Standards in Genomic Sciences (2013) 7:343-356 DOI:10.4056/sigs.3246665 Non contiguous-finished genome sequence and description of Senegalemassilia anaerobia gen. nov., sp. nov. Jean-Christophe Lagier1, Khalid Elkarkouri1, Romain Rivet1, Carine Couderc1, Didier Raoult1 and Pierre-Edouard Fournier1* 1 Aix-Marseille Université, URMITE, Faculté de médecine, Marseille, France *Corresponding author: Pierre-Edouard Fournier ([email protected]) Keywords: Senegalemassilia anaerobia, genome Senegalemassilia anaerobia strain JC110T sp.nov. is the type strain of Senegalemassilia anaer- obia gen. nov., sp. nov., the type species of a new genus within the Coriobacteriaceae family, Senegalemassilia gen. nov. This strain, whose genome is described here, was isolated from the fecal flora of a healthy Senegalese patient. S. anaerobia is a Gram-positive anaerobic coccobacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,383,131 bp long genome contains 1,932 protein- coding and 58 RNA genes. Introduction Classification and features Senegalemassilia anaerobia strain JC110T (= CSUR A stool sample was collected from a healthy 16- P147 = DSMZ 25959) is the type strain of S. anaer- year-old male Senegalese volunteer patient living obia gen. nov., sp. nov. This bacterium was isolat- in Dielmo (rural village in the Guinean-Sudanian ed from the feces of a healthy Senegalese patient. zone in Senegal), who was included in a research It is a Gram-positive, anaerobic, indole-negative protocol. Written assent was obtained from this coccobacillus. Classically, the polyphasic taxono- individual. No written consent was needed from his my is used to classify the prokaryotes by associat- guardians for this study because he was older than ing phenotypic and genotypic characteristics [1]. 15 years old (in accordance with the previous pro- Culturomics is a new subfield of genomics aimed ject approved by the Ministry of Health of Senegal, at studying the microbial repertoire of the gut, and the assembled village population, and as published has already lead to the isolation of many new bac- elsewhere [28]. Both this study and the assent pro- terial species [2]. In parallel, as more than 3,000 cedure were approved by the National Ethics bacterial genomes have been sequenced so far, we Committee of Senegal (CNERS) and the Ethics proposed to integrate genomic data in descrip- Committee of the Institut Fédératif de Recherche tions of new bacterial species [3-15]. IFR48, Faculty of Medicine, Marseille, France The family Coriobacteriaceae was created in 1997, (agreement numbers 09-022 and 11-017). Several in the class Actinobacteria, and currently contains other new bacterial species were isolated from this 13 genera of anaerobic Gram-positive members of specimen using various culture conditions, includ- the normal intestinal microbiota from humans and ing the recently described Anaerococcus animals [16-28]. Among them, Gordonibacter and senegalensis, Alistipes senegalensis, Alistipes Paraeggherthella have occasionally been isolated timonensis, Peptoniphilus timonensis, Clostridium from Crohn’s disease specimens [26]. senegalense, Paenibacillus senegalensis and Bacillus timonensis, Herbaspirillum massiliense, Kurthia Here we present a summary classification and a massiliensis, Brevibacterium senegalense, set of features for S. anaerobia gen. nov., sp. nov. Aeromicrobium massiliense and Cellulomonas strain JC110T together with the description of the massiliensis[3-15]. complete genomic sequencing and annotation. These characteristics support the circumscription The fecal specimen was preserved at -80°C after of the genus Senegalemassilia and the species S. collection and sent to Marseille. Strain JC110T anaerobia. (Table 1) was isolated in February 2011. The Genomic Standards Consortium Senegalemassilia anaerobia gen. nov., sp. nov. The stool was preincubated for 5 days in a blood Different growth temperatures (25, 30, 37, 45°C) culture bottle, and then inoculated onto 5% sheep were tested; no growth occurred at 25°C or 45°C, blood agar and incubated in anaerobic atmosphere weak growth occurred at 30°C, optimal growth was at 37°C. The strain exhibited a nucleotide sequence observed at 37°C. Colonies were transparent and similarity with members of the Coriobacteriaceae smooth with 0.5 mm in diameter on blood-enriched ranging from 85.3% with Atopobium parvulum to Columbia agar and Brain Heart Infusion (BHI) agar. 92.4% with Enterorhabdus mucosicola (Figure 1). Growth of the strain was tested under anaerobic and This value was lower than the 95% 16S rRNA gene microaerophilic