GENETIC DIVERSITY ANALYSIS OF THERMOPHILIC BACTERIA FROM CANDRADIMUKA CRATER IN CENTRAL JAVA EMPLOYING PCR-RFLP OF 16S-rRNA GENE

Abstrak

The specific primers for bacteria (63f and 1387r) were used to amplify the 16S-rRNA genes from total community genomic DNA of thermophilic bacteria. The total community genomic DNA was obtained from muds and water samples of Candradimuka crater, Dieng Plateau, Central Java. PCR products were cloned into vector  pCR*2.1-TOPO (3.9 kb) and transformed into Escherichia coli TOPIC. Two  tetrameric restriction endonucleases  Rsal  and  Hhal  were employed to generate Restriction Fragment Length Polymorphisms (RFLP) paterns. These enzymes yielded 10 and 9 groups of 16S-rRNA profiles or OTU (Operational Taxonomic Units) from 27 16S-rRNA gene clones. Rsal was found to be more discriminative in differentiating the clones than Hhal. Rsal-RFLP indicated that OTU 7 and OTU 3 represented the most abundant clones, i.e. 6 and 5 clones respectively. The distribution of 16S-rRNA gene clones could  indicate relative distribution of  specific groups of  thermophilic  bacteria  in  their  natural habitat. Analysis of diversity at the DNA level could represent both culturable  and  unculturable bacteria in the  environment. Similarity analysis showed that  at level  0.600 there  were 8 different  groups from 10  RFLP  profiles generated by  Rsal  digestion. This study indicated that there were at least 8 groups of different thermophilic bacteria occupying Candradimuka crater. Key words: Thermophiles, 16S-rRNA, Candradimuka crater.