Identification of Bacillus licheniformis from soil using culture-based method and bioinformative tools for ccpA gene / Nursyafiqah Ahmad Ghazali

Bacillus species is a Gram-positive bacterium which usually found in soil. Bacillus species can also be found in mangrove soil. Mangrove soil able to support diverse group of microbial communities due to its characteristics that contain high rate of organic matter. This study aims to isolate Bacillus licheniformis, that have potential in various bioactivity such as plastic degradation and bioremediation. The identification of bacteria was usually done using culture-based methods such as culture media and biochemical tests. However, this method was laborious and time-consuming. Thus, the bioinformative tools were used in this study to specifically identify Bacillus licheniformis. Various genes can be found in Bacillus licheniformis, and one of the genes is ccpA gene. ccpA gene is the specific gene of Bacillus licheniformis that exhibit many functions such as biofilm formation and spore production. Bioinformative tools help analyse specific genes by designing specific primers to identify the ccpA gene of Bacillus licheniformis. Designing a specific primer pair is a critical step in amplifying PCR products. These bioinformative tools used in this study are BLAST, ClustalW, Primer3, and In-silico PCR amplification. Firstly, the nucleotide sequence of the ccpA gene was aligned in BLAST to compare a query DNA sequence with the database of other strains sequences. Next, the unique region within ccpA gene was found based on the alignment sequence using ClustalW. Based on the unique region within the ccpA gene, a pair of primer, (forward and reverse) was designed. The concentration of bacteria was higher in mangrove soil compared to commercialised soil. Morphology and microscopy analysis showed the characteristics of Bacillus species which are Gram positive, rod-shaped and appeared purple when viewed under microscope (1000x total magnification). Further identification using Microgen Bacillus-ID kit showed highest percent probability of Bacillus licheniformis with 99.41% for mangrove soil and 89.97% for commercialised soil. The primer of ccpA gene consists of 60 % G/C content, length of sequence was 16 bp, and the melting temperature was 60 ºC. After that, the primers were tested for its functionality to amplify and also to determine the specificity using in-silico PCR amplification. The outcome showed that the primers could detect Bacillus licheniformis with the expected amplicon size of 207 bp. In conclusion, the identification method of Bacillus licheniformis using bioinformative tools was a practical method for designing specific primers and PCR amplified ccpA gene reactions.