Non-contiguous finished genome sequence and description of Clostridium dakarense sp. nov.

Clostridium dakarense strain FF1T, is the type strain of Clostridium dakarense sp. nov., a new species within the genus Clostridium. This strain, whose genome is described here, was isolated from the fecal flora of a 4-month-old Senegalese child suffering from gastroenteritis. C. dakarense sp. nov. strain FF1T is an obligate anaerobic Gram-positive bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,735,762 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 27.98% and contains 3,843 protein-coding and 73 RNA genes, including 8 rRNA genes.


Introduction
Clostridium dakarense strain FF1 T (= CSUR P243 = DSM 27086), is the type strain of Clostridium dakarense sp. nov. This bacterium is a Grampositive, anaerobic, spore-forming, indole negative bacillus that was isolated from the stool of a 4-month-old Senegalese child suffering from gastroenteritis as part of a "culturomics" study aiming at cultivating individually all species within human feces. The elevated cost and lack of intra-and interlaboratory reproducibility of the "gold standard" of taxonomic tools. i.e. DNA-DNA hybridization and G+C content determination [1], put bacterial taxonomic classification in a precarious state. In addition, the internationally-validated cutoff values of 16S rRNA sequence comparison [2] do not apply to all validly published genera and species. Recently, high throughput genome sequencing and mass spectrometric analyses of bacteria have allowed unprecedented access to a wealth of genetic and proteomic information [3]. As a consequence, we proposed to use a polyphasic approach [4] to describe new bacterial taxa, including genome sequence, MALDI-TOF spectrum and main phenotypic characteristics [5][6][7][8][9][10][11]. The genus Clostridium (Prazmowski, 1880), classified among the Firmicutes, was created in 1880 [12] and consists of obligate anaerobic rod-shaped bacilli capable of producing endospores [12]. More than 180 Clostridium species have been described to date [13]. Members of the genus Clostridium are mostly environmental bacteria or associated with the commensal digestive flora of mammals, but several are major human pathogens, including C. botulinum, C. difficile, C. tetani and C. perfringens [14,15]. A few species, such as C. butyricum and C. pasteurianum, fix nitrogen and have gained importance in agricultural and industrial applications [16,17]. Here we present a summary classification and a set of features for C. dakarense sp. nov. strain FF1 T (= CSUR P243 = DSM 27086) together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the species C. dakarense sp. nov.

