Assessment of genetic diversity of Bacillus spp. isolated from eutrophic fish culture pond

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.

Keywords: Bacillus group; HSP60 gene; ITS; Molecular marker; PCR–RFLP.

Fig. 1

Phylogenetic tree (unrooted) of partial 16S rRNA sequence of 40 isolates. The tree was constructed by Neighbor joining method using Mega program (Mega 5.05); bootstrap analysis was performed (bootstrap values are shown at the nodal branches)

Fig. 2

RFLP of ITS–PCR digested with TaqI. Lane1—C1G (B. subtilis), Lane2—CF19 (B. cereus), Lane3—CF8 (B. amyloliquifaciens), Lane4—CF25 (B. subtilis), Lane5—CF7 (B. cereus), Lane6—CF12 (B. stratosphericus), Lane7—C8M (B. pumilus), Lane8—CF13 (B. altitudinis), Lane9—CF9 (B. altitudinis), Lane10—C11B2 (B. subtiliss), Lane11—C11D (B. subtilis), Lane12—CF16 (B. cereus), LaneM—100 bp DNA ladder (NEB)

Fig. 3

RFLP analysis of partial ITS PCR amplicon digested with TaqI. The dendrogram was derived with VisionWorks^® Life Science Software by using unweighted pair group method

Fig. 4

RFLP of HSP60–PCR digested with PstI. Lane1—C1G (B. subtilis), Lane2—C8M (B. pumilus), Lane3—C11B2 (B. subtilis), Lane4—CF7 (B. cereus), Lane5—CF12 (B. stratosphericus), Lane6—CF8 (B. pumilus), Lane7—CF10 (B. subtilis), Lane8—C2B (B. subtilis), Lane9—CF11 (B. amyloliquifaciens), Lane10—CF13 (B. altitudinis), Lane11—CF9 (B. altitudinis), Lane12—CF25 (B. subtilis), LaneM—100 bp DNA ladder (NEB)

Fig. 5

RFLP analysis of partial 750 bp HSP60 gene amplicon digested with PstI. The dendrogram was derived with VisionWorks^® Life Science Software by using unweighted pair group method