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Non contiguous-finished genome sequence and description of Enorma timonensis sp. nov.
Standards in Genomic Sciences volume 9, pages970–985 (2014)
Enorma timonensis strain GD5T sp. nov., is the type strain of E. timonensis sp. nov., a new member of the genus Enorma within the family Coriobacteriaceae. This strain, whose genome is described here, was isolated from the fecal flora of a 53-year-old woman hospitalized for 3 months in an intensive care unit. E. timonensis is an obligate anaerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,365,123 bp long genome (1 chromosome but no plasmid) contains 2,060 protein-coding and 52 RNA genes, including 4 rRNA genes.
Enorma timonensis strain GD5T (= CSUR P900 = DSM 26111) is the type strain of E. timonensis sp. nov. This bacterium was isolated from the stool of a 53-year-old French woman hospitalized for 3 months into an intensive care unit for a Guillain-Barre syndrome, as part of a culturomics study aiming at cultivating individually all species within human feces [1–3]. It is a Gram-positive, anaerobic, non-endospore forming, indole-negative, rod-shaped bacillus.
The human gut microbiota consists of billions of microorganisms that outnumber the human cells . Advances in DNA sequence-based technologies and the development of 16S ribosomal RNA sequence-based metagenomic methods have been used to explore the complex gut microbial population, which has a crucial role in human health and disease development [5,6]. The currently used strategy for determining the taxonomic status of a bacterial isolate includes comparing it to its phylogenetically closest neighbors in terms of 16S rRNA gene similarity, G + C content and DNA-DNA hybridization (DDH) [7,8]. However, although considered “gold standards” in bacterial taxonomy, these criteria do not apply to all genera [9,10]. The development of high-throughput sequencing methods  enabled the generation of complete genomic sequences for most bacterial species of medical interest (more than 6,000 bacterial genomes sequenced to date). We recently proposed to describe new bacterial species using a polyphasic approach based on their genome sequence, MALDI-TOF spectrum and main phenotypic characteristics [12–34].
Here, we present a summary classification and a set of features for E. timonensis sp. nov. strain GD5T (= CSUR P900 = DSM 26111) as well as the description of the complete genome sequencing and annotation. These characteristics support the circumscription of the species E. timonensis.
The family Coriobacteriaceae (Stackebrandt et al. 1997) was created in 1997  and presently consists of 13 validated genera : Adlercreutzia (Maruo et al. 2008) , Asaccharobacter (Minamida et al. 2008) , Atopobium (Collins and Wallbanks 1993) , Collinsella (Kageyama et al. 1999) , Coriobacterium (Haas and König 1988) , Cryptobacterium (Nakazawa et al. 1999) , Denitrobacterium (Anderson et al. 2000) , Eggerthella (Wade et al. 1999) , Entherorhabdus (Clavel et al. 2009) , Gordonibacter (Würdemann et al. 2009) , Olsenella (Dewhirst et al. 2001) , Paraeggerthella (Würdemann et al. 2009) , Slackia (Wade et al. 1999) , and the recently described new genus Enorma (Mishra et al. 2013) . These microorganisms are anaerobic, Gram-positive, rod-shaped bacteria . Members of the family Coriobacteriaceae are isolated from the fecal microbiota of humans or animals, and may cause infections such as bacteremia, wound infections and periodontal/endodontic infections. Members of this family also interfere with the metabolism of triglycerides, glucose, and glycogen in humans and animals [35–47].
Classification and features
A stool sample was collected from a 53-year-old woman living in Marseille, France and hospitalized for 3 months in an intensive care unit for Guillain-Barre syndrome. She received antibiotics at the time of stool sample collection. The patient gave an informed and signed consent, and the agreement of the local ethics committee of the Institut Federatif de Recherche 48 (Marseille, France) was obtained under agreement 09-022. The fecal specimen was preserved at −80°C after collection. Strain GD5T (Table 1) was isolated in 2012 by anaerobic cultivation at 37°C on 5% sheep blood-enriched Columbia agar (BioMerieux, Marcy l’Etoile, France), after 3 weeks of preincubation of the stool sample with clarified and sterile sheep rumen in an anaerobic blood culture bottle.
The 16S rDNA sequence (GenBank accession number JX424767) of E. timonensis strain GD5T exhibited the highest similarity (95.0%) with its phylogenetically closest published species, Enorma massiliensis (Figure 1). By comparison with the type species of genera from the family Coriobacteriaceae, E. timonensis exhibited a 16S rDNA sequence similarity ranging from 84 to 95%. This value was lower than the 98.7% 16S rDNA gene sequence threshold recommended by Stackebrandt and Ebers to delineate a new species without carrying out DNA-DNA hybridization .
