Briefly, a pipette tip was used to pick one S. aureus subsp. anaerobius selleck chemicals llc colony from a 5% blood agar plate and to spread it on a MTP 384 MALDI-TOF target plate (Br��ker Daltonics). Smears were overlaid with 1.5 ��L of matrix solution (saturated solution of alpha-cyano-4-hydroxycinnamic acid) in 50% acetonitrile, 2.5% tri-fluoracetic-acid and allowed to dry. MALDI-TOF without bacteria was used as a negative control and the positive control consisted of 1.5 ��L of Br��ker Bacterial Test Standard, a protein extract of Escherichia coli DH5alpha. Negative control spots remained negative and the positive control spots were identified as E. coli with score > 2, however, the S. aureus subsp. anaerobius spots yielded a score of 2.1 with the reference spectra of S. aureus subsp. anaerobius (Figure 3).
Figure 3 Reference mass spectrum from S. aureus subsp. anaerobius strain ST1464. Spectra from 4 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 economic importance in animal trade and public health. This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession ANIT00000000. The version described in this paper is the first version, ANIT01000000. It consists of 100 large contigs. Table 2 shows the project information. Table 2 Genome sequencing project information Growth conditions and DNA isolation S. aureus subsp. anaerobius was grown in microaerophilic atmosphere on 5% sheep blood-enriched Columbia agar (bioM��rieux, Marcy l��Etoile, France) at 37��C.
Two hundred microliters of bacterial suspension were diluted into 1mL Tris EDTA buffer and incubated with lysozyme for 30 minutes at 37��C followed by an overnight incubation with Proteinase K at 37��C. DNA was purified by three successive phenol-chloroform extractions and ethanol precipitation at -20��C overnight. After centrifugation, the DNA was resuspended in 52 ��L TE buffer. DNA concentration was measured by the Quant-it Picogreen kit (Invitrogen Saint Aubin, France) on the Genios_Tecan fluorometer at 60 ng/��L. Genome sequencing and assembly Mechanical fragmentation of three ��g of DNA was done on the Covaris device (KBioScience-LGC Genomics, Teddington, UK) using miniTUBE-Red 5Kb.
DNA fragmentation was visualized using an Agilent 2100 BioAnalyzer on a DNA labchip 7500 with an optimal size of 3.2 kb. The library was constructed according to the 454 Titanium paired end protocol (Roche Applied Science, Mannheim, Germany). Circularization and nebulization generated a pattern with an optimum at 5,72 bp. After PCR amplification through 17 cycles followed by double size selection, the single stranded Drug_discovery paired end library was quantified on the Quant-it Ribogreen kit (Invitrogen, Saint Aubin, France) on a Genios Tecan fluorometer at 1620 pg/��L. The library concentration equivalence was calculated as 2.61E+09 molecules/��L.