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

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.