Growth at different temperatures (25, 30, 37, 45°C) was tested. No growth was observed at 25°C or 30°C. Growth occurred at both 37 and 45°C, but optimal growth was observed at 37°C after 48 hours of incubation. Colonies were translucent grey and approximately 0.4 mm in diameter on 5% sheep blood-enriched Columbia agar (BioMerieux). Growth of the strain was tested in blood-enriched Columbia agar under anaerobic and microaerophilic conditions using GENbag anaer and GENbag microaer systems, respectively (BioMerieux), and under aerobic conditions, with or without 5% CO2. Growth was achieved only anaerobically. Gram staining showed Gram-positive and non-sporulated rods (Figure 2). A motility test was negative. Cells grown on agar have a mean diameter of 0.58 µm and a mean length of 1.32µm, and are mostly grouped in short chains or small clumps (Figure 3).
Strain GD5T exhibited neither catalase nor oxidase activities (Table 2). Using an API ZYM strip (BioMerieux), positive reactions were observed for leucine arylamidase, valine arylamidase, cystin arylamidase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, β-glucuronidase, α-glucosidase and β-glucosidase. Negative reactions were observed for acid phosphatase, nitrate reduction, urease alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), trypsin, α-chemotrypsin, acid phosphatase, α-galactosidase, N-actetyl-β-glucosaminidase, α-mannosidase, α-fucosidase. Using an API Rapid ID 32A strip (BioMerieux), positive reactions were observed for proline arylamidase, phenylalanine arylamidase, histidin arylamidase, serine arylamidase. Negative reactions were observed for urease, arginine dihydrolase, tyrosin arylamidase, leucyl-glycyl arylamidase, alanine arylamidase, glycine arylamidase and arginine arylamidase. Using an API 50 CH strip (BioMerieux), negative reactions were recorded for fermentation of glycerol, erythritol, D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose, D-adonitol, methyl-βD-xylopranoside, D-galactose, D-glucose, D-fructose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol, methyl-α-D-xylopyranoside, methyl-α-D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, esculin ferric citrate, salicin, D-cellobiose, D-maltose, D-lactose, D-mellibiose, D-saccharose, D-trehalose, inulin, D-melezitose, D-raffinose, amidon, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-ketogluconate, potassium-5-ketogluconate.
E. timonensis is susceptible to amoxicillin-clavulanic acid, metronidazole, imipenem, vancomycin, rifampicin, gentamicin and resistant to penicillin G, amoxicillin, ceftriaxon, erythromycin, and trimethoprim/sulfamethoxazole.
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, Leipzig, Germany). Twelve distinct deposits were done for strain GD5T from twelve isolated colonies. The twelve GD5T spectra were imported into the MALDI BioTyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against the main spectra of 4,706 bacteria, which were used as reference data, in the BioTyper database. For strain GD5T, no significant score was obtained, thus suggesting that our isolate was not a member of a known species. We added the spectrum from strain GD5T to our database (Figure 4, Figure 5).
Genome sequencing information
Genome project history
The organism was selected for sequencing on the basis of its phylogenetic position and 16S rDNA similarity to E. massiliensis and other members of the family Coriobacteriaceae 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 2nd genome of an Enorma species and the first genome of E. timonensis sp. nov. The GenBank accession number is CAPF00000000 and consists of 105 contigs. Table 3 shows the project information and its association with MIGS version 2.0 compliance .
Growth conditions and DNA isolation
Enorma timonesis sp. nov., strain GD5T (= CSUR P900 = DSM 26111), was grown anaerobically on 5% sheep blood-enriched Columbia agar (BioMerieux) at 37°C. Four Petri dishes were spread and resuspended in 1ml TE buffer prior to being treated with 2.5 µg/µL lysozyme for 30 minutes at 37°C, and then with Proteinase K overnight at 37°C. The DNA was then purified by 3 successive phenol-chloroform extractions followed by an ethanol precipitation at −20°C overnight. Following centrifugation, the DNA was then resuspended in 305 µL TE buffer. The DNA was then concentrated and purified using a QIAamp kit (Qiagen). The yield and concentration was measured by the Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer at 66.5 ng/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 4.4kb. 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 470 bp. After PCR amplification through 17 cycles followed by double size selection, the single stranded paired-end library was then quantified on the Agilent 2100 BioAnalyzer on a RNA pico 6000 LabChip at 136 pg/µL. The library concentration equivalence was calculated as 5.31E+08 molecules/µL. The library was stored at −20°C until further use.
The paired-end library was clonally amplified with 0.5cpb and 2cbp in 2 SV-emPCR with the GS Titanium SV-emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCRs were 9.37 and 14.09%, respectively, in the range of 5 to 20% from 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 282,633 passed filter wells were obtained and generated 102.68Mb with a length average of 363 bp. The globally passed filter sequences were assembled using Newbler with 90% identity and 40bp as overlap. The final assembly identified 5 scaffolds and 105 large contigs (>1,500 bp) generating a genome size of 2.36 Mb which corresponds to a coverage of 43.5 genome equivalents.
