- Open Access
Non contiguous-finished genome sequence and description of Cellulomonas massiliensis sp. nov.
© The Author(s) 2012
Published: 19 December 2012
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.
Cellulomonas massiliensis strain JC225T (= CSUR P160 = DSM 25695) is the type strain of C. massiliensis sp. nov. This bacterium is a motile, Gram-positive, aerobic, indole-negative rod that was isolated from the stool of a healthy Senegalese patient as part of a culturomics study aiming at cultivating all bacterial species within human feces .
The current approach to the classification of prokaryotes, known as polyphasic taxonomy, relies on a combination of phenotypic and genotypic characteristics . However, as more than 3,000 bacterial genomes have been sequenced , and proteomic information is more becoming more readily accessible , we recently proposed that genomic information should be integrated in the description of new bacterial species [5–11].
The genus Cellulomonas was created in 1923 to reclassify several bacteria previously classified as Bacillus species . To date, this genus is made of 19 species [13–24]. The two species that are the most phylogenetically related to C. massiliensis are C. composti  and C. persica . Most of these species were originally solated from environmental samples, notably from habitats enriched in cellulose, such as soil or sugar fields, and occasionally from the rumen and activated sludge. Rare cases of human endocarditis , osteomyelitis , endophtalmitis  and cholecystitis  caused by Cellulomonas species have been reported. To date, members of the genus Cellulomonas have not been described in the normal fecal flora.
Here we present a summary classification and a set of features for C. massiliensis sp. nov. strain JC225T together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the species C. massiliensis.
Classification and features
A stool sample was collected from a healthy 16-year-old male Senegalese volunteer patient living in Dielmo (a rural village in the Guinean-Sudanian zone in Senegal), who was included in a research protocol. Written assent was obtained from this individual; no written consent was needed from his guardians for this study because he was older than 15 years old (in accordance with the previous project approved by the Ministry of Health of Senegal and the assembled village population and as published elsewhere .
Classification and general features of Cellulomonas massiliensis strain JC225T
Species Cellulomonas massiliensis
Type strain JC225T
growth in BHI medium + 5% NaCl
Sample collection time
51 m above sea level
Differential phenotypic characteristics of five Cellulomonas strains†.
G+C content (mol%)
Forest humus soil
Forest humus soil
Genome sequencing information
Genome project history
Paired-end 3 Kb library
454 GS FLX Titanium
Newbler version 2.5.3
Gene calling method
EMBL Date of Release
May 30, 2012
Study of the human gut microbiome
Growth conditions and DNA isolation
Cellumonas massiliensis sp. nov. JC225T (= CSUR P160 = DSM 25695) was grown aerobically on 5% sheep blood-enriched Columbia agar (BioMérieux) at 37°C. Ten petri dishes were spread and resuspended in 3×100µl of G2 buffer (EZ 1 DNA Tissue kit, Qiagen). A first mechanical lysis was performed using glass powder on a Fastprep-24 device (MP Biomedicals, Ilkirch, France) during 2×20 seconds. DNA was then treated with 2.5µg/µL lysozyme (30 minutes at 37°C) and extracted using a BioRobot EZ 1 Advanced XL (Qiagen). The DNA was then concentrated and purified using a Qiamp kit (Qiagen). The yield and the concentration were measured using a Quant-it Picogreen kit (Invitrogen) on a Genios_Tecan fluorometer at 78.9 ng/µl.
Genome sequencing and assembly
Both shotgun sequencing and paired-end sequencing strategies were used (Roche). Both libraries were pyrosequenced on a GS FLX Titanium sequencer (Roche). This project was loaded onto a single 1/4 region of a PTP Picotiterplate (Roche, Meylan, France) for the shotgun library and 2 ×1/4 region for the 3-kb paired-end library. The shotgun library was constructed with 500ng of DNA with the GS Rapid library Prep kit as described by the manufacturer (Roche). For the paired-end library, 5µg of DNA was mechanically fragmented on a Hydroshear device (Digilab, Holliston, MA, USA) with an enrichment size at 3–4 kb. The DNA fragmentation was visualized through an Agilent 2100 BioAnalyzer on a DNA labchip 7500 with an optimal size of 3.216 kb. The library was constructed according to the 454 Titanium paired-end protocol (Roche). Circularization and nebulization were performed and generated a pattern with an optimum at 395 bp. After PCR amplification through 17 cycles followed by double size selection, the single stranded paired-end library was quantified on with a Quant-it Ribogreen kit (Invitrogen) on a Genios Tecan fluorometer at 132pg/µL. The library concentration equivalence was calculated at 6.11E+08 molecules/µL. The libraries were stored at −20°C until further use.
