caribbica using the publicly available ITS1-5.8S-ITS2 sequences, (ii) to evaluate the selected enzymes by in vitro ITS-RFLP analysis of ambiguously identified Cell Cycle inhibitor 55 yeast isolates for species-specific taxonomic assignment, and (iii) to validate the taxonomic assignment by ITS1-5.8S-ITS2 sequencing, mitochondrial DNA (mtDNA)-RFLP and pulsed field gel electrophoresis (PFGE) karyotyping. Methods Yeast isolates and strains The yeast isolates used in the present study are listed in Additional file 1: Table S1. These isolates were obtained from samples collected at different stages of indigenous bamboo shoot fermentation for the production of soibum in Manipur state of North East India [38]. The sample (10 g) was homogenized in 90 mL of sterile
physiological saline (1 g/L bacteriological
peptone, 8.5 g/L NaCl, pH 6.1) using Stomacher® 400 Circulator (Seward, Worthing, West Sussex) at 250 rpm for 3 min. The yeasts were isolated by serial dilution spread-plating of the above homogenate on yeast extract peptone dextrose (YEPD) agar medium (pH 6.5) (HiMedia, Mumbai, India) containing 100 μg/mL each of filter-sterilized ampicillin and tetracycline (Sigma-Aldrich, Bangalore, India), followed by incubation at 30°C for 48 − 72 h under aerobic conditions. All the isolates were purified by sub-culturing twice on the same agar medium and preserved at −80°C in YEPD selleckchem broth containing 10% (v/v) sterile glycerol (Sigma-Aldrich). For short term storage, the cultures were maintained at 4°C on YEPD agar. The type strain C. guilliermondii ATCC 6260 used for comparison was obtained from American Type Culture
Collection. Phenotypic characterization and morphological observation Phenotypic identification of the yeast isolates was carried out using the API 20 C AUX yeast identification system (bioMérieux, New Delhi, India) following manufacturer’s instructions. Teicoplanin Colony and cell morphology of the isolates were studied using SZ-PT stereo binocular RG-7388 ic50 microscope (Olympus, Japan) and BX61 phase contrast microscope (Olympus). In silico analysis and restriction enzyme selection The full length ITS1-5.8S-ITS2 sequences of M. guilliermondii and M. caribbica were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/) and Centraalbureau voor Schimmelcultures (CBS-KNAW) yeast nucleotide databases (http://www.cbs.knaw.nl/Collections/Biolomics.aspx?Table=CBS+strain+database). Type strain sequences of the two species, C. guilliermondii ATCC 6260 [GenBank: AY939792.1] and M. caribbica CBS 9966 (http://www.cbs.knaw.nl/Collections/BioloMICS.aspx?Link=T&TargetKey=14682616000000137&Rec=36291&Revert=F) were subjected to in silico PCR amplification using primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [39] to trim off the untargeted regions on both 5′ and 3′ ends of the sequences using the online Sequence Manipulation Suite (http://www.bioinformatics.org/sms2/pcr_products). Using NEBcutter, version 2.0 (http://tools.neb.