Nitrate reduction ability and ��-galactosidase activities were found by using the API 20 NE kit. T. senegalensis strain JC301T was susceptible to amoxicillin, imipenem, ciprofloxacin and gentamicin but resistant to trimethoprim/sulfamethoxazole and metronidazole. When compared with representative selleckchem Brefeldin A species from the suborder Micrococcineae, T. senegalensis gen. nov., sp. nov. strain JC301T exhibited the phenotypic differences detailed in Table 2. Table 2 Differential characteristics of Timonella senegalensis gen. nov., sp. nov., strain JC301T, Cellulomonas fimi strain ATCC484, Oerskovia turbata strain ATCC 25835 and Sanguibacter keddieii strain ST-74T Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was carried out as previously described [34].
Briefly, a pipette tip was used to pick one isolated bacterial colony from a culture agar plate and spread it as a thin film on a MTP 384 MALDI-TOF target plate (Bruker Daltonics, Germany). Twelve distinct deposits from twelve isolated colonies were performed for strain JC301T. Each smear was overlaid with 2 ��l of matrix solution (a saturated solution of alpha-cyano-4-hydroxycinnamic acid) in 50% acetonitrile, 2.5% trifluoracetic acid and allowed to dry for 5 minutes. Next, measurements were taken with a Microflex spectrometer (Bruker). The spectra were recorded using a positive linear mode for the mass range of 2,000 to 20,000 Da (parameter settings: ion source 1 (ISI), 20kV; IS2, 18.5 kV; lens, 7 kV). A spectrum was obtained after 675 shots with variable laser power.
The time of acquisition was between 30 seconds and 1 minute per spot. The twelve JC301T spectra were imported into the MALDI Bio Typer software (version 2.0, Bruker) and analyzed via standard pattern matching (with default parameter settings) against the main spectra of 2,843 bacteria, including spectra from six validly published species of Sanguibacter, twenty-three validly published species of Cellulomonas and five validly published species of Oerskovia which were used as reference data. The method of identification included the m/z from 3,000 to 15,000 Da. For every spectrum, a maximum of 100 peaks at most were compared with the spectra in database. The resulting score enabled the identification of tested species: a score �� 2 with a validly publsihed species enabled identification at the species level, a score �� 1.
7 but < 2 enabled identification at the genus level, and a score < 1.7 did not enable any identification. No significant MALDI-TOF score was obtained for strain JC301T against the Bruker database, suggesting that our isolate Drug_discovery was not a member of a known genus (Figures 4 and and5).5). We added the spectrum from strain JC301 T to our database. Figure 4 Reference mass spectrum from T. senegalensis strain JC301T. Spectra from 12 individual colonies were compared and a reference spectrum was generated. Figure 5 Gel view comparing T. senegalensis strain JC301T.