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Non-contiguous finished genome sequence and description of Kallipyga massiliensis gen. nov., sp. nov., a new member of the family Clostridiales Incertae Sedis XI
Standards in Genomic Sciences volume 8, pages 500–515 (2013)
Kallipyga massiliensis strain ph2T is the type strain of Kallipyga massiliensis gen. nov., sp. nov., the type species of the new genus Kallipyga within the family Clostridiales Incertae Sedis XI. This strain, whose genome is described here, was isolated from the fecal flora of a 26-year-old woman suffering from morbid obesity. K. massiliensis is an obligate anaerobic coccus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,770,679 bp long genome (1 chromosome but no plasmid) contains 1,575 protein-coding and 50 RNA genes, including 4 rRNA genes.
Kallipyga massiliensis strain ph2T (CSUR=P241, DSM=26229) is the type strain of K. massiliensis gen. nov., sp. nov. This bacterium was isolated from the stool sample of an obese French patient as part of a study aiming at individually cultivating all species occurring within human feces [1–3]. It is a Gram-positive, anaerobic, indole-negative coccus. Defining the taxonomic status of bacterial isolates remains a challenging task. The taxonomic molecular tools currently available, including 16S rRNA sequence similarity, G + C content and DNA-DNA hybridization (DDH) [4,5], although considered as gold standards, have limitations [6,7]. The 16S rRNA sequence similarity and G+C content thresholds do not apply uniformly to all species or genera, and the DDH method lacks intra- and inter-laboratory reproducibility . The advent of high-throughput genome sequencing and proteomic analysis  has granted unprecedented access to exhaustive genetic and protein information for bacterial isolates. We recently proposed a polyphasic approach to describe new bacterial species in which genome sequences and MALDI-TOF spectra are used along with phenotypic characteristics [9–30].
The family Clostridiales Incertae Sedis XI (Garrity and Holt 2001) was created in 2001  and currently includes the 11 following genera: Anaerococcus (Ezaki et al. 2001) , Dethiosulfatibacter (Takii et al. 2007) , Finegoldia (Murdoch and Shah 2000) , Gallicola (Ezaki et al. 2001) , Helcococcus (Collins et al. 1993) , Parvimonas (Tindall and Euzéby 2006) , Peptoniphilus (Ezaki et al. 2001) , Sedimentibacter (Breitenstein et al. 2002) , Soehngenia (Parshina et al. 2003) , Sporanaerobacter (Hernandez-Eugenio et al. 2002)  and Tissierella (Collins and Shah 1986) . Currently, 31 species with validly published names are reported in this family . The species listed in the Clostridiales Incertae Sedis XI are mostly comprised of Gram-positive, obligate anaerobic cocci. Members belonging to this family were identified as pathogens in both humans and animals. In humans, they were often isolated from patients with septic arthritis, necrotizing pneumonia, prosthetic joint infection and other clinical conditions associated with vaginal discharges and ovarian, peritoneal and sacral abscesses [42–46].
Here we present a summary classification and a set of features for K. massiliensis gen. nov., sp. nov., strain ph2T (CSUR=P241, DSM=26229) together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the genus Kallipyga and its type species, K. massiliensis within the Clostridiales Incertae Sedis XI family.
Classification and features
A stool sample was collected from a 26-year-old woman living in Marseille (France). She suffered from morbid obesity and had a body mass index of 48.2 (118.8 kg, 1.57 meter). At the time of stool sample collection she did not take any medication and was not on a diet. The patient gave an informed and signed consent, and the agreement of the ethics committee of the Institut Fédératif de Recherche (IFR48, Faculty of Medicine, Marseille, France) was obtained under reference 09-022. Another four new bacterial species, Alistipes obesi, Peptoniphilus grossensis, P. obesi and Enorma massiliensis [25–27,33], were also isolated from this specimen using various culture conditions. The fecal specimen was preserved at −80°C after collection. Strain ph2 T (Table 1) was isolated in 2011 by anaerobic culture on 5% sheep blood-enriched agar in anaerobic atmosphere at 37°C, following 26 days in a blood culture bottle with rumen and sheep blood. The 16S rRNA nucleotide sequence (GenBank accession number JN837487) of Kallipyga massiliensis strain ph2T was 86.09% similar to Helcococcus sueciensis, the phylogenetically closest species (Figure 1). This value was lower than the 95.0% 16S rRNA gene sequence threshold recommended by Stackebrandt and Ebers (2006) to delineate a new genus without carrying out DNA-DNA hybridization .
