information (ELIXIR). The network was launched by the BMBF in March 2015 and now includes 40 service projects operated by 30 project partners that are organized in eight service centers. The de.NBI staff develops further and maintains almost 100 bioinformatics services for the human, plant and microbial research fields and provides comprehensive training courses to support users with different expertise levels in bioinformatics. In the future, de.NBI will expand its activities to the European level, as the de.NBI consortium was assigned by the BMBF to establish and run the German node of ELIXIR.

Complete Genome Sequence of Streptomyces lavendulae subsp. lavendulae CCM 3239 (Formerly "Streptomyces aureofaciens CCM 3239"), a Producer of the Angucycline-Type Antibiotic Auricin.

Streptomyces lavendulae subsp. lavendulae CCM 3239 produces the angucycline antibiotic auricin and was thought to be the type strain of Streptomyces aureofaciens We report the complete genome sequence of this strain, which consists of a linear chromosome and the linear plasmid pSA3239, and demonstrate it to be S. lavendulae subsp. lavendulae.

The secreted metabolome of Streptomyces chartreusis and implications for bacterial chemistry.

Actinomycetes are known for producing diverse secondary metabolites. Combining genomics with untargeted data-dependent tandem MS and molecular networking, we characterized the secreted metabolome of the tunicamycin producer Streptomyces chartreusis NRRL 3882. The genome harbors 128 predicted biosynthetic gene clusters. We detected >1,000 distinct secreted metabolites in culture supernatants, only 22 of which were identified based on standards and public spectral libraries. S. chartreusis adapts the secreted metabolome to cultivation conditions. A number of metabolites are produced iron dependently, among them 17 desferrioxamine siderophores aiding in iron acquisition. Eight previously unknown members of this long-known compound class are described. A single desferrioxamine synthesis gene cluster was detected in the genome, yet different sets of desferrioxamines are produced in different media. Additionally, a polyether ionophore, differentially produced by the calcimycin biosynthesis cluster, was discovered. This illustrates that metabolite output of a single biosynthetic machine can be exquisitely regulated not only with regard to product quantity but also with regard to product range. Compared with chemically defined medium, in complex medium, total metabolite abundance was higher, structural diversity greater, and the average molecular weight almost doubled. Tunicamycins, for example, were only produced in complex medium. Extrapolating from this study, we anticipate that the larger part of bacterial chemistry, including chemical structures, ecological functions, and pharmacological potential, is yet to be uncovered.

Isolation and whole genome analysis of endospore-forming bacteria from heroin.

Infections caused by endospore-forming bacteria have been associated with severe illness and death among persons who inject drugs. Analysis of the bacteria residing in heroin has thus been biased towards species that affect human health. Similarly, exploration of the bacterial diversity of seized street market heroin correlated with the skin microflora of recreational heroin users insofar as different Staphylococus spp. or typical environmental endospore formers including Bacillus cereus and other Bacilli outside the B. cereus sensu lato group as well as diverse Clostridia were identified. In this work 82 samples of non-street market ("wholesale") heroin originating from the German Federal Criminal Police Office's heroin analysis program seized during the period between 2009 and 2014 were analyzed for contaminating bacteria. Without contact with the end user and with only little contaminations introduced by final processing, adulteration and cutting this heroin likely harbors original microbiota from the drug's original source or trafficking route. We found this drug to be only sparsely populated with retrievable heterotrophic, aerobic bacteria. In total, 68 isolates were retrieved from 49 out of 82 samples analyzed (60% culture positive). All isolates were endospore-forming, Gram-positive Bacilli. Completely absent were non-endospore-formers or Gram-negatives. The three most predominant species were Bacillus clausii, Bacillus (para)licheniformis, and Terribacillus saccharophilus. Whole genome sequencing of these 68 isolates was performed using Illumina technology. Sequence data sets were assembled and annotated using an automated bioinformatics pipeline. Average nucleotide identity (ANI) values were calculated for all draft genomes and all close to identical genomes (ANI>99.5%) were compared to the forensic data of the seized drug, showing positive correlations that strongly warrant further research on this subject.

Complete genome sequence of Streptomyces reticuli, an efficient degrader of crystalline cellulose.

