Non-contiguous finished genome sequence and description of Brevibacterium senegalense sp. nov.

Brevibacterium senegalense strain JC43T sp. nov. is the type strain of Brevibacterium senegalense sp. nov., a new species within the Brevibacterium genus. This strain, whose genome is described here, was isolated from the fecal flora of a healthy Senegalese patient. B. senegalense is an aerobic rod-shaped Gram-positive bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,425,960 bp long genome (1 chromosome but no plasmid) contains 3,064 protein-coding and 49 RNA genes.


Introduction
Brevibacterium senegalense strain JC43 T (= CSUR P155 = DSM 25783) is the type strain of B. senegalense. sp. nov. This bacterium is a nonmotile, rod-shaped, Gram-positive, catalasepositive bacterium that was isolated from the stool of a healthy Senegalese patient as part of a study aiming at cultivating individually all bacterial species within human feces.
Bacterial taxonomy has undergone many changes over recent years. The DNA-DNA hybridization and G+C content criteria, once considered as gold standards [1], were gradually replaced by gene sequencing. In particular, 16S rRNA sequencing has deeply changed the way bacteria and archaea are classified [2]. More recently, the development of high throughput genome sequencing methods and mass spectrometric analyses of bacteria have provided a wealth of genetic and proteomic information [3]. We recently used a polyphasic approach [4] that includes genomic data, MALDI-TOF spectrum and major phenotypic characteristics to describe new bacterial species [5][6][7][8][9][10][11].
The genus Brevibacterium  [12] was created in 1953 to gather short non-sporeforming and non-branching rods. To date, this genus is comprised of Gram-positive, irregular, rodshaped, non-acid-fast bacteria, and contains 31 recognized species with validly published names [13]. Brevibacterium is the type genus of the family Brevibacteriaceae   [14]. Members of the genus Brevibacterium are isolated from human samples, dairy products, poultry and environmental specimens. In humans, they are found on skin surfaces [15], but have also been demonstrated to cause rare cases of bacteremia, endocarditis, pericarditis, brain abscess and peritonitis. These infections have been observed mainly in immunocompromised patients [10][11][12][13][14][15][16][17][18][19], with the exception of two cases of bacteremia in immunocompetent patients with central venous catheters [9,19]. To  Here we present a summary classification and a set of features for B. senegalense sp. nov. strain JC43 T together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the B. senegalense species.

Organism information
A stool sample was collected from a healthy 16year-old male Senegalese volunteer patient living in Dielmo (rural village in the Guinean-Sudanian zone in Senegal), who was included in a research protocol. Written assent was obtained from this individual. No written consent was needed from his guardians for this study because he was older than 15 years old (in accordance with the previous project approved by the Ministry of Health of Senegal Standards in Genomic Sciences and the assembled village population, and as published elsewhere [20]. Both this study and the assent procedure were approved by the National Ethics Committee of Senegal (CNERS) and the Ethics Committee of the Institut Fédératif de Recherche IFR48, Faculty of Medicine, Marseille, France (agreement numbers 09-022 and 11-017)). Several other new bacterial species were isolated from this specimen using various culture conditions, including the recently described Alistipes timonensis, A. senegalensis, Anaerococcus senegalensis, Bacillus timonensis, Clostridium senegalense, Paenibacillus senegalensis, and Peptoniphilus timonensis [5][6][7][8][9][10][11]. The fecal specimen was preserved at -80°C after collection and sent to Marseille. Strain JC43 T ( Table  1) was isolated in December 2010 after inoculation on Brucella agar (BD diagnostic, Heilderberg, Germany), in aerobic atmosphere at 37°C.
The strain exhibited 97.1 and 96.7% nucleotide sequence similarities with B. salitolerans (Guan et al. 2010) and B. album (Tang et al. 2008), respectively, the phylogenetically closest validated Brevibacterium species (Figure 1). These values were lower than the 98.7% 16S rRNA gene sequence threshold recommended by Stackebrandt and Ebers to delineate a new species without carrying out DNA-DNA hybridization [2]. In comparison to 16S sequences in the GenBank database [30], strain JC43 T also exhibited nucleotide sequence similarities greater than 98.7% with uncultured bacterial clones detected in watermiscible metalworking fluids [31] and on clean room surfaces [32]. These bacteria most likely belong to the same species as strains JC43 ( Figure 1).  Different growth temperatures (25, 30, 37, 45°C) were tested; no growth occurred at 45°C, weak growth occurred at 25°C, and optimal growth was observed between 30 to 37°C. Colonies were translucent and smooth, with a diameter of 1 mm on blood-enriched Columbia agar and Brain Heart Infusion (BHI) agar. Growth of the strain was tested under anaerobic and microaerophilic conditions using GENbag anaer and GENbag microaer systems, respectively (BioMérieux), and in the presence of air, of 5% CO 2 and in aerobic conditions. Optimal growth was obtained aerobically and with 5% CO 2. Weak growth was observed under microaerophilic conditions. No growth was observed in an anaerobic atmosphere.

