Non-contiguous finished genome sequence and description of Alistipes timonensis sp. nov.
© The Author(s) 2012
Published: 30 July 2012
Alistipes timonensis strain JC136T sp. nov. is the type strain of A. timonensis sp. nov., a new species within the genus Alistipes. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. A. timonensis is an obligate anaerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,497,779 bp long genome (one chromosome but no plasmid) contains 2,742 protein-coding and 50 RNA genes, including three rRNA genes.
Alistipes timonensis strain JC136T (= CSUR P148 = DSM 25383) is the type strain of A. timonensis sp. nov. This bacterium is a Gram-negative, anaerobic, indole-positive bacillus and was isolated from the stool of a healthy Senegalese patient as part of a “culturomics” study aiming at cultivating individually all species within human feces.
With more than 3,000 genome sequences available, bacterial genomics has revolutionized several aspects of microbiology. To date, taxonomy has remained unaffected by this progress, despite the debate around the definition of bacterial species. Despite its elevated cost, poor reproducibility and inter-laboratory comparability, DNA-DNA hybridization remains the “gold standard” criterion . Even the application of internationally validated cutoff values in 16S rRNA sequence similarity that enabled the taxonomic classification or reclassification of hundreds of taxa, is debated . High throughput genome sequencing and mass spectrometric analyses of bacteria provide access to a wealth of genetic and proteomic information . We propose to use a polyphasic approach  to describe new bacterial taxa that includes their genome sequence, MALDI-TOF spectrum and main phenotypic characteristics (habitat, Gram-stain reaction, culture and metabolic characteristics, and when applicable, pathogenicity).
Here we present a summary classification and a set of features for A. timonensis sp. nov. strain JC136T together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the species A. timonensis.
The genus Alistipes (Rautio et al. 2003) was created in 2003 . To date, this genus, composed of bile-resistant, strictly anaerobic and Gram-negative bacilli, contains five species including A. finegoldii (Rautio et al. 2003) , A. indistinctus (Nagai et al. 2010) , A. onderdonkii (Song et al. 2006) , A. putredinis (Weinberg et al. 1937) Rautio et al. 2003 , and A. shahii (Song et al. 2006) . Pigment production, initially considered as characteristic of Alistipes species, was recently demonstrated to be inconstant . Members of the genus Alistipes are members of the normal human intestinal microbiota, but have also been reported in urine and the mouth , and have occasionally been isolated from abdominal, appendiceal and rectal abscesses, blood cultures from colon cancer patients , and feces from children with irritable bowel syndrome . A. putredinis was also demonstrated to be associated to cruciferous vegetable intake . In addition, A. finegoldii has been suspected to play the role of growth promoter in chickens .
Classification and features
Classification and general features of Alistipes timonensis strain JC136T
Species Alistipes timonensis
Type strain JC136T
Growth in BHI medium + 1% NaCl
Sample collection time
51 m above sea level
Strain 136T exhibited catalase activity but no oxidase activity, and was resistant to 20% bile. Using API Rapid ID 32A, a positive reaction was obtained for α-galactosidase, β-galactosidase, β-glucuronidase, glutamic acid decarboxylase, leucyl glycine arylamidase and alanine arylamidase. Weak reactions were obtained for indole production and N-acetyl-β-glucosaminidase. No mannose and raffinose fermentation were observed. A. timonensis is susceptible to penicillin G, amoxicillin + clavulanic acid, imipeneme, clindamycin, metronidazole and resistant to vancomycin. By comparison with A. senegalensis, strain 136T differed in mannose fermentation and proline arylamidase, arginine arylamidase and glycine arylamidase. By comparison with A. shahii, strain 136T differed in catalase activity and mannose and raffinose fermentation .
Genome sequencing information
Genome project history
One paired end 3-kb library and one Shotgun library
454 GS FLX Titanium
Newbler version 2.5.3
Gene calling method
EMBL Date of Release
February 28, 2012
Study of the human gut microbiome
Growth conditions and DNA isolation
A. timonensis sp. nov. strain JC136T, CSUR P148, DSM 25383, was grown anaerobically on 5% sheep blood-enriched Columbia agar at 37°C. Eight petri dishes were spread and resuspended in 4×100µl of G2 buffer (EZ1 DNA Tissue kit, Qiagen, Hilden, Germany). A first mechanical lysis was performed by glass powder on the Fastprep-24 device (MP Biomedicals, Santa Ana, CA, USA) using 2×20 seconds cycles. DNA was then treated with 2.5 µg/µL lysozyme for 30 minutes at 37°C and extracted using the BioRobot EZ 1 Advanced XL (Qiagen). The DNA concentration was measured at 40 ng/µL using the Genios fluorometer (Tecan, Lyon, France).