Classification and features
A stool specimen was collected from a 4-month-old Senegalese child suffering from gastroenteritis. Informed consent was obtained from the child's parents and approval from the ethics committee from the Institut Federatif de Recherche 48 (Faculté de Médecine, Marseille, France). The fecal specimen was preserved at -20°C after collection and sent to Marseille. Strain FF1 T (Table 1) was isolated in July 2011 by anaerobic cultivation on 5% sheep bloodenriched Columbia agar (BioMerieux, Marcy l'Etoile, France). This strain exhibited a 96.90% 16S rRNA nucleotide sequence similarity with C. lituseburense, the phylogenetically closest validated Clostridium species (Figure 1). Although sequence similarity of the 16S rRNA is not uniform across taxa, this value was lower than the 98.7% threshold recommended by Stackebrandt and Ebers to delineate a new species without carrying out DNA-DNA hybridization [30]. In addition, it was consistent with 16S rRNA identity values observed among validated species within the Clostridium genus that range from 78.4 to 99.8%. , not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [29]. If the evidence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements. Different growth temperatures (25, 30, 37, 45 and 56°C) were tested. Growth was observed between 25 and 37°C, with optimal growth at 37°C after 24 hours of inoculation in anaerobic conditions. Colonies were 1.5 mm in diameter and opaque and smooth appearance on blood-enriched Columbia agar. Growth of the strain was tested under anaerobic and microaerophilic conditions using GENbag anaer and GENbag microaer systems, respectively (BioMerieux), and under aerobic conditions, with or without 5% CO 2 . The strain growth was obtained only in anaerobic conditions. Gram staining showed rod-shaped Gram-positive bacilli able to form spores ( Figure 2). The motility test was positive. Cells grown on agar have a mean diameter of 1.2 µm (Figure 3). Strain FF1 T exhibited neither catalase nor oxidase activities. Using API Rapid ID 32A (BioMerieux, Marcy l'Etoile), a positive reaction were observed for arginine dihydrolase, N-acetyl-β-glucosaminidase and pyroglutamic acid arylamidase. Negative reactions were observed for urease, indole and nitrate reduction. Using API 50 CH (BioMerieux, Marcy l'Etoile), positive reactions were observed for galactose, glucose, maltose and saccharose fermentation and negative reaction were observed for ribose, lactose and fructose. C. dakarense is susceptible to amoxicillin, metronidazole, vancomycin, imipenem and rifampicin and resistant to trimethoprim/ sulfamethoxazole. When compared with representative species from the genus Clostridium, C. dakarense strain FF1 T exhibited the phenotypic differences detailed in Table 2.   Oxygen  Matrix-assisted laser-desorption/ionization timeof-flight (MALDI-TOF) MS protein analysis was carried out as previously described [31]. Briefly, a pipette tip was used to pick one isolated bacterial colony from a culture agar plate, and to spread it as a thin film on a MTP 384 MALDI-TOF target plate (Bruker Daltonics, Leipzig, Germany). Eighteen distinct deposits were made for strain FF T from eighteen isolated colonies. Each smear was overlaid with 2 µL of matrix solution (saturated solution of alpha-cyano-4-hydroxycinnamic acid) in 50% acetonitrile, 2.5% tri-fluoracetic-acid, and allowed to dry for five minutes. Measurements were performed with a Microflex spectrometer (Bruker). Spectra were recorded in the positive linear mode for the mass range of 2,000 to 20,000 Da (parameter settings: ion source 1 (IS1), 20 kV; IS2, 18.5 kV; lens, 7 kV). A spectrum was obtained after 675 shots at a variable laser power. The time of acquisition was between 30 seconds and 1 minute per spot. The eighteen spectra were imported into the MALDI BioTyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against the main spectra of 4,706 bacteria including 216 spectra from validly published species of Clostridium, that are part of the reference data contained in the BioTyper database. The method of identification included the m/z from 2,000 to 20,000 Da. For every spectrum, 100 peaks at most were taken into account and compared with spectra in the database. A score enabled the identification, or not, from the tested species: a score > 2 with a validly published species enabled the identification at the species level, and a score < 1.7 did not enable any identification at the genus level. For strain FF1 T , the maximal obtained score was lower than 1.9, thus suggesting that our isolate was not a member of a known species. We added the spectrum from strain FF1 T to our database for future reference ( Figure 4). Finally, the gel view allows us to highlight the spectrum differences with other members of the genus Clostridium ( Figure 5).  The Gel View displays the raw spectra of all loaded spectrum files arranged in a pseudo-gel like look. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed by a Gray scale scheme code. The color bar and the right y-axis indicate the relation between the color a peak is displayed with and the peak intensity in arbitrary units.

Genome sequencing information Genome project history
The organism was selected for sequencing on the basis of its phylogenetic position and 16S rRNA similarity to other members of the genus Clostridium, and is part of a "culturomics" study of the human digestive flora aiming at isolating all bacterial species within human feces. It was the 94 th genome of a Clostridium species and the first genome of Clostridium dakarense sp. nov. The Genbank accession number is CBTZ00000000 and consists of 257 contigs. Table 3 shows the project information and its association with MIGS version 2.0 compliance [32].

Genome sequencing and assembly
This project was loaded twice on a 1/4 region for the paired-end application and once on a 1/8 region for the shotgun on PTP Picotiterplates. The shotgun library was constructed with 500 ng of DNA as described by the manufacturer (Roche) with the GS Rapid library Prep kit. For the pairedend sequencing, 5 µg of DNA was mechanically fragmented on the Hydroshear device (Digilab, Holliston, MA, USA) with an enrichment size of 3-4kb. The DNA fragmentation was visualized using an Agilent 2100 BioAnalyzer on a DNA labchip 7500, which yield an optimal size of 3.6 kb. The library was constructed according to the 454_Titanium paired-end protocol and manufacturer. Circularization and nebulization were performed and generated a pattern with an optimum at 561 bp. After PCR amplification through 15 cycles followed by double size selection, the single stranded paired end library was then quantified with Quant-it Ribogreen kit (Invitrogen) on the Genios_Tecan fluorometer at 52 pg/µL. The library concentration equivalence was calculated as 1.7E+08 molecules/µL. The library was stored at -20°C until use.
The shotgun library was clonally amplified with 3cpb in 3 emPCR reactions and the paired end library was amplified with lower cpb (1cpb) in 4 emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2. The yield of the emPCR was 5.37% for the shotgun reads and 19.27% for the pairedend reads, according to the quality expected by the range of 5 to 20% from the Roche procedure. A total of 340,000 beads from the 1/8 region of the shotgun reads and 790,000 beads from the 1/4 region of the paired-end reads were loaded on the GS Titanium PicoTiterPlates (PTP Kit 70×75) and sequenced with the GS Titanium Sequencing Kit XLR70.
The runs were performed overnight and then analyzed on the cluster through the gsRunBrowser and gsAssembler_Roche. The global 383,079 passed filter sequences generated 96.50 Mb with a length average of 277 bp. These sequences were assembled using the Newbler software from Roche with 90% identity and 40 bp as overlap. Fourteen scaffolds and 257 large contigs (>1500bp) were obtained, for a genome size of 3,735,762 bp.

