MOLECULAR INVESTIGATION OF A MICROBIAL MAT ASSOCIATED WITH THE GREAT ARTESIAN BASIN

Culture-independent modes of analysis were chosen to investigate a mic robial mat associated with the thermal waters of the Great Artesian Ba sin (GAB), 16S rDNA was amplified from total genomic mat DNA, and used to construct a clone library. Use of plasmid-specific primers to ampl ify the inserts from 92 selected recombinants proved to be an effectiv e approach, and demonstrated that there were four size categories of i nsert: 1500 bp (62% of clones examined), 1400 bp (25% of clones examin ed). 500 bp (5% of clones examined) and 240 bp (8% of clones examined) . Restriction enzyme analysis was evaluated for its ability to group t he 1500 bp and 1400 bp size inserts into operational taxonomic units. Clone inserts were presumptively identified by analysis of partial seq uence data, and each operational taxonomic unit was found to be phylog enetically cohesive. Phylogenetic analyses of the sequence data indica ted the presence of a broad range of bacteria related to the cyanobact eria, Thermus species. thiobacilli, planctomycetes, thermophilic hydro gen oxidisers, thermotogales, clostridia, actinomycetes, and beta and delta subclasses of the proteobacteria. Use of the restriction enzyme analysis protocol also enabled the extent of insert repetition within the library to be monitored, and would thus have eliminated 70% of seq uencing expenses. This is the first time a community analysis has been performed on this extreme environment. (C) 1998 Federation of Europea n Microbiological Societies. Published by Elsevier Science B.V.