conditions using GENbag anaer and sequence threshold recommended by Stackebrandt GENbag microaer systems, respectively (BioMérieux), and Ebers to delineate a new genus [33]. By compar- and in the presence of air, of 5% CO2 and in aerobic ison to the NR database, strain JC110 T also exhibited conditions. Growth only occurred under anaerobic nucleotide sequence similarities greater than 99% conditions. A motility test was positive. Cells grown with uncultured bacterial clones detected in on agar appear as Gram-positive coccobacilli (Figure metagenomic studies of the human gut flora. These 2) and have a diameter ranging from 0.62 to 0.76 µm bacteria are most likely classified within the same (mean of 0.70 µm) and a length ranging from 1.36 to species as strain JC110 (Figure 1). 1.73 µm (mean of 1.56 µm)(Figure 3). Table 1. Classification and general features of Senegalemassilia anaerobia strain JC110T according to the MIGS recommendations [29] MIGS ID Property Term Evidence codea Domain Bacteria TAS [30] Phylum Actinobacteria TAS [31] Class Actinobacteria TAS [16] Current classification Order Coriobacteriales TAS [16,32] Family Coriobacteriaceae TAS [16,32] Genus Senegalemassilia TAS Species Senegalemassilia anaerobia IDA Type strain JC110T Gram stain positive IDA Cell shape coccobacillus IDA Motility motile IDA Sporulation nonsporulating IDA Temperature range mesophile IDA Optimum temperature 37°C IDA MIGS-6.3 Salinity unknown IDA MIGS-22 Oxygen requirement anaerobic IDA Carbon source unknown Energy source unknown MIGS-6 Habitat human gut IDA MIGS-15 Biotic relationship free living IDA MIGS-14 Isolation human feces Pathogenicity unknown Biosafety level 2 MIGS-4 Geographic location Senegal IDA MIGS-5 Sample collection time September 2010 IDA MIGS-4.1 Latitude 13.7167 IDA MIGS-4.1 Longitude – 16.4167 IDA MIGS-4.3 Depth Surface IDA MIGS-4.4 Altitude 51 m above sea level IDA Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, iso- lated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [33]. If the evidence is IDA, then the property was di- rectly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements. 344 Standards in Genomic Sciences Lagier et al. Senegalemassilia anaerobia (JF824809) 62 Uncultured bacterium clone RL181 (DQ798733) sequence amplified from human fecal sample (FP082007) 54 Uncultured bacterium clone TS15 (FJ364506) 99 Uncultured bacterium clone 16slp69 (FJ508678) 53 Uncultured bacterium clone orang1 (EU467470) 100 Coriobacteriaceae bacterium WAL 18889 (GQ433638) 99 Uncultured bacterium clone AP15K (AM406030) Uncultured bacterium clone Z93 (DQ353935) 64 100 Adlercreutzia equolifaciens (AB306661) Asaccharobacter celatus (AB266102) 34 100 Enterorhabdus caecimuris (DQ789120) 82 Enterorhabdus musicola (AM747811) Denitrobacterium detoxificans (U43492) 70 Eggerthella lenta (CP001726) 85 29 Eggerthella sinensis (AY321958) 85 Gordonibacter pamelae (AM886059) 64 Paraeggerthella hongkongensis (AY288517) Slackia isoflavoniconvertens (EU826403) 50 Slackia equolifaciens (EU377663) 40 99 Slackia piriformis (AB490806) 88 81 Slackia faecicanis (AJ608686) Slackia exigua (AF101240) 100 Slackia heliotrinireducens (CP001684) Cryptobacterium curtum (CP001682) Colinsella aerofaciens (AB011816) Coriobacterium glomerans (X79048) 100 Colinsella tanakaei (AB490807) 53 94 Colinsella intestinalis (AB031063) 100 Colinsella stercoris (AB031061) 73 Olsenella profusa (AF292374) 58 Olsenella umbata (FN178463) Olsenella uli (CP002106) 100 Atopobium vaginae (Y17195) 100 Atopobium minutum (X67148) 73 Atopobium fossor (L34620) 55 Atopobium rimae (AF292371) 100 Atopobium parvulum (AF292372) Figure 1. Phylogenetic tree highlighting the phylogenetic position of Senegalemassilia anaerobia strain JC110T relative to other type strains within the Coriobacteriaceae family. GenBank accession numbers are indicated in parentheses. Sequences were aligned using CLUSTALW, and phylogenetic inferences were made using the maximum-likelihood method within the MEGA software. Numbers at the nodes are percentages of bootstrap values (500 repetitions) to generate a majority consensus tree. The scale bar indicates a 1% nucleotide sequence divergence. The red square groups sequences that exhibit degrees of similarity > 99% with S. anaerobia (same species), whereas that in the dashed-line square is 97.2% similar (same genus). http://standardsingenomics.org 345 Senegalemassilia anaerobia gen. nov., sp. nov. Figure 2. Gram staining of S. anaerobia strain JC110T Figure 3. Transmission electron microscopy of S. anaerobia strain JC110T, using a Morgani 268D (Philips) at an