The four JC50T spectra were imported into the MALDI Bio Typer software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against the main spectra of 2,843 bacteria, http://www.selleckchem.com/products/jq1.html including spectra from three validly published Alistipes species used as reference data, in the Bio Typer database. The method of identification included the m/z from 3,000 to 15,000 Da. For every spectrum, 100 peaks at most were taken into account and compared with the spectra in database. A score enabled the presumptive identification, or discrimination, from the tested species: a score �� 2 with a validated species enabled the identification at the species level; a score �� 1.7 but < 2 enabled the identification at the genus level; and a score < 1.7 did not enable any identification.
Spectra were compared with the Bruker database that contained spectra from the three validated Alistipes species. No significant score was obtained, thus suggesting that our isolate was not a member of a known species. We incremented our database with the spectrum from strain JC50T (Figure 4). Figure 4 Reference mass spectrum from A. senegalensis strain JC50T. Spectra from 12 individual colonies were compared and a reference spectrum was generated. Genome sequencing and annotation Genome project history The organism was selected for sequencing on the basis of its phylogenetic position and 16S rRNA similarity to other members of the Alistipes genus, and is part of a ��culturomics�� study of the human digestive flora aiming at isolating all bacterial species within human feces.
It was the second genome of an Alistipes species and the first genome of Alistipes senegalensis sp. nov. A summary of the project information is shown in Table 2. The Genbank accession number is “type”:”entrez-nucleotide”,”attrs”:”text”:”CAHI00000000″,”term_id”:”390170061″,”term_text”:”CAHI00000000″CAHI00000000 and consists of forty contigs. Table 2 shows the project information and its association with MIGS version 2.0 compliance [5]. Table 2 Project information Growth conditions and DNA isolation A. senegalensis sp. nov. strain JC50T, CSUR P156, was grown on blood agar medium at 37��C. Twelve petri dishes were spread and resuspended in 6��100��l of G2 buffer (EZ1 DNA Tissue kit, Qiagen). A first mechanical lysis was performed by glass powder on the Fastprep-24 device (Sample Preparation system) from MP Biomedicals, USA during 2��20 seconds.
DNA was then incubated for a lysozyme treatment (30 minutes at 37��C) and extracted through the BioRobot EZ 1 Advanced XL (Qiagen). The DNA was then concentrated and purified on a Qiamp kit (Qiagen). The yield and the concentration was Cilengitide measured by the Quant-it Picogreen kit (Invitrogen) on the Genios_Tecan fluorometer at 62.7 ng/��l.