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.
ABSTRACT
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.
INTRODUCTION
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
REFERENCES
ALMEIDA LA, ARAUJO R. 2013. Highlights on molecular identification of closely related species. Infect Genet Evol 13:67-75.
ALTSCHUL SF, MADDEN TL, SCHAFFER AA, ZHANG Z, MILLER W, LIPMAN DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389-3402.
AANNIZ T, OUDGHIRI M, MELLOUL M, SWINGS J, ELFAHIME E, IBIJBIJEN J, ISMAILI M, AMAR M. 2015. Thermophilic bacteria in Moroccan hot springs, salt marshes and desert soils. Braz J Microbiol 46(2):443-453.
Advanced Studies in Biology 3(3):103-110.
BEFFA T, BLANC M, LYON PF, ARAGNO M. 1996. Isolation of Thermus strains from hot composts (60ºC to 80ºC). Appl. Environ. Microbiol 62:1723-1727.
BERTHIER B, EHRLICH SD. 1998. Identification within two groups of closely related lactobacilli using PCR primers that target the 16S/23S rRNA spacer region. FEMS Microbiology Letters 161(1):97-106.
BROWN TA. 2002. Understanding a Genome Sequence. In Genomes 2. Chapter 7 2nd ed. Oxford: Wiley-Liss; https://www.ncbi.nlm.nih.gov/books/NBK21136
DE SOUZA AN, LELIS M, MARTINS L. 2001. Isolation, properties and kinetics of growth of thermophilic Bacillus. Brazilian Journal of Microbiology 32:271-275.
DEB P, TALUKDAR SA, MOHSINA SARKER PK, ABU SAYEM SM. 2013. Production and partial characterization of extracellular amylase enzyme from Bacillus amyloliquefaciens. SpringerPlus 2:154.
DUNLAP CA, KIM SJ, KWON SW, ROONEY AP. 2015. Phylogenetic analysis shows that Bacillus amyloliiquefaciens subsp. plantarum is a later heterotypic synonym of Bacillus methylotrophicus. International Journal of Systematic and Evolutionary Microbiology 65:2104-2109.
HAKI G, RAKSHIT S. 2003. Developments in industrially important thermostable enzymes: a review. Bioresource Technology 89:17-34.
HUANG CY, PATEL BK, MAH RA, BARESI L. 1998. Caldicellulosiruptor owensensis sp. nov., an anaerobic, extremely thermophilic, xylanolytic bacterium. Int. J. Syst. Bacteriol 48:91-97.
KRISTAJANSSON JK. 1989. Thermophilic organisms as sources of thermostable enzymes. Trends in Biotechnology 7:349-353.
MARTEINSSON VT, BIRRIEN JL, JEANTHON C, PRIEUR D. 1996. Numerical taxonomic Bacillus isolated from three geographically separated deep-sea hydrothermal vents. FEMS Microbiology Ecology 21:255-266.
MEDDEB-MOUELHI F, MOISANA JK, BEAUREGARD M. 2014. A comparison of plate assay methods for detecting extracellular cellulase and xylanase activity. Enzyme and Microbial Technology (66):16-19.
PANDEY A, RAMACHANDRAN S. 2006. General Introduction In Enzyme Technology: In Pandey A, Webb C, Soccol CR, Larroche C eds. New Delhi: Asiatech Publishers Inc. p. 1-10.
RAINCY FA, FRITZE D, STACKCBRANDT E. 1994. The phylogenetic diversity of thermophilic members of the genus Bacillus as revealed by 16S rDNA analysis. FEMS Microbiology Letters 115:205-212.
RAKSHIT S. 2006. Thermozymes. In Pandey at al. eds. New Delhi: Asiatech Publishers Inc. p. 603-614.
REHMAN HU, SIDDIQUE NN, AMAN A, NAWAZ MA, BALOCH AH, QADER SA. 2015. Morphological and molecular based identification of pectinase producing Bacillus licheniformis from rotten vegetable. Journal of Genetic Engineering and Biotechnology 13:139-144.
SIDDALINGESHWARA KG , UDAY J, HUCHESH CH, PUTTARAJU HP, KARTHIC J , UDIPTA KM, PRAMOD T, VISHWANATHA T. 2010. Screening and characterization of protease from Bacillus sp . International Journal of Applied Biology and Pharmaceutical Technology 1(2):575-581. 205
YAMAMOTO S, HARAYAMA S. 1995. PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Appl and Environmental Microbiol 61(3):1104-1109.
