Non-contiguous finished genome sequence and description of Holdemania massiliensis sp. nov.
© The Author(s) 2013
Published: 20 December 2013
Holdemania massiliensis strain AP2T sp. nov. is the type strain of H. massiliensis sp. nov., a new species within the genus Holdemania. This strain, whose genome is described here, was isolated from the fecal flora of a 21-year-old French Caucasian female suffering from severe restrictive anorexia nervosa. H. massiliensis is a Gram-positive, anaerobic bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,795,625 bp-long genome (one chromosome but no plasmid) contains 3,461 protein-coding and 49 RNA genes, including 3 rRNA genes.
KeywordsHoldemania massiliensis genome culturomics taxono-genomics
Holdemania massiliensis strain AP2T (= CSUR P195 = DSM 26143) is the type strain of H. massiliensis sp. nov. This bacterium is a Gram-positive, non-spore-forming, indole negative, anaerobic and non-motile bacillus that was isolated from the stool of a 21-year-old woman suffering from anorexia as part of a “culturomics” study aiming to individually cultivate all species within human feces [1–3].
The current prokaryotic species classification, known as polyphasic taxonomy, is based on a combination of genomic and phenotypic properties . The number of sequenced genomes is increasing exponentially and in parallel with the decreasing cost of sequencing. To date, more than 6,000 bacterial genomes have been published and approximately 25,000 genomes project are anticipated to be completed in a near future . We recently proposed to integrate genomic information in the taxonomic framework and description of new bacterial species [6–27].
Here we present a summary classification and a set of features for H. massiliensis sp. nov. strain AP2T (= CSUR P195 = DSM 26143), together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the species H. massiliensis. The genus Holdemania (Willems et al. 1997) was created in 1997 on the basis of 16S rDNA gene sequence, biochemical tests, fatty acid and cell wall murein analysis . To date, this genus includes a single species, H. filiformis, which was isolated from feces of healthy humans .
Classification and features
Classification and general features of Holdemania massiliensis strain AP2T according to the MIGS recommendations 
Species Holdemania massiliensis
Type strain AP2T
Sample collection time
0 m above sea level
Differential characteristics of Holdemania massiliensis strain AP2T, Holdemania filiformis strain ATCC 51649, Solobacterium moorei strain CCUG 39336 and Erysipelothrix rhusiopathiae strain ATCC 19414T.
Cell diameter (µm)
Hydrolysis of gelatin
G+C content (mol%)
animal feces, environmental, human infection
Genome sequencing information
Genome project history
One 454 paired end 3-kb library
454 GS FLX Titanium
Newbler version 2.5.3
Gene calling method
Genbank Date of Release
July 31, 2012
Study of the human gut microbiome
Growth conditions and DNA isolation
H. massiliensis sp. nov. strain AP2T, (= CSUR P195= DSM 26143), was grown anaerobically on Columbia agar medium at 37°C. Five Petri dishes were spread and resuspended in 3x100µl of G2 buffer (EZ1 DNA Tissue kit, Qiagen). A first mechanical lysis was performed using glass powder in the Fastprep-24 device (Sample Preparation system, MP Biomedicals, USA) for 2×20 seconds. DNA was treated with 2.5 µg/µL of lysozyme (30 minutes at 37°C) and extracted using the BioRobot EZ 1 Advanced XL (Qiagen). The DNA was concentrated and purified on a Qiamp kit (Qiagen). The yield and the concentration of DNA was 69.3 ng/µl as measured by using Quant-it Picogreen kit (Invitrogen) on the Genios Tecan fluorometer
Genome sequencing and assembly
DNA (5 µg) was mechanically fragmented for the paired-end sequencing, using a Covaris device (Covaris Inc., Woburn, MA, USA) with an enrichment size of 3–4 kb. The DNA fragmentation was visualized through an Agilent 2100 BioAnalyzer on a DNA Labchip 7500 which yielded an optimal size of 3.4 kb. The library was constructed using the 454 GS FLX Titanium paired-end rapid library protocol (Roche, Meylan, France). Circularization and nebulization were performed which generated a pattern of optimal size of 589 bp. PCR amplification was performed for 17 cycles followed by double size selection. The single-stranded paired-end library was quantified using a Quant-it Ribogreen Kit (Invitrogen) using the Genios Tecan fluorometer. The library concentration equivalence was calculated as 1.42× 1010 molecules/µL. The library was stored at −20°C until further use.
For the shotgun sequencing, DNA (500 ng) was mechanically fragmented using a Covaris device (Covaris Inc.) as described by the manufacturer. The DNA fragmentation was visualized using an Agilent 2100 BioAnalyzer on a DNA Labchip 7500 which yielded an optimal size of 1.7 kb. The library was constructed using the GS Rapid library Prep kit (Roche) and quantified using a TBS 380 mini fluorometer (Turner Biosystems, Sunnyvale, CA, USA). The library concentration equivalence was calculated as 2.8× 109 molecules/µL. The library was stored at −20°C until further use.