By comparison to the Genbank database , strain ph2T also exhibited a nucleotide sequence similarity greater than 98.7% with 16 sequences from uncultured bacteria from the human skin microbiome . These bacteria are most likely classified within the same species as strain ph2T.
Different growth temperatures (25, 30, 37, 45°C) were tested; no growth occurred at 25°C and 30°C, growth occurred between 37°C and 45°C, and optimal growth was observed at 37°C. Colonies were bright grey with a diameter of 1.0 mm on 5% blood-enriched Columbia 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, with or without 5% CO2. Optimal growth was obtained anaerobically. No growth was observed under aerobic and microaerophilic conditions. Gram staining showed Gram-positive cocci (Figure 2). A motility test was negative. Cells grown on agar are Gram-positive, have a diameter in electron microscopy ranging from 0.57µm to 0.78µm (mean, 0.67 µm, Figure 3) and are mostly grouped in pairs, short chains or small clumps.
Strain ph2T exhibited neither catalase or oxidase activities. Using API 32A (BioMerieux), nitrate reduction, indole formation and urease production were negative. A positive reaction was obtained for α-galactosidase, arginine dihydrolase and arginine arylamidase, α-glucosidase and β-glucosidase. Strain ph2T did not ferment mannose or raffinose. Negative reactions were observed for β-galactosidase, β-galactosidase-6-phosphate, α-arabinosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, glutamic acid decarboxylase, proline arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase, and serine arylamidase. Using an API Zym (BioMerieux), positive reactions were observed for esterase lipase, leucine arylamidase, α-glucosidase, β-glucosidase and acid phosphatase. Negative reactions were obtained for esterase, lipase, valine and cysteine arylamidase, trypsine, α-chymotrypsine, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Using an API 50CH (BioMerieux), K. massiliensis weakly fermented D-ribose, D-glucose, D-fructose and aesculin. By comparison with its closest phylogenetic neighbors, K. massiliensis differed from Finegoldia magna in α-galactosidase and α-glucosidase production, D-ribose and D-fructose fermentation. It also differed from Helcococcus kunzii in oxygen requirement, α-galactosidase and leucine arylamidase production, D-ribose, D-glucose and esculin utilization. It differed from Parvimonas micra in alkaline phosphatase, glutamyl glutamic acid arylamidase, β-glucosidase, phenylalanine arylamidase and histidine arylamidase production and D-glucose fermentation. It differed from Peptoniphilus indolicus in α-galactosidase, indole, α-glucosidase, β-glucosidase, dihydrolase phenylalanine, phenylalanine arylamidase and histidine arylamidase production, and D-glucose fermentation (Table 2).
K. massiliensis is susceptible to amoxicillin, amoxicillin-clavulanic acid, gentamicin 500, penicillin, imipenem, vancomycin, rifampicin and nitrofurantoin, but resistant to ciprofloxacin, metronidazole, gentamicin 10, trimethoprim/sulfamethoxazole, ceftriaxon, erythromycin and doxycycline.
Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was carried out as previously described  using a Microflex spectrometer (Bruker Daltonics, Germany). Twelve distinct deposits were done for strain ph2T from 12 isolated colonies. The twelve ph2T spectra were imported into our database and compared to spectra from 3,769 bacteria using the MALDI BioTyper software (version 2.0, Bruker). A score enabled the presumptive identification and discrimination of the tested species from those in a database: a score ≥ 2 with a validly published species enabled the identification at the species level; a score ≥ 1.7 but < 2 enabled the identification at the genus level; and a score < 1.7 did not enable any identification. For strain ph2T, no significant score was obtained, suggesting that our isolate was not a member of any known species or genus (Figures 4 and 5). A broader study incorporating MALDI-TOF and 16S rDNA and genomic DNA identity data may be conducted to define taxonomic criteria at the family level.