Genome annotation
Open Reading Frames (ORFs) were predicted using Prodigal [33] with default parameters but the predicted ORFs were excluded if they spanned a sequencing gap region. The predicted bacterial protein sequences were searched against the GenBank database [34] and the Clusters of Orthologous Groups (COG) databases using BLASTP. The tRNAScanSE tool [35] was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer [36] and BLASTn against the GenBank database. Lipoprotein signal peptides and numbers of transmembrane helices were predicted using SignalP [37] and TMHMM [38] respectively. ORFans were identified if their BLASTP E-value was lower than 1e -03 for alignment length greater than 80 amino acids. If alignment lengths Standards in Genomic Sciences were smaller than 80 amino acids, we used an Evalue of 1e-05. Such parameter thresholds have already been used in previous works to define ORFans. Artemis [39] was used for data management and DNA Plotter [40] was used for visualization of genomic features. Mauve alignment tool was used for multiple genomic sequence alignment and visualization [41].

Genome properties
The genome of C. dakarense sp. nov. strain FF1 T is 3,735,762 bp long (1 chromosome, but no plasmid) with a 27,98% G + C content of ( Figure 6 and Table 4). Of the 3,916 predicted genes, 3,843 protein-coding genes, and 73 were RNAs. Eight rRNA genes (one 16S rRNA, one 23S rRNA and six 5S rRNA) and 65 predicted tRNA genes were identified in the genome. A total of 2,769 genes (72.05%) were assigned a putative function (by COG or NR blast). Two hundred ninety-eight genes were identified as ORFans (7.75%). The remaining 515 genes were annotated as hypothetical proteins (13, 40%). The distribution of genes into COGs functional categories is presented in Table 4. The properties and the statistics of the genome are summarized in Tables 4 and 5. a The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome

Comparison with the genomes from other Clostridium species
The genome sequence of Clostridium sp. is currently available for more than seventy-five Clostridium species. Here we compared the genome sequence of C. dakarense strain FF1 T with than those of C.  The total is based on the total number of protein coding genes in the annotated genome.

Conclusion
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Clostridium dakarense sp. nov. which contains strain FF1 T . This bacterium strain has been isolated from the fecal flora of a 4-months-old Senegalese child suffering from gastroenteritis.

Description of Clostridium senegalense sp. nov.
Clostridium dakarense (da.kar.e′n.se. L. gen. neutr. n. dakarense, pertaining to, or originating from Dakar, the capital of Senegal, where the type strain was isolated).
Colonies were 1.5 mm in diameter on bloodenriched Columbia agar and Chocolate agar + PolyViteX. Cells are rod-shaped with a mean diameter of 1.2 μm. Optimal growth is achieved anaerobically. No growth is observed in aerobic conditions. Growth occurs between 25-37°C, with optimal growth observed at 37°C, in medium 5% sheep blood-enriched Columbia agar. Cells stain Gram-positive, are endospore-forming, and motile. Catalase, oxidase, urease, indole and nitrate reduction activity are absent. Arginine dihydrolase, Nacetyl-β-glucosanimidase and pyroglutamic acid arylamidase activity are present. Cells are susceptible to amoxicillin, metronidazole, vancomycin, imipenem and rifampicin but resistant to trimethoprim/sulfamethoxazole.
The G+C content of the genome is 27.98%. The 16S rRNA gene sequence and whole-genome shotgun sequence of C. dakarense strain FF1 T (= CSUR P243 = DSM 27086) are deposited in GenBank under accession numbers KC517358 and CBTZ00000000, respectively. The type strain FF1 T (= CSUR P243 = DSM 27086) was isolated from the fecal flora of a 4-months-old child in Dakar, Senegal.