We report the complete, GC-rich genome sequence of the melanin producer Streptomyces reticuli Tü 45 (S. reticuli) that targets and degrades highly crystalline cellulose by the concerted action of a range of biochemically characterized proteins. It consists of a linear 8.3 Mb chromosome, a linear 0.8 Mb megaplasmid, a linear 94 kb plasmid and a circular 76 kb plasmid. Noteworthy, the megaplasmid is the second largest known Streptomyces plasmid. Preliminary analysis reveals, among others, 43 predicted gene clusters for the synthesis of secondary metabolites and 456 predicted genes for binding and degradation of cellulose, other polysaccharides and carbohydrate-containing compounds.

Revisiting Corynebacterium glyciniphilum (ex Kubota et al., 1972) sp. nov., nom. rev., isolated from putrefied banana.

A strain of a species of the genus Corynebacterium, designated AJ 3170(T), was isolated during the 1980s from putrefied bananas. Since then, there have been no further updates on the description of the strain or its phylogenetic classification. However, phylogenetic analysis of this strain using 16S rRNA and in silico DNA-DNA hybridization has confirmed that it is a member of the genus Corynebacterium and that strain AJ 3170(T) clusters with Corynebacterium variabile DSM 44702(T), Corynebacterium terpenotabidum Y-11(T) and Corynebacterium nuruki S6-4(T) in one subgroup. Furthermore, a combination of enzymatic, chemical, and morphological characterization techniques was applied in order to describe strain AJ 3170(T) further. The strain grew well at pH values of 6-10 and at temperatures of 30-41 °C. The major fatty acids were C16 : 0 (42.15 %), C18 : 1ω9c (41.6 %) and C18 : 0 10-methyl (TBSA) (8.56 %). The whole-cell sugars were determined to comprise galactose, arabinose and ribose. On the basis of this phenotypic, chemotaxonomic and phylogenetic characterization, it is proposed that strain AJ 3170(T) represents a novel species, for which the name Corynebacterium glyciniphilum sp. nov. is proposed; the type strain is AJ 3170(T) ( = DSM 45795(T) = ATCC 21341(T)).

Complete genome sequence of Corynebacterium vitaeruminis DSM 20294T, isolated from the cow rumen as a vitamin B producer.

We report the complete genome sequence of Corynebacterium vitaeruminis DSM 20294(T) which was identified as the producer of B vitamins in the rumen of cows. The genome of C. vitaeruminis DSM 20294(T) consists of a single replicon, the chromosome with a size of 2,931,780 bp and a G+C content of 65.53%. The genome encodes for 2,580 protein coding genes, among them those for a complete pathway to synthesize biotin.

Genome sequence of the squalene-degrading bacterium Corynebacterium terpenotabidum type strain Y-11(T) (= DSM 44721(T)).

Corynebacterium terpenotabidum Takeuchi et. al 1999 is a member of the genus Corynebacterium, which contains Gram-positive and non-spore forming bacteria with a high G+C content. C. terpenotabidum was isolated from soil based on its ability to degrade squalene and belongs to the aerobic and non-hemolytic Corynebacteria. It displays tolerance to salts (up to 8%) and is related to Corynebacterium variabile involved in cheese ripening. As this is a type strain of Corynebacterium, this project describing the 2.75 Mbp long chromosome with its 2,369 protein-coding and 72 RNA genes will aid the G enomic E ncyclopedia of Bacteria and Archaea project.

Reconstruction of the lipid metabolism for the microalga Monoraphidium neglectum from its genome sequence reveals characteristics suitable for biofuel production.