Genome sequencing information
Genome project history
The organism was selected for sequencing on the basis of its phylogenetic position and 16S rRNA similarity to members of the family Clostridiales Incertae Sedis XI and is part of a study of the human digestive flora aiming at isolating all bacterial species within human feces [1–3]. It was the thirty-sixth genome from the family Clostridiales Incertae Sedis XI to be sequenced and the first genome of K. massiliensis gen. nov., sp. nov. The GenBank accession number is CAHC00000000 and consists of 22 contigs. Table 3 shows the project information and its association with MIGS version 2.0 compliance .
Growth conditions and DNA isolation
Kallipyga massiliensis gen. nov., sp. nov., strain ph2T (CSUR= P241, DSM=26229) was grown anaerobically on 5% sheep blood-enriched Columbia agar at 37°C. Three petri dishes were spread and the bacteria cultivated were resuspended in 3 × 100µl of G2 buffer (EZ1 DNA Tissue kit, Qiagen). A first mechanical lysis was performed by glass powder on the Fastprep-24 device (MP Biomedicals, USA) using 2 × 20 seconds cycles. DNA was then treated with 2.5µg/µL lysozyme for 30 minutes at 37°C and extracted using the BioRobot EZ1 Advanced XL (Qiagen). The DNA was then concentrated and purified on a QIAamp kit (Qiagen). The yield and concentration was measured by the Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer at 78.2ng/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 3.179kb. A 3kb paired-end library was constructed according to the 454 GS FLX Titanium paired-end protocol (Roche). Circularization and nebulization were performed and generated a pattern with an optimal at 600 bp. After PCR amplification through 17 cycles followed by double size selection, the single stranded paired-end library was quantified on the Quant-it Ribogreen kit (Invitrogen) on the Genios Tecan fluorometer at 58 pg/µL. The library concentration equivalence was calculated as 1.77E+08 molecules/µL. The library was stored at −20°C until further use.
The paired-end library was clonally amplified with 0.5 cpb and 1 cbp in 2 SV-emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR were essentially the same at 12.3 and 12%, in the range of 5 to 20% recommended by the Roche procedure.
Approximately 790,000 beads were loaded on 1/4 region of a GS Titanium PicoTiterPlate PTP Kit 70x75 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 gsAssembler (Roche). A total, of 261,794 passed filter wells were obtained and generated 90.68 Mb with a length average of 346 bp. The global passed filter sequences were assembled using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 3 scaffolds and 22 large contigs (> 1,500 bp) generating a genome size of 1.77 Mb which corresponds to a coverage of 51.23× genome equivalent.
Open Reading Frames (ORFs) were predicted using Prodigal  with default parameters. However, the predicted ORFs were excluded if they spanned a sequencing gap region. The predicted bacterial protein sequences were searched against the GenBank  and Clusters of Orthologous Groups (COG) databases using BLASTP. The tRNAs and rRNAs were predicted using the tRNAScan-SE  and RNAmmer  tools, respectively. 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-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. Artemis  and DNA Plotter  were used for data management and visualization of genomic features, respectively. Mauve alignment tool (version 2.3.1) was used for multiple genomic sequence alignment . To estimate the mean level of nucleotide sequence similarity at the genome level between K. massiliensis and four other members of the family Clostridiales Incertae Sedis XI (Table 6), orthologous proteins were detected using the Proteinortho software  and genomes compared two by two. For each pair of genomes, we determined the mean percentage of nucleotide sequence identity among orthologous ORFs using BLASTn.
The genome is 1,770,679 bp long (one chromosome, no plasmid) with a G+C content of 51.40% (Figure 6 and Table 4). Of the 1,625 predicted chromosomal genes, 1,575 were protein-coding genes and 50 were RNAs. A total of 1,238 genes (76.18%) were assigned a putative function. Forty-two genes were identified as ORFans (2.66%) and the remaining genes were annotated as hypothetical proteins. The properties and statistics of the genome are summarized in Tables 4 and 5. The distribution of genes into COGs functional categories is presented in Table 5.
