Non-contiguous finished genome sequence and description of Brevibacterium senegalense sp. nov.
© The Author(s) 2012
Published: 19 December 2012
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.
Brevibacterium senegalense strain JC43T (= CSUR P155 = DSM 25783) is the type strain of B. senegalense. sp. nov. This bacterium is a non-motile, rod-shaped, Gram-positive, catalase-positive 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 , were gradually replaced by gene sequencing. In particular, 16S rRNA sequencing has deeply changed the way bacteria and archaea are classified . 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 . We recently used a polyphasic approach  that includes genomic data, MALDI-TOF spectrum and major phenotypic characteristics to describe new bacterial species [5–11].
The genus Brevibacterium (Breed 1953)  was created in 1953 to gather short non-spore-forming and non-branching rods. To date, this genus is comprised of Gram-positive, irregular, rod-shaped, non-acid-fast bacteria, and contains 31 recognized species with validly published names . Brevibacterium is the type genus of the family Brevibacteriaceae (Breed 1953) . Members of the genus Brevibacterium are isolated from human samples, dairy products, poultry and environmental specimens. In humans, they are found on skin surfaces , 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, with the exception of two cases of bacteremia in immunocompetent patients with central venous catheters [15,16]. To date, only four Brevibacterium species have been detected in human infection, including B. epidermidis (Collins et al. 1983) [15,17,18], B. casei (Collins et al. 1983) [16,19], B. iodinum (Collins et al. 1981) and B. otitidis (Pascual et al. 1996).
Here we present a summary classification and a set of features for B. senegalense sp. nov. strain JC43T together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the B. senegalense species.
Classification and general features of Brevibacterium senegalense strain JC43T according to the MIGS recommendations 
Species Brevibacterium senegalensis
Type strain JC43T
Sample collection time
Latitude - Longitude
51 m above sea level
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% CO2 and in aerobic conditions. Optimal growth was obtained aerobically and with 5% CO2. Weak growth was observed under microaerophilic conditions. No growth was observed in an anaerobic atmosphere.
Strain JC43T exhibited catalase activity but not oxidase activity. Using the API CORYNE system(BioMérieux), positive reactions were observed for nitrate reduction, pyrrolidonyl arylamidase, alkaline phosphatase, α-glucosidase. A weak reaction was observed for gelatin hydrolysis. Negative reactions were observed for urease, pyrazinamidase, β-glucuronidase, β-galactosidase, α-glucosidase, N-acetyl-β-glucosaminidase, β-glucosidase (aesculin hydrolysis), and acid production from D-ribose, D-glucose, D-xylose, D-mannitol, maltose, D-lactose, sucrose and glycogen. Using API ZYM (BioMérieux), positive reactions were observed for esterase (C4), esterase lipase (C8), leucine arylamidase and acid and alkaline phosphatase. Negative reactions were observed for valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, naphtol-AS-BI-phosphohydrolase, lipase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. B. senegalense is susceptible to penicillin G, amoxicillin, imipenem, ciprofloxacin, rifampin, gentamicin, doxycycline and vancomycin but resistant to trimethoprim/sulfamethoxazole and metronidazole. By comparison to B. salitolerans  and B. album , B. senegalense strain JC43T differed in growth temperature, gelatin hydrolysis, pyrazinamidase, acid production from D-ribose, and nitrate reduction. In addition, B. senegalense also differed from the former species in β-glucosidase (aesculin hydrolysis) activity , and from the latter species in motility, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin and naphtol-AS-BI-phosphohydrolase activities .
Genome sequencing and annotation
Genome project history
High quality draft
One shotgun, one paired-end 3-kb
454 GS FLX Titanium
Newbler version 2.5.3
Gene calling method
EMBL date of Release
February 2, 2012
Study of the human gut microbiot
Growth conditions and DNA isolation
B. senegalense sp. nov. strain JC43T (CSUR = P155, DSM = 25783) was grown aerobically on 5% sheep blood-enriched Columbia agar at 37°C. Seven petri dishes were spread and resuspended in 3x100µl of G2 buffer (EZ1 DNA Tissue kit, Qiagen). A first mechanical lysis was performed by glass powder on the Fastprep-24 device (Sample Preparation system; MP Biomedicals, USA) using 2x20 seconds cycles. DNA was then treated with 2.5µg/µL lysozyme (30 minutes at 37°C) and extracted through the BioRobot EZ 1 Advanced XL (Qiagen). The DNA was then concentrated and purified on a Qiamp kit (Qiagen). The yield and the concentration was measured by the Quant-it Picogreen kit (Invitrogen) on the Genios_Tecan fluorometer at 68,1 ng/µl.
