1. Identification of actino-like bacterial endosymbionts in Philippine molluscs potentially producing bioactive compounds by 16S ribosomal RNA gene isolation, polymerase chain reaction amplification, and sequence analysis Eugene John F. Balmores Jr., Mary Anne A. Ammon, Gisela P. Concepcion Marine Natural Products Laboratory, Marine Science Institute, University of the Philippines INTRODUCTION Identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. Sequencing of the well-conserved 16S ribosomal RNA gene has emerged as a preferred genetic technique. Many studies had proven the use of 16S rRNA gene sequence data for better identifying poorly described, rarely isolated, or phenotypically aberrant strains and can even lead to recognition of novel pathogens and noncultured bacteria. Routine comparison of 16S rRNA gene with sequences of known bacteria in a database allows faster identification of the bacterium in question. METHODOLOGY RESULTS 16s rRNA gene amplified through PCR Results of PCR step as seen in Figure 2 shows that the expected size of 16S rRNA gene, 1200 basepairs, was amplified through the forward (63f) and reverse (1387r) primers. Candidate identity of bacterial isolates using the 16S rRNA gene sequences Results show that sequences aligned with bacterial 16S rRNA genes from NCBI database. Table 2 shows the top hits after sequence alignment of the PCR product sequences. The length of sequences that aligned is seen as the number of nucleotides. Overlapping nucleotides represents DNA similar to both forward and reverse sequences. Sample Code Source 532H-I-1a- 18a Teredo mindanensis anterior caecum 532H-I-1a- 18a Teredo mindanensis anterior caecum 532H-I-1a- 18b Teredo mindanensis anterior caecum 532H-I-1a- 18b Teredo mindanensis anterior caecum 472x-Ib-01Volema myristica hepatopancreas 496-001bErgalatax margariticola hepatopancreas 526-01aLienardia rubida venom duct 526w-R-3a- 001 Lienardia rubida venom duct Figure 2. Five uL of the PCR product was used for running in 0.8% agarose gel at 135 V for 17 minutes. Lanes 1: molecular weight marker; 2: 532H-I-1a-18a; 3: 532H-I-1a-18a; 4: 532H-I-1a-18b; 5: 532H-I-1a-18b; 6: 472x-Ib-01; 7: 496-001b; 8: 526-01a; 9: 526w-R-3a-001; 10: negative control SampleForward SequenceReverse SequenceCombined Sequence 532H-I-1a-18a99 %: Streptomyces hygroscopicus subsp. angustmyceticus (942 nucleotides) 99%: Streptomyces hygroscopicus subsp.angustmyceticus (938 nucleotides) 99%: Streptomyces hygroscopicus subsp. angustmyceticus (1263 nucleotides, 618 nucleotides overlap) 532H-I-1a-18b-ISP299 %: Streptomyces hygroscopicus subsp.angustmyceticus (938 nucleotides) 99%: Streptomyces hygroscopicus subsp.angustmyceticus (941 nucleotides) 99%: Streptomyces hygroscopicus subsp.angustmyceticus (1263 nucleotides, 608 nucleotides overlap) 532H-I-1a-18b-R2A99 %: Streptomyces hygroscopicus subsp.angustmyceticus (942 nucleotides) 99%: Streptomyces hygroscopicus subsp.angustmyceticus (938 nucleotides) 99%: Streptomyces hygroscopicus subsp.angustmyceticus (1266 nucleotides, 615 nucleotides overlap) 472x-Ib-0199 %: Streptomyces hygroscops subsp. angustmyceticus (944 nucleotides) Unreliable sequence99%: Streptomyces hygroscopicus subsp. angustmyceticus (1263 nucleotides, 618 nucleotides overlap) 496-001bUnreliable sequence95%: Streptomyces tubercidicus (937 nucleotides) 99%: Streptomyces sp. (1247 nucleotides, 615 nucleotides overlap) 526-01aUnreliable sequence100%: Actinomadura sp. (937 nucleotides) 99%: Actinomadura sp. (1273 nucleotides) DISCUSSION The most reliable information for the 16S rRNA gene sequence of a given bacterium is that obtained by contig assembly, or a double stranded DNA coverage of the gene. A 100% sequence alignment to a known bacterial 16S rRNA gene of such sequence can ascertain an isolates identity. In the same way, a 96% alignment of a highly reliable data can suggest a new bacterium altogether. As in the case of the isolate 532H-I, the 618 nucleotide overlap between the forward and reverse sequences can be considered useful in the identity of the isolate. This overlap can be used to design internal primers that can extend the gene sequence towards the 3 or 5 end. In the case of the next three isolates, the unreliable sequence signifies chromatograms that have mixed nucleotide signals. Identity of the isolate cannot be readily determined even though the opposite sequence is reliable. A chimeric sequence can be a source of such discrepancy and this can be resolved by resequencing the PCR products or repeating the PCR amplification altogether. Using a set of primers to facilitate contig assembly is the best approach to sequence the whole 16S rRNA gene. ACKNOWLEDGEMENT This study is funded by NIH through the PMS-ICBG Project. RESEARCH OBJECTIVES This study aims to identify bacterial isolates from Philippine molluscs using 16S rRNA gene sequence. This study, more importantly, is part of a more encompassing objective of studying bacterial biodiversity among the molluscs found in the Philippine seas and discovering which among these bacteria are capable of producing antimicrobial, anticancer and neuroactive compounds. Table 1. Source of bacterial isolates was obtained from the PMS-ICBG database. 1 2 3 4 5 6 7 8 9 10 Sequence Analysis Alignment with NCBI Database through BLAST 2.2.22 Sequencing Macrogen Inc. (Korea) PCR Products Purification Exonuclease and Shrimp Alkaline Phosphatase Template: genomic DNA of bacteria Primers: 63f (5- CAG GCC TAA CAC ATG CAA GTC -3) and 1387 r (5- GGG CGG WGT GTA CAA GGC -3). Profile Initial denaturation: 95 C, 5 minutes Denaturation: 95 C, 30 seconds Annealing: 51 C, 45 seconds Extension: 72 C, 1 minute 30 seconds (30 cycles) Final Extension: 72 C, 7 minutes Genomic Extraction ZymoResearch Fungal-Bacterial Kit® Figure 1. Plates of pure isolates. A: 532H-I-1a-18a; B: 472x-Ib-01; C: 496-001b; D: 526-01a AB C D PCR Amplification of 16S rRNA gene 1 1 1 1