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

Abstract

ABSTRACTBacteria produced-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 bacteriumBacillus subtilisare particularly sensitive to the growth-inhibitory effects ofd-tyrosine due to the absence ofd-aminoacyl-tRNA deacylase, an enzyme that prevents misincorporation ofd-tyrosine and otherd-amino acids into nascent proteins. We isolated spontaneous mutants ofB. subtilisthat survive in the presence of a mixture ofd-leucine,d-methionine,d-tryptophan, andd-tyrosine. Whole-genome sequencing revealed that these strains harbored mutations affecting tRNATyrcharging. 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 thetyrSriboswitch, as well as by a mutation that transformed a tRNAPheinto atyrSriboswitch 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 tRNATyr, thus facilitating the exclusion ofd-tyrosine from protein biosynthesis in cells that lackd-aminoacyl-tRNA deacylase.IMPORTANCEProteins are composed ofl-amino acids. Mischarging of tRNAs withd-amino acids or the misincorporation ofd-amino acids into proteins causes toxicity. This work reports on mutations that confer resistance tod-amino acids and their mechanisms of action.