Survey and Validation of tRNA Modifications and Their Corresponding Genes in Bacillus subtilis sp Subtilis Strain 168

Extensive knowledge of both the nature and position of tRNA modifications in all cellular tRNAs has been limited to two bacteria,Escherichia coliandMycoplasma capricolum.Bacillus subtilissp subtilis strain 168 is the model Gram-positive bacteria and the list of the genes involved in tRNA modifications in this organism is far from complete. Mass spectrometry analysis of bulk tRNA extracted fromB. subtilis, combined with next generation sequencing technologies and comparative genomic analyses, led to the identification of 41 tRNA modification genes with associated confidence scores. Many differences were found in this model Gram-positive bacteria when compared toE. coli. In general,B. subtilistRNAs are less modified than those inE. coli, even if some modifications, such as m(1)A22 or ms(2)t(6)A, are only found in the model Gram-positive bacteria. Many examples of non-orthologous displacements and of variations in the most complex pathways are described. Paralog issues make uncertain direct annotation transfer fromE. colitoB. subtilisbased on homology only without further experimental validation. This difficulty was shown with the identification of theB. subtilisenzyme that introduces psi at positions 31/32 of the tRNAs. This work presents the most up to date list of tRNA modification genes inB. subtilis, identifies the gaps in knowledge, and lays the foundation for further work to decipher the physiological role of tRNA modifications in this important model organism and other bacteria.