Philippine Science Letters
Philipp. Sci. Lett. 2014 7 (1) 1-6
available online: January 18, 2014
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Received: November 18, 2013
Revised: January 7, 2014
Accepted: January 8, 2014
This study was conducted as an initial approach to come up with biotechnological solutions to address problems associated with oil spill and waste oil contamination. Oil sludge sample from an oil refinery in Manila (Philippines) was used to inoculate minimal broth supplemented with bunker oil in order to isolate putative bunker oil-degrading bacteria. A bacterial isolate identified as a strain of Acinetobacter baumannii based on 16s rRNA gene sequence exhibited significant bunker oil utilization in minimal broth and minimal agar plate with bunker oil and was designated as the oil sludge Acinetobacter baumannii strain OS1. Six primers alkMF1, alkMF2, alkMF3, alkMR1, alkMR2, alkMR3 were designed and used in PCR in order to detect and amplify the alkane1-monooxygenase gene (alkM) that codes for an enzyme involved in hydrocarbon degradation. Primer sets alkMF1/alkMR1, alkMF2/alkMR2, alkMF1/alkMR2, and alkMF3/alkMR3 amplified the expected 715-bp, 807-bp, 1,340-bp, and 506-bp fragments, respectively. These primers can be used in screening procedures (including a metagenomic approach) to identify bacteria that possess alkM. Sequence analysis of the different amplicons resulted in the elucidation of the complete sequence of strain OS1 alkM gene (GenBank: accession no. KC888016). BLAST analysis revealed 99% sequence similarity of Acinetobacter baumannii strain OS1 alkM gene to alkM reported for Acinetobacter baumannii AB307 0294 (GenBank: accession no CP001172). A fourteen-nucleotide variation expected to result in 3 amino acid differences was observed. The amplification and isolation of the complete alkM gene from strain OS1 will pave the way for cloning and expression of the gene in an appropriate host cell.