Genomic comparison of K. massiliensis and other members of the family Clostridiales Incertae Sedis XI.
Currently, 35 genomes are available for members of the family Clostridiales Incertae Sedis XI. Here, we compared the genome sequence of K. massiliensis strain ph2T with those of Finegoldia magna strain ATCC 29328, Helcococcus kunzii strain ATCC 51366, Peptoniphilus indolicus strain ATCC 29427 and Parvimonas micra strain ATCC 33270. The draft genome of K. massiliensis (1.77Mb) is smaller than all other genomes except P. micra (1.70Mb) and exhibits a higher G+C content (51.40) (Table 6A). The gene content of K. massiliensis is also lower than the other four genomes used for comparison (Table 6B). In addition, K. massiliensis shared 653, 549, 592 and 548 orthologous genes with F. magna, H. kunzii,P. indolicus and P. micra respectively. The average nucleotide sequence identity ranged from 58.61 to 69.17% among Clostridiales Incertae Sedis XI family species, and from 58.61 to 59.97% between K. massiliensis and other species, thus confirming its new genus status (Table 6B).
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Kallipya massiliensis gen. nov., sp. nov., that contains the strain ph2T. This bacterium has been found in France.
Description of Kallipyga gen. nov.
Kallipyga (cal.li.pi’ga N.L. fem. N. Kallipyga of the Greek epithet kallipygos, said of a statue of Aphrodite having beautifully proportioned buttocks).
Gram-positive cocci. Strictly anaerobic. Mesophilic. Non-Motile. Does not exhibit catalase, oxidase and indole production nor nitrate reduction. Positive for α-galactosidase, arginine dihydrolase, arginine arylamidase, and α- and β-glucosidase. Habitat: human digestive tract. Type species: Kallipyga massiliensis.
Description of Kallipyga massiliensis gen. nov., sp. nov.
Kallipyga massiliensis (mas.il’ien’sis. L. gen. fem. n. massiliensis, of Massilia, the Latin name of Marseille where was cultivated strain ph2T). It has been isolated from the feces of an obese French patient.
Gram-positive cocci. Strictly anaerobic. Mesophilic. Optimal growth at 37°C. Non-motile and non-sporulating. Colonies are bright grey with 1.0 mm in diameter on blood-enriched Columbia agar. Cells are cocci with a diameter ranging from 0.57 µm to 0.78 µm with a mean diameter of 0.67. Catalase and oxidase activities are negative. Nitrate reduction and indole production are absent. Negative reactions were observed for β-galactosidase, β-galactosidase-6-phosphate, α-arabinosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, glutamic acid decarboxylase, proline arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase, and serine arylamidase, mannose and raffinose fermentation, esterase, lipase, valine and cystine arylamidase, trypsine, α-chymotrypsine, naphthol-AS-BI-phosphohydrolase, β-galactosidase, β-Glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Positive reactions were observed for α-galactosidase, arginine dihydrolase and arginine arylamidase, α and β-glucosidase, esterase lipase, leucine arylamidase and acid phosphatase. Cells weakly oxidized D-ribose, D-glucose, D-fructose and aesculin. Cells are susceptible to amoxicillin, amoxicillin-clavulanic acid, gentamicin 500, penicillin, imipenem, vancomycin, rifampin and nitrofurantoine. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers JN837487 and CAHC00000000, respectively. The G+C content of the genome is 51.40%. The type strain ph2T (= CSUR P241 = DSM 26229) was isolated from the fecal flora of an obese patient in France.
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he authors thank the Xegen Company (www.xegen.fr) for automating the genomic annotation process. This study was funded by the Mediterranee-Infection Foundation.
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Hugon, P., Ramasamy, D., Robert, C. et al. Non-contiguous finished genome sequence and description of Kallipyga massiliensis gen. nov., sp. nov., a new member of the family Clostridiales Incertae Sedis XI. Stand in Genomic Sci 8, 500–515 (2013). https://doi.org/10.4056/sigs.4047997
- Kallipyga massiliensis e]genome e]culturomics e]taxonogenomics