Integrated morpho-biochemical, molecular, phylogenetic, and thermodynamic characterization of Bacillus isolates from...

Soil microorganisms represent an invaluable reservoir of taxonomic diversity and functional traits with immense potential for sustainable agriculture, environmental management, and microbial biotechnology. In the present study, eight bacterial isolates were characterized from soils collected from three ecologically distinct regions of India.

Comprehensive morpho-biochemical characterization revealed that all representative isolates (AMU-UP-1, AMU-K-1, and AMU-MY-1) were Gram-positive, catalase-positive, and Voges-Proskauer-positive, whereas the Methyl Red test differentiated isolate AMU-K-1 from AMU-UP-1 and AMU-MY-1, indicating metabolic heterogeneity. Antimicrobial susceptibility analysis demonstrated clear inhibition zones against tetracycline, amoxicillin, and azithromycin in agar well diffusion assays; however, broth microdilution assays revealed substantial variability in antibiotic resistance. Molecular characterization generated high-quality ~1500 bp 16S rRNA amplicons, producing sequencing reads of 1,048 bp (AMU-K-1) and 1,102 bp (AMU-UP-1). BLAST analysis identified AMU-K-1 as closely related to Bacillus safensis (GenBank: PZ619164), whereas AMU-UP-1 exhibited 100% sequence identity to multiple closely related taxa within the Bacillus subtilis/ Bacillus velezensis species complex (GenBank: PZ619172), precluding unambiguous species-level resolution based on 16S rRNA gene data alone. Phylogenetic reconstruction using the Tamura 3-parameter model with invariant sites (T92+I) confirmed their evolutionary placement within well-supported Bacillus lineages, while nucleotide substitution analysis demonstrated a pronounced transition bias over transversions across more than 1,300 aligned nucleotide positions. Computational RNA secondary structure prediction further revealed highly stable ribosomal conformations, with minimum free energies of -530.80 kcal mol⁻¹ for AMU-K-1 and -523.80 kcal mol⁻¹ for AMU-UP-1. Notably, AMU-K-1 exhibited greater in silico ensemble diversity (357.82) than AMU-UP-1 (151.53), suggesting differences in predicted conformational flexibility; however these computational properties translate to physiological adaptability require direct experimental verification.

The integration of phenotypic, biochemical, molecular, phylogenetic, antimicrobial, and thermodynamic analyses provides a comprehensive characterization of indigenous Bacillus isolates and establishes a valuable resource for future exploitation in plant growth promotion, biological control, environmental bioremediation, and microbial biotechnology.