| Summary: | The microflora of two acidic environments was investigated using analysis of 16S rDNA amplified by the polymerase chain reaction (PCR) from environmental DNA. These environments had different chemical characteristics from most of the acidic environments studied by others. The first sample site, a coal spoil (Birch Coppice, Warwickshire), might have been expected to produce niches enriched in humic matter. The second, comprising geothermal vents on the Island of Vulcano, was unusual for natural acidic environments since it was saline. Three vent regions of different temperatures (30°C, 45°C and 80°C) were examined. Prior to the 16S rDNA analysis of the sites, a brief investigation into selection of a suitable method of DNA extraction was carried out. A bead-beating method and a chemical lysis/freeze-thaw method were compared. With regard to clone types found via each method, there was little qualitative difference. DNA was extracted from the two sites and 16S rRNA genes were amplified by PCR. PCR products were ligated and competent E. coli cells were transformed to produce clone libraries. Restriction fragment length polymorphisms (RFLPs) were examined and representatives of each RFLP type were sequenced and analysed with reference to RNA gene sequence data bases. The coal spoil clone library was dominated by sequences related to those from uncultured actinobacteria, particularly those found previously in peat bogs and various soils. Representatives of some well-known acidophiles were also found (e.g. Leptospirillum species). The clone bank from the saline, geothermal site DNA comprised sequences from acidophiles capable of growth at the respective temperatures of different samples. The lowest temperature samples produced sequences from a novel Acidithiobacillus species and also indicated a novel species probably related to Thiobacillus prosperus (which was isolated previously from Vulcano). A high temperature sample gave sequences from archaeal acidophiles, Acidianus brierleyi and, previously isolated from Vulcano, Acidianus infernus and Thermoplasma volcanium. Where the clone banks revealed the presence of novel organisms, attempts were made to isolate and characterise them. The novel actinobacteria did not appear to grow in laboratory enrichment cultures. The novel Acidithiobacillus species and two novel Thiobacillus prosperus-like species were characterised.
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