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 3–4 kb. 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.
Open Reading Frames (ORFs) were predicted using Prodigal  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 tRNAScanSE tool  was used to find tRNA genes, whereas ribosomal RNAs were found using RNAmmer . Transmembrane domains and signal peptides were predicted using TMHMM  and SignalP , 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.
Nucleotide content and gene count levels of the genome
% of totala
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
Genes with function prediction
Genes assigned to COGs
Genes with peptide signals
Genes with transmembrane helices
Number of genes associated with the 25 general COG functional categories
% of totala
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, mitosis and meiosis
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Genomic comparison with B. linens and B. mcbrellneri
Currently, two draft genomes from Brevibacterium species are available. By comparison with B. linens strain BL2 (GenBank accession number AAGP00000000) and B. mcbrellneri strain ATCC 49030 (ADNU00000000) B. senegalense strain JC43T has a smaller genome than the former (3.42 Mb vs 4.37Mb) but larger than the latter (2.56Mb). B. senegalense also has a higher G+C content than the other two genomes (70.00% vs 62.8% and 58.00%, respectively); it has a smaller number of predicted genes (3,114) than B. linens (4,054) but greater than B. mcbrellneri (2,437). Finally, at the genome level, B. senegalense exhibited percentages of nucleotide sequence similarity of 86.28% (range 70.01–100%) and 70.19% (range 86.09–100%) with B. linens and B. mcbrellneri, respectively.
Phenotypic differences observed between B. senegalense strain JC43T, B. salitolerans strain YIM90718 and B. album strain TRM415.
B. senegalense JC43T
B. album TRM415
B. salitolerans YIM90718
T° of growth
Esterase lipase C8
β-glucosidase (aesculin hydrolysis)
Acid production for
Description of Brevibacterium senegalense sp. nov.
Brevibacterium senegalense (se.ne.gal.e′n.se L. gen. neutr. n. senegalense, pertaining to, or originating from Senegal, the country from which the specimen that enabled isolation of B. senegalense was isolated.)
Colonies are translucent, smooth and have a diameter of 1 mm on blood-enriched Columbia agar and Brain Heart Infusion (BHI) agar. Cells are rod-shaped and occur mostly in small clumps. Their length and width range from 0.83 to 3.86 µm (mean, 2.55 µm) and 0.57 to 0.78 µm (mean, 0.68 µm), respectively. Optimal growth is achieved aerobically with or without CO2. Weak growth is observed under microaerophilic conditions. No growth is observed under anaerobic conditions. Growth occurs between 30–37°C. Cells stain Gram-positive, are non-endospore-forming, and non-motile. Catalase, nitrate reduction, pyrrolidonyl arylamidase, alkaline phosphatase, α-glucosidase, gelatin hydrolysis, esterase (C4), esterase lipase (C8), leucine arylamidase and acid and alkaline phosphatase activities are present. Urease, pyrazinamidase, β-glucuronidase, β-galactosidase, α-glucosidase, N-acetyl-β-glucosaminidase, β-glucosidase (aesculin hydrolysis), acid production from D-ribose, D-glucose, D-xylose, D-mannitol, maltose, D-lactose, sucrose and glycogen, valine aylamidase, cystine aylamidase, trypsin, α-chymotrypsin, naphtol-AS-BI-phosphohydrolase, lipase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase activities are absent. Oxidase activity is absent. Cells are susceptible to penicillin G, amoxicillin, imipenem, ciprofloxacin, rifampin, gentamicin, doxycycline and vancomycin, but resistant to trimethoprim/sulfamethoxazole and metronidazole.
The G+C content of the genome is 70.00%. The 16S rRNA and genome sequences are deposited in EMBL under accession numbers JF824806 and CAHK00000000, respectively.
The type strain JC43T (= CSUR P 155 = DSM 25783) was isolated from the fecal flora of a healthy patient in Senegal.
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