Impact of gene concatenation number on phylogenetic resolution in the Bacillus genus

Abstract

Bacillus species are commonly found in diverse natural environments and are valued for their broad range of applications in medicine, industry, agriculture, and environmental management. To date, more than 433 species have been described. The species of these Bacillus are identified mostly through 16S rRNA sequences. The 16S rRNA sequences of certain Bacillus spp. like B. subtilis, B. amyloliquefaciens and B. velezensis, are more than 99% similar to each other and pose challenges in unraveling Bacillus species taxonomy resulting in misinterpretation of Bacillus genus in biodiversity, environmental monitoring or microbial product marketing. In this study, we designed multilocus sequencing analysis MLSA from the alignment of 16S rRNA gene together with a set of five conserved housekeeping loci, consisting of rpoD, glpF, ptA, purH, and pycA, and which were used to construct phylogenetic trees and infer evolutionary relationships. The results indicated that phylogenetic trees constructed using concatenated sequences of four and above genes provided higher resolution, with more extended branches and significantly improved bootstrap support, compared with trees constructed using three or fewer genes. MLSA scheme approach offers novel prospects for understanding the evolutionary patterns within the genus Bacillus. Interestingly, some species also had different phylogenetic placements than traditionally assumed, for example, B. atrophaeus clustered more closely with B. subtilis and B. amyloliquefaciens, whereas B. velezensis were placed on separate branches distinct from B. amyloliquefaciens.