BACKGROUND: Microalgae are gaining importance as sustainable production hosts in the fields of biotechnology and bioenergy. A robust biomass accumulating strain of the genus Monoraphidium (SAG 48.87) was investigated in this work as a potential feedstock for biofuel production. The genome was sequenced, annotated, and key enzymes for triacylglycerol formation were elucidated. RESULTS: Monoraphidium neglectum was identified as an oleaginous species with favourable growth characteristics as well as a high potential for crude oil production, based on neutral lipid contents of approximately 21% (dry weight) under nitrogen starvation, composed of predominantly C18:1 and C16:0 fatty acids. Further characterization revealed growth in a relatively wide pH range and salt concentrations of up to 1.0% NaCl, in which the cells exhibited larger structures. This first full genome sequencing of a member of the Selenastraceae revealed a diploid, approximately 68 Mbp genome with a G + C content of 64.7%. The circular chloroplast genome was assembled to a 135,362 bp single contig, containing 67 protein-coding genes. The assembly of the mitochondrial genome resulted in two contigs with an approximate total size of 94 kb, the largest known mitochondrial genome within algae. 16,761 protein-coding genes were assigned to the nuclear genome. Comparison of gene sets with respect to functional categories revealed a higher gene number assigned to the category "carbohydrate metabolic process" and in "fatty acid biosynthetic process" in M. neglectum when compared to Chlamydomonas reinhardtii and Nannochloropsis gaditana, indicating a higher metabolic diversity for applications in carbohydrate conversions of biotechnological relevance. CONCLUSIONS: The genome of M. neglectum, as well as the metabolic reconstruction of crucial lipid pathways, provides new insights into the diversity of the lipid metabolism in microalgae. The results of this work provide a platform to encourage the development of this strain for biotechnological applications and production concepts.

Complete genome sequence of Saccharothrix espanaensis DSM 44229(T) and comparison to the other completely sequenced Pseudonocardiaceae.

BACKGROUND: The genus Saccharothrix is a representative of the family Pseudonocardiaceae, known to include producer strains of a wide variety of potent antibiotics. Saccharothrix espanaensis produces both saccharomicins A and B of the promising new class of heptadecaglycoside antibiotics, active against both bacteria and yeast. RESULTS: To better assess its capabilities, the complete genome sequence of S. espanaensis was established. With a size of 9,360,653 bp, coding for 8,501 genes, it stands alongside other Pseudonocardiaceae with large genomes. Besides a predicted core genome of 810 genes shared in the family, S. espanaensis has a large number of accessory genes: 2,967 singletons when compared to the family, of which 1,292 have no clear orthologs in the RefSeq database. The genome analysis revealed the presence of 26 biosynthetic gene clusters potentially encoding secondary metabolites. Among them, the cluster coding for the saccharomicins could be identified. CONCLUSION: S. espanaensis is the first completely sequenced species of the genus Saccharothrix. The genome discloses the cluster responsible for the biosynthesis of the saccharomicins, the largest oligosaccharide antibiotic currently identified. Moreover, the genome revealed 25 additional putative secondary metabolite gene clusters further suggesting the strain's potential for natural product synthesis.

Pangenomic study of Corynebacterium diphtheriae that provides insights into the genomic diversity of pathogenic isolates from cases of classical diphtheria, endocarditis, and pneumonia.

Corynebacterium diphtheriae is one of the most prominent human pathogens and the causative agent of the communicable disease diphtheria. The genomes of 12 strains isolated from patients with classical diphtheria, endocarditis, and pneumonia were completely sequenced and annotated. Including the genome of C. diphtheriae NCTC 13129, we herewith present a comprehensive comparative analysis of 13 strains and the first characterization of the pangenome of the species C. diphtheriae. Comparative genomics showed extensive synteny and revealed a core genome consisting of 1,632 conserved genes. The pangenome currently comprises 4,786 protein-coding regions and increases at an average of 65 unique genes per newly sequenced strain. Analysis of prophages carrying the diphtheria toxin gene tox revealed that the toxoid vaccine producer C. diphtheriae Park-Williams no. 8 has been lysogenized by two copies of the ω(tox)(+) phage, whereas C. diphtheriae 31A harbors a hitherto-unknown tox(+) corynephage. DNA binding sites of the tox-controlling regulator DtxR were detected by genome-wide motif searches. Comparative content analysis showed that the DtxR regulons exhibit marked differences due to gene gain, gene loss, partial gene deletion, and DtxR binding site depletion. Most predicted pathogenicity islands of C. diphtheriae revealed characteristics of horizontal gene transfer. The majority of these islands encode subunits of adhesive pili, which can play important roles in adhesion of C. diphtheriae to different host tissues. All sequenced isolates contain at least two pilus gene clusters. It appears that variation in the distributed genome is a common strategy of C. diphtheriae to establish differences in host-pathogen interactions.

