Non contiguous-finished genome sequence and description of Bacillus timonensis sp. nov.
© The Author(s) 2012
Published: 30 July 2012
Bacillus timonensis strain MM10403188T sp. nov. is the type strain of a proposed new species within the genus Bacillus. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. B. timonensis is an aerobic Gram-negative rod shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,632,049 bp long genome (1 chromosome but no plasmid) contains 4,610 protein-coding and 74 RNA genes, including 5 rRNA genes.
Bacillus timonensis strain MM10403188T (= CSUR P162 = DSM 25372) is designated as the type strain of B. timonensis, a new Gram-negative aerobic, indole-positive bacillus that was isolated from the stool of a healthy Senegalese patient as part of a “culturomics” study aiming at cultivating individually all species within human feces.
To date, DNA-DNA hybridization and G+C content determination  remain the gold standard methods for the definition of bacterial species, despite the development of 16S rRNA PCR and sequencing which have deeply changed bacterial taxonomy . Over recent years, high throughput genome sequencing provided a wealth of genetic information . In an effort to include genomic data in bacterial taxonomy we recently used a polyphasic approach  that includes genomic data, MALDI-TOF spectrum and main phenotypic characteristics to describe new bacterial species [5,6].
Here we present a summary classification and a set of features for B. timonensis sp. nov. strain MM10403188T together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the species B. timonensis.
The genus Bacillus (Cohn 1872) was created in 1872 . To date, this genus, mostly comprised of Gram-positive, motile, and spore-forming bacteria, is made of 256 species and 7 subspecies with validly published names . Members of the genus Bacillus are ubiquitous bacteria isolated from various environments including soil, fresh and sea water, food, and occasionally from humans in whom they are either pathogens, such as B. anthracis and B. cereus, or opportunists in immunocompromised patients . Apart from anthrax, caused by B. anthracis , and toxi-infections caused by B. cereus, Bacillus species may be involved in a variety of aspecific human infections, including cutaneous, ocular, central nervous system or bone infections, pneumonia, endocarditis and bacteremia .
Classification and features
Classification and general features of Bacillus timonensis strain MM10403188T
Species Bacillus timonensis
Type strain MM10403188T
growth in BHI medium + 5% NaCl
Sample collection time
51 m above sea level
Strain MM10403188T exhibited oxidase activity but not catalase activity, and was positive for indole. Using API 50CH, a positive reaction was obtained for L-arabinose, D-lactose, D-melibiose, D-trehalose, D-saccharose, and D-turanose fermentation. A weak reaction was obtained for aesculin. Other tests were negative. Using API-ZYM, positive reactions were obtained for esterase, α-chimotrypsine, β-glucorinidase, and α- and β-glucosinidase. B. timonensis was susceptible to penicillin G, amoxicillin, vancomycin, gentamicin, erythromycin, doxycyclin, rifampicin, and ciprofloxacin but resistant to trimethoprim/sulfamethoxazole.
By comparison with B. humi, B. timonensis differed in Gram staining, in culture atmosphere, as B. humi was able to grow anaerobically, in catalase activity, in spore forming capacity, in indole production, and in carbohydrate metabolism, notably for arbutin, salicin, L-arabinose, melibiose, turanose, and trehalose .
Genome sequencing information
Genome project history
454 GS shotgun and paired-end 3-kb libraries
454 GS FLX Titanium
Newbler version 2.5.3
Gene calling method
Genbank Date of Release
February 28th, 2012
NCBI project ID
Study of the human gut microbiome
Growth conditions and DNA isolation
B. timonensis sp. nov. strain MM10403188T, CSUR P162, DSM 25372, was grown aerobically on 5% sheep blood-enriched BHI agar at 37°. Four petri dishes were spread and growth from the plates was resuspended in 3x500µl of TE buffer and stored at 80°C. Then, 500µl of this suspension were thawed, centrifuged 3 minutes at 10,000 rpm 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 using the BioRobot EZ1 Advanced XL (Qiagen). The DNA was then concentrated and purified using the Qiamp kit (Qiagen). The yield and the concentration was measured by the Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer at 50ng/µl.
Genome sequencing and assembly
DNA (5 µg) was mechanically fragmented on a Hydroshear device (Digilab, Holliston, MA, USA) with an enrichment size at 3–4kb. The DNA fragmentation was visualized through the Agilent 2100 BioAnalyzer on a DNA labchip 7500 with an optimal size of 3.345kb. The library was constructed according to the 454 GS FLX Titanium paired-end protocol. Circularization and nebulization were performed and generated a pattern with an optimum at 492 bp. After PCR amplification through 15 cycles followed by double size selection, the single stranded paired end library was then quantified on the Quant-it Ribogreen kit (Invitrogen) on the Genios Tecan fluorometer at 339 pg/µL. The library concentration equivalence was calculated as 12,6E+08 molecules/µL. The library was stored at −20°C until further use.