The shotgun library was clonally amplified with 3 cpb in 3 emPCR reactions and the 3-kb paired-end library was amplified with 0.5cpb in 4 emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yield of the shotgun emPCR reactions was 10.13%, and the yields of the paired-end emPCRs was 8.6%, in the range of 5 to 20% from the Roche procedure.
Approximately 790,000 beads for both the shotgun and paired-end libraries were loaded on the GS Titanium PicoTiterPlate PTP Kit 70×75 and sequenced with the GS FLX Titanium Sequencing Kit XLR70 (Roche). The runs were performed overnight and then analyzed on the cluster through the gsRunBrowser and Newbler Assembler (Roche). A total of 255,758 and 256,082 passed filter wells were obtained for the shotgun and paired-end strategies, respectively, and generated 86.75 and 78.45 Mb of DNA sequence with length averages of 339 and 313 bp, respectively. The filter passed sequences were assembled using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 250 contigs (>200 bp) arranged into 5 scaffolds and generated a genome size of 3.40 Mb.
Open Reading Frames (ORFs) were predicted using Prodigal  with default parameters but the predicted ORFs were excluded if they were spanned 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 tRNAscan-SE tool  was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer . Transmembrane domains and signal peptides were predicted using TMHMM  and SignalP , 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 been used in previous works to define ORFans. To estimate the mean level of nucleotide sequence similarity at the genome level between C. massiliensis and C. flavigena and C. fimi (EMBL accession numbers CP001964 and CP002666, respectively), the only two available genomes from validly published Cellulomonas species to date, we compared the ORFs only using BLASTN at a query coverage of ≥ 70% and a minimum nucleotide length of 100 bp.
Nucleotide content and gene count levels of the genome
% of totala
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
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 the genomes from other Cellulomonas species
Here, we compared the genome sequence of C. massiliensis strain JC225T with those of C. flavigena strain 134T  and C. fimi strain ATCC 484T (EMBL accession number CP002666). The draft genome sequence of C. massiliensis has a smaller size than those of C. flavigena and C. fimi (3.40 vs 4.12 and 4.26 Mb, respectively), a lower G+C content (71.22 vs 74.3 and 74.7, respectively), and a smaller number of predicted genes (3,131 vs 3,788 and 3,863, respectively). In addition, C. massiliensis shared a mean 88.75% (range 70.01-100%) and 89.61% (range 70.07-100%) sequence similarity with C. flavigena and C. fimi, respectively, at the genome level.
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Cellulomonas massiliensis sp. nov. that contains the strain JC225T. This bacterium has been found in Senegal.
Description of Cellulomonas massiliensis sp. nov.
Cellulomonas massiliensis (ma.si.li.e′n.sis. L. gen. masc. n. massiliensis, of Massilia, the Latin name of Marseille where was isolated C. massiliensis).
Colonies are transparent and smooth with a diameter of 1 mm on blood-enriched Columbia agar and Brain Heart Infusion (BHI) agar. Cells are rod-shaped with a diameter and length ranging from 0.37 to 0.60 µm (mean of 0.48 µm), and from 0.55 to 1.4 µm (mean of 0.95 µm), respectively. Optimal growth is achieved aerobically. Weak growth is observed with 5% CO2 and under microaerophilic conditions. No growth is observed under anaerobic conditions. Growth occurs between 30–37°C, with optimal growth at 37°C. Cells stain Gram-positive, are non-endospore forming, and are motile. Catalase, oxidase, aesculin hydrolysis and β-galactosidase activities are present. Indole production, nitrate reduction, glucose fermentation, arginine dihydrolase, urease, gelatin hydrolysis, and glucose, arabinose, mannose, mannitol N-acetyl-glucosamine, maltose, gluconate, caprate, adipate, malate, citrate, and phenyl-acetate assimilation activities are absent. Cells are susceptible to amoxicillin, imipenem, ciprofloxacin and gentamicin, but resistant to trimethoprim/sulfamethoxazole and metronidazole. The 16S rRNA and genome sequences are deposited in Genbank and EMBL under accession numbers JN657218 and CAHD00000000, respectively. The G+C content of the genome is 71.22%. The type strain JC225T (= CSUR P160 = DSM 25695) was isolated from the fecal flora of a healthy patient in Senegal.
The authors thank Mr. Julien Paganini at Xegen Company for automating the genomic annotation process.
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