Towards the Molecular Characterisation of a Sclerotium rolfsii Strain with Expanding Biotechnological Potential (poster)

R. POMA; J.I. FARIÑA; O.E. MOLINA; L.I.C. FIGUEROA

Salt Lake City

Congreso; 102nd GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY; 2002

ASM

Last years of research on the biotechnological potential of Sclerotium rolfsii ATCC 201126 support the idea that it constitutes a valuable source for miscellaneous fungal metabolites of industrial interest such as the biopolymer scleroglucan and also, diverse exocellular enzymes (e.g. amylases, xylanases, laminarinases) with known application in many different industrial areas. The consequent increasing interest focused on this strain and, the potential use of genetic manipulation as a future tool, led us to this first approach to the genetic characterisation. Sequence divergence in the nuclear ribosomal noncoding Internal Transcribed Spacers (ITS), bounded by the 18S and 5.8S genes, was explored as a potential source of characters for studying the relatedness of the strain with other Sclerotium representatives. This analysis involved PCR amplification of ITS regions and their subsequent analysis by Restriction Fragment Length Polymorphism (ITS-RFLP). Several extraction protocols tested for DNA isolation rendered suitable amounts of high MW genomic DNA, such as those performed with frozen mycelium grown either on solid plates covered with cellophane or from liquid cultures, regardless the amount of biomass used (30-200 mg wet weight). Better yields involved biomass disruption with liquid N2, phenol:chloroform extraction and double DNA precipitation with isopropanol and absolute ethanol in the presence of Na-acetate. The use of glass beads did not improve DNA release from cells. The results obtained in the ITS-RFLP analysis (with restriction enzymes AluI, HpaII, RsaI and MboI) evidenced a different pattern from those belonging to other characterised Sclerotium strains reported so far, which might be likely related to the natural source of the isolate. Likewise, by the use of optimised conditions for protoplast formation, the preparation of intact chromosomal DNA was also performed. The subsequent separation of chromosome-sized DNAs by Pulsed Field Gel Electrophoresis (PFGE) provided electrophoretic karyotype information and was intended to estimate the chromosomal arrangement and genome size of the strain.