Standards in Genomic Sciences (2012) 7:258-270 DOI:10.4056/sigs.3316719 Non contiguous-finished genome sequence and description of Cellulomonas massiliensis sp. nov. Jean-Christophe Lagier1, Dhamodharan Ramasamy1, Romain Rivet1, Didier Raoult1 and Pierre-Edouard Fournier1* 1Aix-Marseille Université, Faculté de médecine, Marseille, France *Corresponding author: Pierre-Edouard Fournier ([email protected]) Keywords: Cellulomonas massiliensis, genome Cellulomonas massiliensis strain JC225T sp. nov. is the type strain of Cellulomonas massiliensis sp., a new species within the genus Cellulomonas. This strain, whose genome is described here, was isolated from the fecal flora of a healthy Senegalese patient. C. massiliensis is an aerobic rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,407,283 bp long genome contains 3,083 protein-coding and 48 RNA genes. Introduction Cellulomonas massiliensis strain JC225T (= CSUR date, members of the genus Cellulomonas have not P160 = DSM 25695) is the type strain of C. been described in the normal fecal flora. massiliensis sp. nov. This bacterium is a motile, Here we present a summary classification and a Gram-positive, aerobic, indole-negative rod that set of features for C. massiliensis sp. nov. strain was isolated from the stool of a healthy Senegalese JC225T together with the description of the com- patient as part of a culturomics study aiming at plete genomic sequencing and annotation. These cultivating all bacterial species within human fe- characteristics support the circumscription of the ces [1]. species C. massiliensis. The current approach to the classification of pro- karyotes, known as polyphasic taxonomy, relies Classification and features on a combination of phenotypic and genotypic A stool sample was collected from a healthy 16- characteristics [2]. However, as more than 3,000 year-old male Senegalese volunteer patient living bacterial genomes have been sequenced [3], and in Dielmo (a rural village in the Guinean-Sudanian proteomic information is more becoming more zone in Senegal), who was included in a research readily accessible [4], we recently proposed that protocol. Written assent was obtained from this genomic information should be integrated in the individual; no written consent was needed from description of new bacterial species [5-11]. his guardians for this study because he was older The genus Cellulomonas was created in 1923 to than 15 years old (in accordance with the previ- reclassify several bacteria previously classified as ous project approved by the Ministry of Health of Bacillus species [12]. To date, this genus is made Senegal and the assembled village population and of 19 species [13-24]. The two species that are the as published elsewhere [28]. most phylogenetically related to C. massiliensis are Both this study and the assent procedure were C. composti [17] and C. persica [21]. Most of these approved by the National Ethics Committee of species were originally solated from environmen- Senegal (CNERS) and the Ethics Committee of the tal samples, notably from habitats enriched in cel- Institut Fédératif de Recherche IFR48, Faculty of lulose, such as soil or sugar fields, and occasionally Medicine, Marseille, France (agreement numbers from the rumen and activated sludge. Rare cases 09-022 and 11-017). Several other new bacterial of human endocarditis [25], osteomyelitis [25], species were isolated from this specimen using endophtalmitis [26] and cholecystitis [27] caused various culture conditions, including the recently by Cellulomonas species have been reported. To described Anaerococcus senegalensis, Bacillus The Genomic Standards Consortium Lagier et al. timonensis, Alistipes senegalensis, Alistipes cultivated from cattle farm compost [17]. This timonensis,Clostridium senegalense, Paenibacillus value was lower than the 98.7% 16S rRNA gene senegalensis and Peptoniphilus timonensis [5-11], sequence threshold recommended by thus suggesting that the human digestive flora is Stackebrandt and Ebers to delineate a new species far from being fully known. The fecal specimen without carrying out DNA-DNA hybridization [39]. was preserved at -80°C after collection and sent to By comparison to the Genbank database [40] Marseille. Strain JC225 (Table 1) was isolated in strain JC225T also exhibited a nucleotide sequence May 2011 by passive filtration of the stool and similarity greater than 99.5% with Cellulomonas aerobic incubation