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 tRNAScanSE  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. The 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 E. timonensis and five other members of the family Coriobacteriaceae (Table 6), we used the Average Genomic Identity Of gene Sequences (AGIOS) home-made software. Briefly, this software combines the Proteinortho software  for detecting orthologous proteins between genomes compared two by two, then retrieves the corresponding genes and determines the mean percentage of nucleotide sequence identity among orthologous ORFs using the Needleman-Wunsch global alignment algorithm. Enorma timonensis strain GD5T was compared to E. massiliensi strain phIT (GenBank accession number CAGZ00000000), C. aerofaciens strain ATCC 25986 (AAVN00000000), C. tanakei strain YIT 12063 (ADLS00000000) and C. glomerans strain PW2 (NC_015389).
The genome is 2,365,123 bp long (1 chromosome, no plasmid) with a 65.8% G+C content (Figure 6 and Table 4). Of the 2,060 predicted chromosomal genes, 2,006 were protein-coding genes and 52 were RNAs, including a complete rRNA operon, an additional 5S rRNA and 48 tRNAs. A total of 1,384 genes (67.18%) were assigned a putative function. Fifty-five genes were identified as ORFans (2.74%) and the remaining genes were annotated as hypothetical proteins. The properties and statistics of the genome are summarized in Tables 3 and 4. The distribution of genes into COGs functional categories is presented in Table 5.
Genome comparison of E. timonensis with other members of the Coriobacteriaceae family
We compared the genome of E. timonensis strain GD5T with those of E. massiliensis phI, Collinsella aerofaciens strain ATCC 25986, Collinsella tanakaei strain YIT 12063 and Coriobacterium glomerans strain PW2 (Table 6).
The draft genome sequence of E. timonensis strain GD5T is smaller than those of C. aerofaciens and C. tanakaei (2.36, 2.43 and 2.48 Mb, respectively), but larger than those of E. massiliensis and C. glomerans (2.26 and 2.11 Mb, respectively). The G+C content of E. timonensis is larger than those of E. massiliensis, C. aerofaciens, C. tanakaei and C. glomerans (65.80, 62.0, 60.54, 60.23 and 60.40%, respectively). The gene content of E. timonensis is smaller to those of E. massiliensis, C. glomerans and C. tanakaei (2,006, 2,159 and 2,195, respectively) but larger than those of C. aerofaciens and C. tanakaei (1,901 and 1,768, respectively). The distribution of genes into COG categories was not entirely similar in all compared genomes (Figure 7).
In addition, E. timonensis shared 1,109, 1,026, 880 and 1,077 orthologous genes with E. massiliensis, C. aerofaciens, C. glomerans and C. tanakaei respectively. The average genomic nucleotide sequence identity ranged from 66.37 to 79.44% among Coriobacteriaceae family members, and from 66.01 to 79.44% between E. timonensis and other species (Table 6 and Table 7).
On the basis of phenotypic, phylogenetic and genomic analyses (taxono-genomics), we formally propose the creation of Enorma timonensis sp. nov. that contains strain GD5T. This bacterium has been found in France.
Description of Enorma timonensis sp. nov.
Enorma timonensis (ti.mo.nen’sis. L. gen. masc. timonensis, of Timone, the name of the hospital where strain GD5T was cultivated). Colonies are translucent grey and 0.4 mm in diameter on blood-enriched Columbia agar. Cells are rod-shaped with a mean diameter of 0.58 µm and a mean length of 1.32 µm. Optimal growth is achieved in anaerobic conditions. No growth is observed in aerobic or microaerophilic conditions. Growth occurs between 37–45°C, with optimal growth being observed at 37°C on blood-enriched Columbia agar. Cells are Gram-positive, non-endospore forming, and non-motile. Cells are negative for catalase and oxidase. Using an API ZYM strip, positive reactions are observed for leucine arylamidase, valine arylamidase, cystin arylamidase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, β-glucuronidase, α-glucosidase and β-glucosidase. Negative reactions are observed for acid phosphatase, nitrate reduction, urease alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), trypsin, α-chemotrypsin, acid phosphatase, α-galactosidase, N-actetyl-β-glucosaminidase, α-mannosidase, α-fucosidase. Using an API Rapid ID 32A strip, positive reactions are observed for proline arylamidase, phenylalanine arylamidase, histidin arylamidase, serine arylamidase. Negative reactions are observed for urease, arginine dihydrolase, tyrosin arylamidase, leucyl-glycyl arylamidase, alanine arylamidase, glycine arylamidase and arginine arylamidase. Using an API 50 CH strip, fermentation or assimilation was not observed.
Cells are susceptible to amoxicillin-clavulanic acid, metronidazole, imipenem, vancomycin, rifampicin, gentamicin and resistant to penicillin G, amoxicillin, ceftriaxon, erythromycin, and trimethoprim/sulfamethoxazole. The 16S rDNA and genome sequences are deposited in GenBank under accession numbers JX424767 and CAPF00000000, respectively. The G+C content of the genome is 65.8%. The habitat of the organism is the human digestive tract. The type strain GD5T (= CSUR P900 = DSM 26111) was isolated from the fecal flora of a 53-year old French patient hospitalized in an intensive care unit. This strain has been found in Marseille, France.
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The authors thank the Xegen Company for automating the genomic annotation process. This study was funded by the Mediterranee Infection Foundation.
These 2 authors contributed equally