Abstract
The genus Bacillus comprises of a diverse group with a wide range of nutritional requirements and physiological and metabolic diversity. Their role in nutrient cycle is well documented. 16S rDNA sequences do not always allow the species to be discriminated. In this study 40 Bacillus spp. obtained from fish culture pond and 10 culture type strains were analysed for their genomic diversity by PCR–RFLP of intergenic spacer region of 16S-23S and HSP60 genes. TaqI digestion of PCR products amplified by ITS PCR did not render distinctive RFLP patterns. Numerical analysis of ITS PCR–RFLP pattern differentiated the isolates into 11 clusters. Same species were found to be grouped in different clusters. But PstI digested PCR products amplified from HSP60 gene of the isolates showed distinctive RFLP patterns. The dendrogram constructed from HSP60 PCR–RFLP delineated the isolates into 11 clusters also. All the clusters, except cluster I grouped only one type of species. The results showed that Bacillus spp. could be clearly distinguished by PCR–RFLP of HSP60 gene. Therefore, the HSP60 gene is proposed as an additional molecular marker for discrimination of Bacillus group.
Highlights
It is generally accepted that the mechanism of mineral phosphate solubilisation by phosphate solubilising bacteria (PSB) strains is associated with the release of low molecular weight organic acids which through their hydroxyl and carboxyl groups chelate the cations bound to phosphate, thereby converting it into soluble forms (Chen et al 2006)
We examined the genetic relationship of Bacillus spp. based on PCR–RFLP profile of HSP60 gene and compared with intergenic transcribed spacer regions (ITS) PCR RFLP in order to evaluate the use of an alternative gene as a marker for molecular microbial ecology
Comparison of the 16S ribosomal RNA (rRNA) gene sequences has been useful in phylogenetic studies at the genus level, its use has been questioned in studies at the species level
Summary
It is generally accepted that the mechanism of mineral phosphate solubilisation by phosphate solubilising bacteria (PSB) strains is associated with the release of low molecular weight organic acids which through their hydroxyl and carboxyl groups chelate the cations bound to phosphate, thereby converting it into soluble forms (Chen et al 2006). The use of molecular methods has revolutionised their identification, by improving the quality and effectiveness of this identification (Dellaglio et al 1991). The intergenic transcribed spacer regions (ITS), located between the 16S and 23S (ITS-1) and 23S and 5S (ITS-2) ribosomal genes, are thought to be under less evolutionary pressure and, may provide greater genetic variation than rRNA genes. These characteristics make ITS regions a potentially valuable tool for taxonomic and typing purposes, and their use as such has received increased attention (Osorio et al 2004).
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