Api zym and api 20ne strips

Chemotaxonomic markers of the strains and their close phylogenomic neighbors were determined using standard thin-layer chromatographic procedures. To this end, isomers of diaminopimelic acid (A2pm) (Schleifer and Kandler, 1972 (link)) and polar lipid (Bligh and Dyer, 1959 (link); Tindall et al., 2007 (link)) patterns were carried out. Cellular fatty acids of the strains were extracted and analyzed by gas chromatography (Agilent 6890N) following the standard protocols of the microbial identification (MIDI) system (Sasser, 1990 ). Fatty acids were identified by a GC–MS run on an Agilent GC–MS 7000D instrument (Vieira et al., 2021 (link)). Isoprenoid quinones were extracted, separated by HPLC, and identified by using both a DAD and high-resolution mass spectrometer, according to the work of Schumann et al. (2021) (link). Biochemical and enzymatic properties of the strains and their phylogenomic neighbors were determined using API-ZYM and API 20NE strips, as instructed by the manufacturer (bioMerieux, Lyon, France).

Unless stated otherwise, the strains were observed on TSA for their cultural, morphological, and physiological characteristics. Gram staining of strains T3-5-0-4, N1-5-1-14 and N5-1-1-5 was conducted with a Gram staining kit (Sigma-Aldrich, St. Louis, MO, USA). Cultures were conducted at 4, 10, 15, 20, 25, 30, 37, 42, and 50 °C to determine the optimal temperature range for growth. The optimal pH range for growth was carried out using trypticase soy broth (TSB, Difco), with pH adjustments ranging from 4.5 to 10.0 (at intervals of 0.5 units) [31 (link)]. The NaCl requirement and tolerance were investigated by NaCl-free TSB supplemented with 0–10% (w/v) NaCl at 1% intervals. The growth tests were performed on Reasoner’s 2A (R2A, Difco) agar, Marine Agar 2216 (MA, Difco), nutrient agar (NA, Difco), and TSA at 25 °C for 14 days. The morphology and motility of cells were observed after 48 h of incubation at 25 °C by transmission electron microscopy (JEM-1230; JEOL) as well as phase-contrast microscopy (HFX; Nikon, Tokyo, Japan). The activity of constitutive enzymes, utilization of carbon source, and other physiological properties were analyzed by the API ZYM and API 20NE strips (bioMérieux, Marcy l’Etoile, France), and BIOLOG GEN III MicroPlate system (BIOLOG, Hayward, CA, USA).