Non contiguous-finished genome sequence and description of Peptoniphilus timonensis sp. nov.
© The Author(s) 2012
Published: 10 October 2012
Peptoniphilus timonensis strain JC401T sp. nov. is the type strain of P. timonensis sp. nov., a new species within the Peptoniphilus genus. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. P. timonensis is an obligate Gram-positive anaerobic coccus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,758,598 bp long genome (1 chromosome, no plasmid) contains 1,922 protein-coding and 22 RNA genes, including 5 rRNA genes.
Peptoniphilus timonensis strain JC401T (= CSUR P165= DSM 25367) is the type strain of P. timonensis sp. nov. This bacterium is a Gram-positive, anaerobic, indole-positive coccus 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.
Since the early days of bacterial taxonomy, defining a bacterial species has been a matter of debate. Currently, the availability of a wide array of molecular methods, notably 16S rRNA and full genome sequencing, offers a possibility to base the description of new species on other methods than the “gold standard” of DNA-DNA hybridization . In particular, sequence similarity of the 16S rRNA, although neither uniform across taxa nor necessarily predictive, enabled the taxonomic classification or reclassification of many taxa , and genome sequencing has provided access to the complete genetic information of bacteria . As a consequence, we based our description of P. timonensis sp. nov. on a polyphasic approach  including their genome sequence and main phenotypic characteristics (habitat, Gram-stain reaction, culture and metabolic characteristics, MALDI-TOF spectrum, and when applicable, pathogenicity).
Here we present a summary classification and a set of features for P. timonensis sp. nov. strain JC401T together with the description of the complete genomic sequencing and annotation. These characteristics support the creation of the P. timonensis species.
The genus Peptoniphilus (Ezaki et al. 2001) was created in 2001  and consist of species that are non-saccharolytic, butyrate-producing, non-motile gram-positive anaerobic cocci and use peptones and oligopeptide as major energy source . To date, the genus Peptoniphilus contains eight species namely P. asaccharolyticus, P. harei, P. indolicus, P. ivorii, P. lacrimalis , P. gorbachii, P. olsenii , P. methioninivorax . Members of the genus Peptoniphilus have mostly been isolated from various human clinical specimens such as vaginal discharges, ovarian, peritoneal, sacral and lachrymal gland abscesses . P. indolicus causes summer mastitis in cattle .
Classification and general features of Peptoniphilus timonensis strain JC401T according to the MIGS recommendation 
Family XI Incertae sedis
Species Peptoniphilus timonensis
Type strain JC401T
Growth in BHI medium + 1% NaCl
Sample collection time
51 m above sea level
Strain JC401T exhibited a catalase activity but no oxidase activity. Using API Rapid ID 32A, positive reactions were obtained for α galactosidase, arginine arylimidase, tyrosine arylamidase, histidine arylamidase, serine arylamidase and indole production. Weak reactions were observed for leucine arylamidase and phenylalanine arylamidase. P. timonensis is susceptible to penicillin G, imipeneme, amoxicillin + clavulanic acid, vancomycin, clindamycin and metronidazole.
Genome sequencing information
Genome project history
One 454 paired end 3-kb library
454 GS FLX Titanium
Newbler version 2.5.3
Gene calling method
NCBI project ID
Genbank Date of Release
January 30, 2012
Study of the human gut microbiome
Growth conditions and DNA isolation
P. timonensis sp. nov. strain JC401T (CSUR P165, DSM 25367) was grown anaerobically on 5% sheep blood-enriched Columbia agar at 37°C. Six petri dishes were spread and resuspended in 6x100µl 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) during 2x20 seconds. 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 123.3ng/µl.
Genome sequencing and assembly
Five µg of DNA was mechanically fragmented on the 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 2.47 kb. The library was constructed according to the 454 GS FLX Titanium paired end protocol. Circularization and nebulization were performed. After PCR amplification through 15 cycles followed by double size selection, the single stranded paired end library profile was visualized on an Agilent 2100 RNA Pico 6000 Labchip with an optimal at 568bp. Then the library was quantified on the Quant-it Ribogreen kit (Invitrogen) on the Genios_Tecan fluorometer at 890 pg/µL. The library concentration equivalence was calculated as 2.87E+09 molecules/µL. The library was stored at −20°C until further use.
The shotgun library was clonal amplified with 0.25 and 0.5cpb in 2 emPCR reactions per conditions with the GS Titanium SV emPCR Kit (Lib-L) v2. The yields of the emPCR were 2.79% and 10.79% respectively in the range of 5 to 20% from the Roche procedure.
Approximately 790,000 beads for a ¼ region and 340000 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, 193,186 passed filter wells were obtained and generated 37.47Mb with a length average of 190 bp. The passed filter sequences were assembled Using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 7 scaffolds and 97 large contigs (>1500bp) generating a genome size of 1.76 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 Peptoniphilus species, we compared the ORFs only using BLASTN and the following parameters: 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
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 Peptoniphilus species
Draft genome sequences are currently available for six species. Here we compared the genome sequence of P. timonensis strain JC401T with those of P. harei strain ACS-146-V-Sch2b, P. indolicus strain ATCC BAA-1640 and P. lacrimalis strain 315-B.
The draft genome sequence of P. timonensis is larger than P. lacrimalis (1.76 Mb and 1.69 Mb, respectively) and smaller than P. indolicus and P. harei (2.2 Mb and 1.8 Mb, respectively). The G+C content of P. timonensis is comparable to P. lacrimalis (30.7 and 29.91% respectively) but smaller than those of P. indolicus and P. harei (32.29 and 34.44% respectively). Additionally, P. timonensis has more predicted genes than P. harei and P. lacrimalis (1,922, 1,724 and 1,589 respectively) and lesser than P. indolicus (2,269). The genes assigned to COGs of P. timonensis are comparable to P. harei (1,368 and 1,381 respectively) greater than P. lacrimalis (1,192) and lesser than P. indolicus (1,690). However, the distribution of genes into COG categories (Table 4) was almost similar in all the four genomes.
In addition, P. timonensis shared a mean 86.49% (range 77.75 to 99.15%), 85.54% (range 77.36 to 99.13) and 82.80% (range 77.43 to 95.39) sequence similarity with P. harei, P. lacrimalis and P. indolicus respectively at the genome level.
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Peptoniphilus timonensis sp. nov. that contains the strain JC401T. This strain has been found in Senegal.
Description of Peptoniphilus timonensis sp. nov.
Peptoniphilus timonensis (tim.on.en’sis. L. gen. masc. n. timonensis, of Timone, the name of the hospital where strain JC401T was cultivated. Isolated from stool from an asymptomatic Senegalese patient. P. timonensis is an anaerobic Gram-positive bacterium. Grows on axenic medium at 37°C in an anaerobic atmosphere. Strain JC401T exhibited a catalase activity but no oxidase activity. Positive reactions were obtained for α galactosidase, arginine arylimidase, tyrosine arylamidase, histidine arylamidase, serine arylamidase and indole production. Weak reactions were observed for leucine arylamidase and phenylalanine arylamidase. Positive for indole. P. timonensis is susceptible to penicillin G, imipeneme, amoxicillin + clavulanic acid, vancomycin, clindamycin and metronidazole. Non-motile. The G+C content of the genome is 30.7%. The type strain is JC401T (= CSUR P165= DSM 25367).
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