The shotgun library was clonally amplified with 1 and 2 cpb in two emPCR reactions each, and the paired-end library was amplified with 0.5 cpb in three emPCR reactions using the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR were 6.8 and 9.8%, respectively, for the shotgun library, and 11.29% for the paired-end library. These yields fall into the expected 5 to 20% range according to Roche protocol.
For each library, approximately 790,000 beads for a quarter region were loaded on the GS Titanium PicoTiterPlate PTP kit and sequenced with the GS FLX Titanium Sequencing Kit XLR70 (Roche). The run was performed overnight and analyzed on a cluster using the gsRunBrowser and Newbler assembler (Roche). For the shotgun sequencing, 188,659 passed-filter wells were obtained. The sequencing generated 129.3 Mb with an average length of 685 bp. For the paired-end sequencing, 106,675 passed-filter wells were obtained. The sequencing generated 35 Mb with an average length of 262 bp. The passed-filter sequences were assembled using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 8 scaffolds and 66 contigs (>1,500 bp) and generated a genome size of 3.79 Mb which corresponds to a coverage of 54.25 genome equivalents.
Open Reading Frames (ORFs) were predicted using Prodigal  with default parameters, but the predicted ORFs were excluded if they were spanning 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 tRNAScanSE tool  was used to find tRNA genes, whereas ribosomal RNAs were found by using RNAmmer  and BLASTn against the GenBank database. 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. Ortholog sets composed of one gene from each of the four genomes H. massiliensis strain AP2T, Holdemania filiformis strain ATCC 51649 (GenBank accession number ACCF00000000), Solobacterium moorei strain F0204 (AECQ00000000), and Erysipelothrix rhusiopathiae strain Fujisawa (AP012027) were identified using Proteinortho software (version 1.4)  by using cut-off values of 30% protein identity and an E-value of 1e−05. The average percentages of nucleotide sequence identity between corresponding orthologous sets were determined using the Needleman-Wunsch algorithm global alignment technique. Artemis  was used for data management and DNA Plotter  was used for visualization of genomic features. The Mauve alignment tool was used for multiple genomic sequence alignment and visualization .
Nucleotide content and gene count levels of the genome
% of totala
Genome size (bp)
DNA coding region (bp)
DNA G+C content (bp)
Number of replicons
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
Genome comparison with Holdemania filiformis, Solobacterium moorei and Erysipelothrix rhusiopathiae
Here, we compared the genome of H. massiliensis strain AP2T, with those of H. filiformis strain ATCC 51649 (GenBank accession number ACCF00000000), S. moorei strain F0204 (AECQ00000000), and E. rhusiopathiae strain Fujisawa (AP012027).
The draft genome of H. massiliensis is comparable in size to that of H. filiformis (3.79 and 3.80 Mb, respectively) and larger in size than those of S. moorei and E. rhusiopathiae (2.01 and 1.79 Mb, respectively). The G+C content of H. massiliensis is smaller than that of H. filiformis (47.10 and 50.18%, respectively) but higher than those of S. moorei and E. rhusiopathiae (36.80 and 36.60%, respectively).
The numbers of orthologous proteins shared between genomes (upper right)*
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Holdemania massiliensis sp. nov. that contains the strain AP2T. This bacterial strain has been found in Marseille, France.
Description of Holdemania massiliensis sp. nov.
Holdemania massiliensis (mas.si.li.en’sis. L. masc. adj. massiliensis of Massilia, the Roman name of Marseille, France, where the type strain was isolated).
Colonies were 0.2 mm in diameter on blood-enriched Columbia agar and Brain Heart Infusion agar. Cells are rod-shaped with a mean diameter of 0.57 µm and a mean length of 1.75 µm. Strictly anaerobic. Growth occurs between 25 and 45°C, with optimal growth observed at 37°C. Cells stain Gram-positive, and are non-motile. Cells are positive for β-galactosidase, α-glucosidase, α-fucosidase and pyroglutamic acid arylamidase. Positive reactions were observed for glycerol, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, inositol, D-mannitol, D-sorbitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, D-lactose, D-saccharose, D-melezitose, gentiobiose, D-tagatose and potassium gluconate. Cells are susceptible to amoxicillin, metronidazole, vancomycin, clindamycin and imipenem. The G+C content of the genome is 47.10%. The 16S rRNA and genome sequences are deposited in GenBank under accession numbers JX101683 and CALK00000000, respectively.
The type strain AP2T (= CSUR P195 = DSM 26143) was isolated from the fecal flora of French Caucasian female suffering from severe restrictive anorexia nervosa .
The 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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