on Brain Heart Infusion agar at sp. strain 3335BRRJ isolated from clean room en- 37°C. This strain exhibited a nucleotide sequence vironments (Genbank accession number similarity of 98.3% with Cellulomonas composti FJ200382). This bacterium is most likely classified (Kang et al 2007), the phylogenetically closest val- within the same species as strain JC225 T (Figure idated Cellulomonas species (Figure 1) that was 1). Table 1. Classification and general features of Cellulomonas massiliensis strain JC225T MIGS ID Property Term Evidence codea Current classification Domain Bacteria TAS [29] Phylum Actinobacteria TAS [30] Class Actinobacteria TAS [31] Order Actinomycetales TAS [31-34] Family Cellulomonadaceae TAS [31,34-37] Genus Cellulomonas TAS [12,32] Species Cellulomonas massiliensis IDA Type strain JC225T IDA Gram stain positive IDA Cell shape rod IDA Motility positive IDA Sporulation nonsporulating IDA Temperature range mesophilic IDA Optimum temperature 37°C IDA MIGS-6.3 Salinity growth in BHI medium + 5% NaCl IDA MIGS-22 Oxygen requirement aerobic IDA Carbon source galactose NAS Energy source chemoorganotrophic NAS MIGS-6 Habitat human gut IDA MIGS-15 Biotic relationship free living IDA Pathogenicity unknown NAS Biosafety level 2 MIGS-14 Isolation human feces 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, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [38]. If the evidence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements. http://standardsingenomics.org 259 Cellulomonas massiliensis sp. nov. 81 C. uda (X83801) 31 C. gelida (X79461) 33 C. iranensis (AF064702) C. composti (AB166887) 26 90 C. massiliensis (JN657218) 99 Cellulomonas sp. strain 3335BRRJ (FJ200382) C. persica (AF064701) 21 42 C. phragmiteti (AM902253) 52 C. flavigena (CP001964) 35 C. terrae (AY884570) 77 C. xylanilytica (AY303668) C. humiferus (X82449) 31 27 C. chitinilytica (AB268586) 22 C. aerilata (EU560979) 50 C. fimi (X83803) 100 40 C. biazotea (X83802) C. cellasea (X83804) 65 C. hominis (X82598) C. denverensis (AY501362) 31 Micrococcus luteus (AJ536198) 47 Oerskovia turbata (X79454) 51 Paraoerskovia marina (AB445007) C. bogoriensis (X92152) 73 C. carbonis (HQ702749) Actinotalea fermentans (X79458) 52 Tropheryma whipplei (AF251035) Figure 1. Phylogenetic tree highlighting the position of Cellulomonas massiliensis strain JC225T relative to other type strains within the genus Cellulomonas and other members of the family Cellulomonadaceae. GenBank accession numbers are indicated in parentheses. Sequences were aligned using CLUSTALW, and phylogenetic inferences obtained using the maximum-likelihood method within the MEGA soft- ware. Numbers at the nodes are bootstrap values obtained by repeating the analysis 500 times to gener- ate a majority consensus tree. The scale bar indicates a 1% nucleotide sequence divergence. Different growth temperatures (25, 30, 37, 45°C) growth was achieved aerobically. Weak growth were tested; no growth occurred at 25°C or 45°C, was observed under microaerophilic condition growth occurred between 30 and 37°C, and opti- and with 5% CO2. No growth was observed under mal growth was observed at 37°C. Colonies were anaerobic conditions. Gram staining showed transparent and smooth with a diameter of 1 mm Gram-positive rods. A motility test was positive. on blood-enriched Columbia agar and Brain Heart Cells grown on agar are Gram-positive (Figure 2), Infusion (BHI) agar. Growth of the strain was test- with a diameter and length ranging from 0.37 to ed under anaerobic and microaerophilic condi- 0.60 µm (mean, 0.48 µm), and from 0.55 to 1.4 µm tions using GENbag anaer and GENbag microaer (mean, 0.95 µm), respectively, in electron micros- systems, respectively (BioMérieux), and in the copy, (Figure 3). presence of air, with or without 5% CO2. Optimal 260 Standards in Genomic Sciences Lagier et al. Figure 2. Gram staining of C. massiliensis strain JC225T Figure 3. Transmission electron microscopy of C. massiliensis strain JC225T, using a Morgani 268D (Philips) at an operating voltage of 60kV. The scale bar represents 200 nm. http://standardsingenomics.org 261 Cellulomonas massiliensis sp. nov. Strain JC225T exhibited catalase and oxidase activ- malate, citrate, and phenyl-acetate assimilation. C. ities. Using the API 20 NE system (BioMérieux), a massiliensis is susceptible to amoxicillin, positive reaction was obtained for aesculin hy- imipenem, gentamicin, and ciprofloxacin but