Non contiguous-finished genome sequence and description of Peptoniphilus grossensis sp. nov.
© The Author(s) 2012
Published: 19 December 2012
Peptoniphilus grossensis strain ph5T sp. nov., is the type strain of Peptoniphilus grossensis sp. nov., a new species within the Peptoniphilus genus. This strain, whose genome is described here, was isolated from the fecal flora of a 26-year-old woman suffering from morbid obesity. P. grossensis strain ph5 is a Gram-positive obligate anaerobic coccus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,101,866-bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 33.9% and contains 2,041 protein-coding and 29 RNA genes, including 3 rRNA genes.
Peptoniphilus grossensis strain ph5T (= CSUR P184 = DSM 25475), is the type strain of Peptoniphilus grossensis sp. nov. This bacterium is a Gram-positive, spore-forming, indole positive, anaerobic coccoid bacterium that was isolated from the stool of a 26-year-old woman suffering from morbid obesity.
Since 1995 and the sequencing of the first bacterial genome, that of Haemophilus influenzae, more than 3,000 bacterial genomes have been sequenced . This was permitted by technical improvements as well as increased interest in having access to the complete genetic information encoded by bacteria. At the same time, biological tools for defining new bacterial species have not evolved, and DNA-DNA hybridization is still considered the gold standard  despite its drawbacks and the taxonomic revolution that has resulted from the comparison of 16S rDNA sequences . In this manuscript, we propose and describe a new Peptoniphilus species using genomic and phenotypic information  to.
Gram-positive anaerobic cocci (GPAC) are part of the commensal flora of humans and animals, and are also commonly associated with a variety of human infections [5,6]. Extensive taxonomic changes have occurred in this group of bacteria, especially in clinically-important genera such as Finegoldia, Micromonas, and Peptostreptococcus . The genus Peptostreptococcus was divided into three genera: Peptoniphilus (Ezaki et al., 2001), Anaerococcus (Ezaki et al., 2001) and Gallicola (Ezaki et al., 2001). The genus Peptoniphilus includes the following butyrate-producing, non-saccharolytic species that use peptone and amino acids as major energy sources: P. asaccharolyticus, P. gorbachii, P. harei, P. indolicus, P. ivorii, P. lacrimalis , P. olsenii  and P. methioninivorax .
Members of the genus Peptoniphilus have mostly been isolated from various human clinical specimens such as vaginal discharges, ovarian, peritoneal, sacral and lacrymal gland abscesses . In addition, P. indolicus causes summer mastitis in cattle .
Here we present a summary classification and a set of features for P. grossensis sp. nov. strain ph5T (= CSUR P184 = DSM 25475), together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the species P. grossensis.
Classification and features
Classification and general features of Peptoniphilus grossensis strain ph5T according to the MIGS recommendations 
Family Clostridiales family XI Incertae sedis
Species Peptoniphilus grossensis
Type strain: ph5
growth in BHI medium + 5% NaCl
Sample collection time
0 m above sea level
Strain ph5 exhibited neither catalase nor oxidase activities but indole production was observed. Using an API Rapid ID 32A strip (BioMerieux), a positive reaction was observed for Mannose fermentation, arginine arylamidase, tyrosine arylamidase, histidine arylamidase and leucine arylamidase. Strain ph5 was susceptible to penicillin G, amoxicillin, ceftriaxon, cefalexin, imipenem fosfomycin, erythromycin, doxycyclin, rifampin, vancomycin and metronidazole, but resistant to ciprofloxacin and cotrimoxazole.
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
Genbank Date of Release
May 30, 2012
Study of the human gut microbiome
Growth conditions and DNA isolation
P. grossensis strain ph5T (= CSUR P184 = DSM 25475), was grown on blood agar medium at 37°C. Six petri dishes were spread and resuspended in 6×100µl 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) from MP Biomedicals, USA) using 40 seconds cycles. DNA was then treated with 2.5 µg/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 a Genios_Tecan fluorometer at 62 ng/µl.
Genome sequencing and assembly
DNA (5µg) was mechanically fragmented on the Hydroshear device (Digilab, Holliston, MA, USA) with an enrichment size of 3–4 kb. The DNA fragmentation was visualized using an Agilent 2100 BioAnalyzer on a DNA labchip 7500 to yield an optimal size of 3.16 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 628 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 34pg/µL. The library concentration equivalence was calculated as 9.93E+08 molecules/µL. The library was held at −20°C until use.
The shotgun library was clonally amplified with 0.5 and 1 cpb in 2 emPCR reactions per condition with the GS Titanium SV emPCR Kit (Lib-L) v2. The yields of the emPCR at 0.5cpb and 1 cpb were of 9.63% and 22.35%, respectively. A total of 790,000 beads for a ¼ region and 790,000 beads for a 1/8 region 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 176,029 passed filter sequences generated 56.24 Mb with a length average of 319 bp. These sequences were assembled using the Newbler software from Roche with 90% identity and 40 bp as overlap. Seventy-seven large contigs (>1500bp) were obtained, for a genome size of 2.1Mb which corresponds to a coverage of 26.78× genome equivalent.
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. Lipoprotein signal peptides and numbers of transmembrane helices were predicted using SignalP  and TMHMM  respectively. ORFans were identified if their BLASTP E-value was lower than 1e-3 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: a query coverage of ≥ 70% and a minimum nucleotide length of 100 bp. Artemis  were used for data management and DNA Plotter  was used for visualization of genomic features. Mauve alignment tool was used for multiple genomic sequence alignment and visualization .
Nucleotide content and gene count levels of the genome
% of totala
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
Number of replicons
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 genomes from other Peptoniphilus species
The genomes from only three validated Peptoniphilus species are currently available. Here, we compared the genome sequence of P. grossensis strain ph5T with those of P. harei strain ACS-146-V-Sch2b, P. duerdenii strain ATCC BAA-1640, P. lacrimalis strain 315-B, as well as P. timonensis strain JC401T that we recently studied.
Description of Peptoniphilus grossensis sp. nov.
Peptoniphilus grossensis (gro.sen′sis. L. gen. masc. n. grossensis, of gros, the French adjective for fat, as the strain was isolated from an obese patient).
Colonies are 1 mm in diameter on blood-enriched Columbia agar and Brain Heart Infusion (BHI) agar. Cells are coccoid with a mean diameter of 1.2 µm, occurring mostly in pairs, short chains or small clumps. Growth is only achieved anaerobically. The optimal growth temperature is 37°C. Cells are Gram-positive, endospore-forming, and non-motile. Cells are negative for catalase and positive for indole production. Acid is produced from mannose. Positive reactions are observed for arginine arylamidase, tyrosine arylamidase, histidine arylamidase and leucine arylamidase. Cells are susceptible to penicillin G, amoxicillin, ceftriaxone, cefalexin, imipenem, fosfomycin, erythromycin, doxycyclin, rifampicin, vancomycin, metronidazole, but resistant to ciprofloxacin and cotrimoxazole. The G+C content of the genome is 33.9%. The genome and 16SrRNA sequences are deposited in GenBank under accession numbers CAGX00000000 and JN837491, respectively. The type strain ph5T (= CSUR P184 = DSM 25475) was isolated from the fecal flora of an obese French patient.
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