Genome sequencing and annotation 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 Brevibacterium genus, and is part of a study aiming at isolating all bacterial species within human feces. It was the third genome of a Brevibacterium species. The genome EMBL accession number is CAHK00000000 and consists of 80 contigs. Table 2 shows the project information and its association with MIGS version 2.0 compliance.

Genome sequencing and assembly
A shotgun library and a 3kb paired end library were pyrosequenced on the 454 Roche Titanium sequencing platform. This project was loaded on one 1/4 region region of PTP Picotiterplate (Roche, Meylan, France) for the shotgun library and 4 × 1/4 region for the 3-kb paired-end library. The shotgun library was constructed with 500 ng of DNA with the GS Rapid library Prep kit as described by the manufacturer (Roche). For the paired-end library, 5µg of DNA was mechanically fragmented on a Hydroshear device (Digilab, Holliston, MA, USA) with an enrichment size at 3-4kb. DNA fragmentation was visualized using an Agilent 2100 BioAnalyzer on a DNA labchip 7500 with an optimal size of 3.692 kb. The library was constructed according to the 454 Titanium paired-end protocol (Roche). Circularization and nebulization were performed and generated a pattern with an optimum of 510 bp. After PCR amplification through 15 cycles followed by double size selection, the single stranded paired-end library was then quantified using a Quant-it Ribogreen kit (Invitrogen) on a Genios Tecan fluorometer at 245 pg/µL. The library concentration equivalence was calculated at 8.80E+08 molecules/µL. The libraries were stocked at -20°C until further use. The shotgun library was clonally amplified with 3 cpb in 3 emPCR reactions and the 3-kb paired-end library was amplified with 1 cpb in 10 emPCR reactions and 0.25 cpb in 4 emPCR with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yield of the shotgun emPCR reactions was higher than expected at 24%, but the yields of the two types of paired-end emPCR were 16.7% and 11.01%, respectively, in the range of 5 to 20% from the Roche procedure.
The libraries were loaded on the GS Titanium PicoTiterPlate PTP Kit 70×75 and sequenced with the GS FLX Titanium Sequencing Kit XLR70 (Roche). The runs were performed overnight and then analyzed on the cluster through the gsRunBrowser and Newbler Assembler (Roche). A total of 752,121 passed filter wells were obtained and generated 203.1 Mb of sequence with an average length of 265 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 80 contigs (>500 bp) arranged into 16 scaffolds and generated a genome size of 3.42 Mb.

Genome annotation
Open Reading Frames (ORFs) were predicted using Prodigal [36] with default parameters but the predicted ORFs were excluded if they were spanning a sequencing GAP region. The predicted bacterial protein sequences were searched against the GenBank database and the Clusters of Orthologous Groups (COG) database using BLASTP. The Standards in Genomic Sciences tRNAScanSE tool [37] was used to find tRNA genes, whereas ribosomal RNAs were found using RNAmmer [38]. Transmembrane domains and signal peptides were predicted using TMHMM [39] and SignalP [40], 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 were smaller than 80 amino acids, we used an E-value of 1e-05. To estimate the mean level of nucleotide sequence similarity at the genome level between B. senegalense, B. linens (GenBank accession number AAGP00000000) and B. mcbrellneri (ADNU00000000) we compared the ORFs only using BLASTN at a query coverage of ≥ 70% and a minimum nucleotide length of 100 bp.

Genome properties
The genome is 3,425,960 bp long (1 chromosome, but no plasmid) with a 70.00% G+C content (Table  3 and Figure 5). Of the 3,114 predicted genes, 3,065 were protein-coding genes and 49 were RNAs, including 3 rRNA operons (5S, 16S and 23S rRNA) and 40 tRNAs. A total of 2,077 genes (66.7%) were assigned a putative function. The distribution of genes into COGs functional categories is presented in Table 4 and Figure 5. The properties and the statistics of the genome are summarized in Tables 3 and 4. Figure 5. Graphical circular map of the Brevibacterium senegalense genome. From outside to the center: genes on the forward strand (colored by COG categories), genes on the reverse strand (colored by COG categories), RNA genes (tRNAs, green; rRNAs, red), G+C content, GC skew.

Conclusion
On the basis of phenotypic (Table 5), phylogenetic and genomic analyses, we formally propose the creation of Brevibacterium senegalense sp. nov. that contains the strain JC43 T . This bacterium originated from Senegal.