Biodiversity of thermophiles in Sungai Klah and Ulu Slim Hot Springs

Abstract

The biodiversity of thermophiles in Sungai Klah and Ulu Slim Hot Springs was determined using 16S rRNA based culture independent method. Genomic DNA was isolated from soil and water samples collected from the two hot springs. PCR amplification of the bacterial 16S rDNA was done using 27F/1492R and 27F-10/1505R- 10 primer sets for conventional and mini PCR respectively. The primers A571F /U1204R were used for archaeal amplifications. TA cloning method was used. Purifed PCR products were ligated to pGEM-T cloning vector and subsequently transformed into E.coli DH5a. A total of 160 transformed colonies were chosen for restriction analysis by Msp I and Hha I restriction digestion. Digested plasmids were visualized by UV illumination upon 3 % gel electrophoresis and differentiated according to the DNA band patterns obtained. Sixty representative clones were initially sequenced, using T7/M13F as forward primers; this was followed by phylogenetic determination by BLAST. A total of 31 samples were sequenced using the reverse primers M13R/SP6. Sequences were assembled into full length 16S rRNA sequence using MEGA 4.1. Upon removal of sequence artefacts by Ballerophon program, 27 clones were used for phylogenetic analysis. The Sungai Klah and Ulu Slim clone sequences clustered as six main phyla; Aquificae, Crenarchaeota, Euryarchaeota, Firmicutes, Proteobacteria, and Thermodesulfobacteria. Nine clone sequences were considered unaffiliated. It can be concluded that archaea and hyperthermophilic bacteria are present in the two Malaysian hot springs studied. Sungai Klah had a richer diversity of thermophiles than Ulu Slim Hot Spring. Thermophiles belonging to all the above phyla were detected in Sungai Klah Hot Spring. Ulu Slim Hot Spring in contrast, consisted of mostly hypethermophilic bacteria and archaea. The phylum Thermodesulfobacteria could only be detected by Mini PCR, this phylum was not detected by conventional PCR. Thus Mini PCR broadened the scope of the sequences or phyla detected.