Main content area

Identification and Characterization of Mutations Conferring Resistance to d-Amino Acids in Bacillus subtilis

Leiman, Sara A., Richardson, Charles, Foulston, Lucy, Elsholz, Alexander K. W., First, Eric A., Losick, Richard
Journal of bacteriology 2015 v.197 no.9 pp. 1632-1639
Bacillus subtilis, Gram-positive bacteria, acids, gene expression, mechanism of action, mutants, mutation, peptides, peptidoglycans, protein synthesis, proteins, sequence analysis, toxicity, transfer RNA, tyrosine
Bacteria produce d -amino acids for incorporation into the peptidoglycan and certain nonribosomally produced peptides. However, d -amino acids are toxic if mischarged on tRNAs or misincorporated into protein. Common strains of the Gram-positive bacterium Bacillus subtilis are particularly sensitive to the growth-inhibitory effects of d -tyrosine due to the absence of d -aminoacyl-tRNA deacylase, an enzyme that prevents misincorporation of d -tyrosine and other d -amino acids into nascent proteins. We isolated spontaneous mutants of B. subtilis that survive in the presence of a mixture of d -leucine, d -methionine, d -tryptophan, and d -tyrosine. Whole-genome sequencing revealed that these strains harbored mutations affecting tRNA ᵀʸʳ charging. Three of the most potent mutations enhanced the expression of the gene (tyrS) for tyrosyl-tRNA synthetase. In particular, resistance was conferred by mutations that destabilized the terminator hairpin of the tyrS riboswitch, as well as by a mutation that transformed a tRNA ᴾʰᵉ into a tyrS riboswitch ligand. The most potent mutation, a substitution near the tyrosine recognition site of tyrosyl-tRNA synthetase, improved enzyme stereoselectivity. We conclude that these mutations promote the proper charging of tRNA ᵀʸʳ, thus facilitating the exclusion of d -tyrosine from protein biosynthesis in cells that lack d -aminoacyl-tRNA deacylase. IMPORTANCE Proteins are composed of l -amino acids. Mischarging of tRNAs with d -amino acids or the misincorporation of d -amino acids into proteins causes toxicity. This work reports on mutations that confer resistance to d -amino acids and their mechanisms of action.