The shotgun library was clonally amplified with 3cpb and the paired-end library was amplified with lower cpb (1 cpb) in 4 emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR was 5.97% for the shotgun and 15.92% for the paired end as expected by the range of 5 to 20% from the Roche procedure.
Approximately 790,000 beads for a 1/4 region and 340,000 beads for a 1/8 region were loaded on the GS Titanium PicoTiterPlate PTP Kit 70×75 and sequenced with the GS FLX Titanium Sequencing Kit XLR70 (Roche). The run was performed overnight and then analyzed on the cluster through the gsRunBrowser and Newbler assembler (Roche). For the shotgun sequencing, 112,962 passed filter wells were obtained and generated 34.48Mb with a length average of 322 bp. For the shotgun sequencing, 213,882 passed filter wells were obtained and generated 50.6 Mb with a length average of 236 bp. The passed filter sequences were assembled Using Newbler with 90% identity and 40bp as overlap. The final assembly identified 11 scaffolds and 89 contigs (>1500bp) generating a genome size of 4.6 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) databases using BLASTP. The tRNAScanSE tool  was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer  and BLASTn against the GenBank database. 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. Such parameter thresholds have already been used in previous works to define ORFans.
To estimate the mean level of nucleotide sequence similarity at the genome level between Bacillus species, we compared the ORFs only using BLASTN and the following parameters: 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
Translation, ribosomal structure and biogenesis
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
Comparison with the genomes from other Bacillus species
Genome sequences are currently available for more than 25 validly named Bacillus species. Here we compared the genome sequence of B. timonensis strain MM10403188T with that of B. licheniformis strain ATCC 14580, the most closely related phylogenetic neighbor for which the genome sequence is available. The draft genome sequence of B. timonensis is larger than B. licheniformis (4.6 Mb and 4.2 Mb, respectively) but its G+C content is lower (37.30 and 46.19%, respectively). B. timonensis has more predicted genes than B. licheniformis (4,684 and 4,356, respectively), and more genes assigned to COGs (3,399 and 3,130, respectively). However, the distribution of genes into COG categories (Table 4) was highly similar in both genomes. In addition, B. timonensis shared a mean 86.10% (range 76.4-93%) sequence similarity with B. licheniformis at the genome level.
Although the degree of 16S rRNA similarity was elevated (98.2%) between strain MM10403188 and B. humi strain DSM 16318, both strains exhibited several phenotypic and genomic differences, and we formally propose the creation of Bacillus timonensis sp. nov. that contains the strain MM10403188T. This strain has been found in Senegal.
Description of Bacillus timonensis sp. nov.
Bacillus timonensis (tim.on.en′sis. L. gen. masc. n. timonensis, of Timone, the name of the hospital where strain MM10403188T was cultivated.) Isolated from stool from an asymptomatic Senegalese patient. B. timonensis is an aerobic Gram-negative bacterium. Grows on axenic medium at 37°C in an aerobic atmosphere. Colonies were 3 mm in diameter on blood-enriched BHI agar. Cells grown on agar are sporulated and have a mean diameter of 0.66 µm. A positive reaction was obtained for L-arabinose, D-lactose, D-melibiose, D-trehalose, D-saccharose, and D-turanose fermentation. Positive reactions were obtained for oxidase, esterase, α-chimotrypsine, β-glucorinidase, and α- and β-glucosinidase activity. No catalase activity was exhibited. Positive for indole. By comparison with B. humi, B. timonensis differs in Gram staining, in culture atmosphere, as B. humi grows anaerobically, in catalase activity, in spore forming capacity, in indole production, and in carbohydrate metabolism, notably for arbutin, salicin, L-arabinose, melibiose, turanose, and trehalose. B. timonensis is susceptible to penicillin G, amoxicillin, vancomycin, gentamicin, erythromycin, doxycyclin, rifampicin, and ciprofloxacin but resistant to trimethoprim/sulfamethoxazole. Motile. The G+C content of the genome is 37.30%. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers JF824810 and CAET00000000, respectively. The type strain MM10403188T (= CSUR P162 = DSM 253720) was isolated from the fecal flora of a healthy patient from Senegal.
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