Genome sequence of the halotolerant bacterium Corynebacterium halotolerans type strain YIM 70093(T) (= DSM 44683(T)).

Corynebacterium halotolerans Chen et al. 2004 is a member of the genus Corynebacterium which contains Gram-positive bacteria with a high G+C content. C. halotolerans, isolated from a saline soil, belongs to the non-lipophilic, non-pathogenic corynebacteria. It displays a high tolerance to salts (up to 25%) and is related to the pathogenic corynebacteria C. freneyi and C. xerosis. As this is a type strain in a subgroup of Corynebacterium without complete genome sequences, this project describing the 3.14 Mbp long chromosome and the 86.2 kbp plasmid pCha1 with their 2,865 protein-coding and 65 RNA genes will aid the Genomic Encyclopedia ofBacteria andArchaea project.

Whole genome sequencing of environmental Vibrio cholerae O1 from 10 nanograms of DNA using short reads.

Multiple Displacement Amplification (MDA) of DNA using φ29 (phi29) DNA polymerase amplifies DNA several billion-fold, which has proved to be potentially very useful for evaluating genome information in a culture-independent manner. Whole genome sequencing using DNA from a single prokaryotic genome copy amplified by MDA has not yet been achieved due to the formation of chimeras and skewed amplification of genomic regions during the MDA step, which then precludes genome assembly. We have hereby addressed the issue by using 10 ng of genomic Vibrio cholerae DNA extracted within an agarose plug to ensure circularity as a starting point for MDA and then sequencing the amplified yield using the SOLiD platform. We successfully managed to assemble the entire genome of V. cholerae strain LMA3984-4 (environmental O1 strain isolated in urban Amazonia) using a hybrid de novo assembly strategy. Using our method, only 178 out of 16,713 (1%) of contigs were not able to be inserted into either chromosome scaffold, and out of these 178, only 3 appeared to be chimeras. The other contigs seem to be the result of template-independent non-specific amplification during MDA, yielding spurious reads. Extraction of genomic DNA within an agarose plug in order to ensure circularity of the extracted genome might be key to minimizing amplification bias by MDA for WGS.

Comparative analysis of two complete Corynebacterium ulcerans genomes and detection of candidate virulence factors.

BACKGROUND: Corynebacterium ulcerans has been detected as a commensal in domestic and wild animals that may serve as reservoirs for zoonotic infections. During the last decade, the frequency and severity of human infections associated with C. ulcerans appear to be increasing in various countries. As the knowledge of genes contributing to the virulence of this bacterium was very limited, the complete genome sequences of two C. ulcerans strains detected in the metropolitan area of Rio de Janeiro were determined and characterized by comparative genomics: C. ulcerans 809 was initially isolated from an elderly woman with fatal pulmonary infection and C. ulcerans BR-AD22 was recovered from a nasal sample of an asymptomatic dog. RESULTS: The circular chromosome of C. ulcerans 809 has a total size of 2,502,095 bp and encodes 2,182 predicted proteins, whereas the genome of C. ulcerans BR-AD22 is 104,279 bp larger and comprises 2,338 protein-coding regions. The minor difference in size of the two genomes is mainly caused by additional prophage-like elements in the C. ulcerans BR-AD22 chromosome. Both genomes show a highly similar order of orthologous coding regions; and both strains share a common set of 2,076 genes, demonstrating their very close relationship. A screening for prominent virulence factors revealed the presence of phospholipase D (Pld), neuraminidase H (NanH), endoglycosidase E (EndoE), and subunits of adhesive pili of the SpaDEF type that are encoded in both C. ulcerans genomes. The rbp gene coding for a putative ribosome-binding protein with striking structural similarity to Shiga-like toxins was additionally detected in the genome of the human isolate C. ulcerans 809. CONCLUSIONS: The molecular data deduced from the complete genome sequences provides considerable knowledge of virulence factors in C. ulcerans that is increasingly recognized as an emerging pathogen. This bacterium is apparently equipped with a broad and varying set of virulence factors, including a novel type of a ribosome-binding protein. Whether the respective protein contributes to the severity of human infections (and a fatal outcome) remains to be elucidated by genetic experiments with defined bacterial mutants and host model systems.