Construction of metagenomic library of thermophilic microorganisms from Mt.Makiling [Los Baños, Laguna, Philippines] mudspring as potential source of restriction enzymes

Mud samples from Mt. Makiling Mudspring were collected and several methods of direct DNA isolation from these samples were tried. Considerable amounts of DNA were extracted only from the settled pond water using the Bio 101 Fast DNA Spin Kit. Degenerate primers for genes encoding three restriction enzymes, namely Sau96a, Eco RV and Pst I were designed and the isolated DNA was used as template in PCR. A number of bands was observed instead of the expected one band for each gene. This may be due to the use of degenerate primers that can possibly amplify non-specific segments of the DNA or it could also be due to the presence of related sequences present in the DNA mixture. The PCR products were ligated with Promega's pGEM-T Easy vector, transformed into Escherichia coli and the inserts sequenced. BLAST results showed no significant homology to known genes. Restriction enzymes are produced and isolated from specific bacterial and fungal organisms. The bacterial or fungal genes coding for these enzymes may not really be in the DNA mixture isolated. Due to the difficulty of directly isolating high molecular weight DNA from the soil, the construction of the BAC library looked impossible. Instead, the isolated DNA was digested and ligated into the plasmid Bluescript (pBS). A number of clones were obtained and sequenced. Just like the results obtained from the sequences of the PCR products, most clones showed no significant homology to any known gene. It is possible that the sequences are so unique that they have not yet been characterized from any other organism, thus the absence of significant hits with the DNA databases. GeneMark.hmm prokaryotic gene production gene prediction software showed that nine of the 25 clones analyzed have 1-3 short (84-294 bases) coding segments and the rest of the clones have noncoding sequences. A study of the 16S rRNA profile of the community from mudspring was done using the following primers pairs: Universal Primer (533F and 1392R, 519F and 1392R), Eubacterial Primer (27F and 1392R), Bacterial Primer (11F and 1492R), Archaeal Primer (21Fa and 1392R, 23FPL and 1391R). PCR product was obtained using the universal primers pair 519F and 1392R and Archaeal primer pair 23FPL and 1391R from DNA directly isolated from mudspring and from the DNA of an enrichment culture using Nutrient Broth (NB). One hundred and one clones were selected after double digestion experiments using Dra-EcoR1 and Xho-EcoR1 restriction enzymes pairs and sequenced by microgen. Most of the clones were identified to be comparable to Sulfolobus tokodai, Solfolobus solfataricus and some clones were noted to be same as some uncultured archaeon gene for 16S rRNA. Clones from amplicons resulting from the use of Eubacterial primers are awaiting sequencing.