Matrix-assisted laser-desorption/ionization but time-of-flight (MALDI-TOF) MS protein analysis was carried out as previously described [23]. Briefly, a pipette tip was used to pick one isolated bacterial colony from a culture agar plate, and to spread it as a thin film on a MTP 384 MALDI-TOF target plate (Bruker Daltonics, Leipzig, Germany). Four distinct deposits were done for strain MM10403188 from four isolated colonies. Each smear was overlaid with 2��L of matrix solution (saturated solution of alpha-cyano-4-hydroxycinnamic acid) in 50% acetonitrile, 2.5% tri-fluoracetic-acid, and allowed to dry for five minutes. Measurements were performed with a Microflex spectrometer (Bruker). Spectra were recorded in the positive linear mode for the mass range of 2,000 to 20,000 Da (parameter settings: ion source 1 (IS1), 20 kV; IS2, 18.

5 kV; lens, 7 kV). A spectrum was obtained after 675 shots at a variable laser power. The time of acquisition was between 30 seconds and 1 minute per spot. The four MM10403188 spectra were imported into the MALDI BioTyper software (version 2.0, Bruker) and analyzed by standard pattern matching (with default parameter settings) against the main spectra of 3,769 bacteria including 129 spectra from 98 Bacillus species, notably B. humi, used as reference data, in the BioTyper 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 spectra in the database.

A score enabled the presumptive identification and discrimination of the tested species from those in the database: 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. For strain MM10403188T, the obtained score was 1.2, thus suggesting that our isolate was not a member of a known species. We incremented our database with the spectrum from strain MM10403188 (Figure 4). The spectrum was made available online in our free-access URMS database [24]. Figure 4 Reference mass spectrum from B. timonensis strain MM10403188T. Spectra from 12 individual colonies were compared and a reference spectrum was generated.

Genome sequencing information Genome project history The organism was selected for sequencing on the basis of its phylogenetic position and 16S rRNA similarity to Brefeldin_A other members of the genus Bacillus, and is part of a ��culturomics�� study of the human digestive flora aiming at isolating all bacterial species within human feces. It was the 60th genome of a Bacillus species and the first genome of Bacillus timonensis sp. nov. A summary of the project information is shown in Table 2. The Genbank accession number is “type”:”entrez-nucleotide”,”attrs”:”text”:”CAET00000000″,”term_id”:”379025437″,”term_text”:”CAET00000000″CAET00000000 and consists of 146 contigs.