Genome sequencing and assembly
Both a shotgun and 3-kb paired-end sequencing were performed. The shotgun library was constructed with 500 ng of DNA with the GS Rapid library Prep kit (Roche). For the paired-end sequencing, 5 µg of DNA was mechanically fragmented on a Hydroshear device (Digilab, Holliston, MA, USA) with an enrichment size at 3–4kb. The DNA fragmentation was visualized using the 2100 BioAnalyzer (Agilent, Massy, France) on a DNA labchip 7500 with an optimal size of 3.393 kb. The library was constructed according to the 454 GS FLX Titanium paired-end protocol. Circularization and nebulization were performed and generated a pattern with an optimal size of 423 bp. After PCR amplification through 15 cycles followed by double size selection, the single stranded paired-end library was then quantified using the Genios fluorometer (Tecan) at 205 pg/µL. The library concentration equivalence was calculated as 8,87E+08 molecules/µL. The library was stored at −20°C until further use.
The shotgun and paired-end libraries were clonally-amplified with 3 cpb and 1cpb, respectively, in 2×8 emPCR reactions with the GS Titanium SV emPCR Kit (Lib-L) v2 (Roche). The yields of the emPCR were 9.3% and 8.9%, respectively. For each sequencing method, approximately 340,000 beads were loaded on the GS Titanium PicoTiterPlate PTP Kit 70×75 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 Newbler assembler (Roche). A total of 201,692 passed filter wells were obtained and generated 70.71 Mb with a length average of 325 bp. The passed filter sequences were assembled using Newbler with 90% identity and 40 bp as overlap. The final assembly identified 9 scaffolds and 23 contigs (>1,500bp).
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 GenBank. 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. To estimate the mean level of nucleotide sequence similarity at the genome level between Alistipes species, we compared the ORFs only using BLASTN and the following parameters: 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
Translation, ribosomal structure and biogenesis
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 other Alistipes genomes
To date, the complete genomes from A. senegalensis strain JC50T (GenBank accession number CAHI00000000), A. shahii strain WAL 8301 (GenBank accession number FP929032) and the unfinished genome from Alistipes sp. strain HGB5 (AENZ00000000) are available. A. timonensis has a smaller genome than A. senegalensis and A. shahii but a bigger genome than Alistipes sp. strain HGB5 (3,497,779 bp vs 4,017,609, 3,763,317 bp and 3,464,615, respectively), a higher number of genes than A. shahii but smaller than A. senegalensis and Alistipes sp. strain HGB5 (2,742 vs 2,563, 3,163 and 2,955 genes, respectively), a higher ratio of genes assigned to COGs (64.00% vs 58.56%, 58.9% and 62.53%, respectively), and a higher G+C content (58.82% vs 57.33%, 58.4% and 57%, respectively). In addition, A. timonensis shared mean nucleotide sequence similarities at the genome level of 92.18% (range 72.16 to 100%), 88.72% (range 77.86 to 100%) and 85.9% (range 77.4 to 100%), with A. senegalensis strain JC50T, A. shahii strain WAL 8301 and Alistipes sp. strain HGB5, respectively.
On the basis of phenotypic, phylogenetic and genomic analyses, we formally propose the creation of Alistipes timonensis sp. nov. that contains the strain JC136T. This bacterium has been cultivated from an healthy Senegalese individual, from whom was also cultivated A. senegalensis strain JC50T, thus suggesting that the fecal flora from humans may contain several undescribed bacterial species that may be isolatable through diversification of culture conditions.
Description of Alistipes timonensis sp. nov.
Alistipes timonensis (tim.on.en’sis. L. gen. masc. n. timonensis, of Timone, the name of the hospital where strain JC136T was isolated).
Colonies are 0.2 to 0.3 mm in diameter and produce brown pigment on blood-enriched Columbia agar and Brain Heart Infusion (BHI) agar. Cells are rod-shaped with a mean diameter of 0.62 µm. Optimal growth is achieved anaerobically. No growth is observed in aerobic or microaerophilic conditions. Growth occurs between 30–37°C, with optimal growth observed at 37°C, in BHI medium + 5% NaCl. Cells stain Gram negative and are non-motile. Catalase, α-galactosidase, β-galactosidase, β-glucuronidase, glutamic acid decarboxylase, leucyl glycine arylamidase, N-acetyl-β-glucosaminidase and alanine arylamidase activities are present. Indole production is also present. Oxidase activity is absent. Cells are susceptible to penicillin G, amoxicillin + clavulanic acid, imipeneme and clindamycin and metronidazole. The G+C content of the genome is 58.82%. The 16S rRNA and genome sequence are deposited in GenBank under accession numbers JF824799 and CAEG00000000, respectively.
A. timonensis is an obligate anaerobic Gram-negative bacterium. Grows on axenic medium at 37°C in an anaerobic atmosphere. Not motile.
The type strain JC136T (= CSUR P148 = DSM 25383) was isolated from the fecal flora of a healthy patient in Senegal.
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