Species Identification of Thermo-tolerant Bacillus Isolates Using 16S rDNA,  gyraseB Gene (gyrB) and Enzyme Gene Sequence Analysis

Cynthia T. Hedreyda1,* and Rosario G. Monsalud2

1National Institute of Molecular Biology and Biotechnology,
University of the Philippines Diliman, Quezon City, Philippines 1101
2National Institute of Molecular Biology and Biotechnology,
University of the Philippines Los Baños, College Laguna Philippines 4031

*Corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it.


Twenty four thermo-tolerant Bacillus isolates that tested positive in preliminary enzyme plate assays were subjected to 16S rDNA sequence analysis, which revealed that identification results were not consistent with conventional biochemical identification in eighteen isolates. Identification inconsistencies were resolved in sixteen isolates by gyrB sequence analysis that gave single species identification, consistent with 16S rDNA sequence analysis. One isolate was identified as B. subtilis based on similar results from the conventional approach and 16S rDNA analysis. Ambiguous identification was observed in seven isolates with16S rDNA and gyrB sequences exhibiting 96-100% sequence identity with two or more closely related Bacillus species. Four isolates with ambiguous identification exhibited significant 16S rDNA and gyrB sequence identity with a group of Bacillus that includes B. cereus, B. thuringiensis, and B. anthracis. Each of three remaining isolates with ambiguous identification exhibited significant rDNA and/or gyrB sequence identity with a different group, a group of bacteria that includes B. vietnamensis and B. aquimaris, a group with B. safensis and B. pumilus and another with B. methylotrophicus and B. amyloliquefaciens. Enzyme gene-targeted polymerase chain reaction (PCR) amplified partial gene sequences of at least one of the enzymes protease, cellulase, amylase, and phytase in each of fourteen isolates.  The enzyme genes exhibited 98-99% sequence identity with genes reported in the database for Bacillus species that matched the identification results. Additional phenotypic and molecular markers that could distinguish closely related Bacillus species are necessary to resolve ambiguous identification.


Thermo-tolerance is an important characteristic that is often desired for bacterial strains used in industry. This is because during the bioprocessing of materials that makes use of bacteria (Haki and Rakshit 2003), increased temperature is either produced or used in the bioconversion of raw materials to come up with the desired quality and quantity of the products. High temperature during bioprocessing is one of the problems that need to be addressed when using microbial enzymes in industry. It is either the bacteria could not survive the increased temperature or the bacterial enzymes undergo protein denaturation with increased temperature (Pandey and Ramachandran 2006). To address this problem, it is necessary to isolate thermo-tolerant . . . read more


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