Arwa Al-Dilaimi

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.

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

BackgroundCorynebacterium 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.ResultsThe 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.ConclusionsThe 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.

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

BackgroundThe 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.ResultsTo 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.ConclusionS. 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.

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.

The secreted metabolome of Streptomyces chartreusis and implications for bacterial chemistry

Significance Bacterial secondary metabolites are of great relevance to human society and the environment. To this day, investigations of secreted metabolites focus on single compounds, compound classes, or compounds with specific bioactivities. Comparing the supernatants of Streptomyces chartreusis cultivated in different media, using liquid chromatography–coupled tandem MS, we detected a great diversity of highly regulated compounds surpassing genome-based expectations. Guided by molecular networking, a new polyether ionophore was identified and subsequently purified and characterized. The approach presented here provides a basis for structure analysis for molecules produced in amounts too low for standard methods of structure elucidation. Simultaneously, it facilitates the differential analysis of secreted metabolomes, providing insights into the chemical profiles under different cultivation conditions. 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.

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.

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 GenomicEncyclopedia ofBacteria andArchaea project.

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

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 GenomicEncyclopedia ofBacteria andArchaea project.

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)).