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

<jats:title>ABSTRACT</jats:title><jats:p>Bacteria produce<jats:sc>d</jats:sc>-amino acids for incorporation into the peptidoglycan and certain nonribosomally produced peptides. However,<jats:sc>d</jats:sc>-amino acids are toxic if mischarged on tRNAs or misincorporated into protein. Common strains of the Gram-positive bacterium<jats:named-content content-type="genus-species">Bacillus subtilis</jats:named-content>are particularly sensitive to the growth-inhibitory effects of<jats:sc>d</jats:sc>-tyrosine due to the absence of<jats:sc>d</jats:sc>-aminoacyl-tRNA deacylase, an enzyme that prevents misincorporation of<jats:sc>d</jats:sc>-tyrosine and other<jats:sc>d</jats:sc>-amino acids into nascent proteins. We isolated spontaneous mutants of<jats:named-content content-type="genus-species">B. subtilis</jats:named-content>that survive in the presence of a mixture of<jats:sc>d</jats:sc>-leucine,<jats:sc>d</jats:sc>-methionine,<jats:sc>d</jats:sc>-tryptophan, and<jats:sc>d</jats:sc>-tyrosine. Whole-genome sequencing revealed that these strains harbored mutations affecting tRNA<jats:sup>Tyr</jats:sup>charging. Three of the most potent mutations enhanced the expression of the gene (<jats:italic>tyrS</jats:italic>) for tyrosyl-tRNA synthetase. In particular, resistance was conferred by mutations that destabilized the terminator hairpin of the<jats:italic>tyrS</jats:italic>riboswitch, as well as by a mutation that transformed a tRNA<jats:sup>Phe</jats:sup>into a<jats:italic>tyrS</jats:italic>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<jats:sup>Tyr</jats:sup>, thus facilitating the exclusion of<jats:sc>d</jats:sc>-tyrosine from protein biosynthesis in cells that lack<jats:sc>d</jats:sc>-aminoacyl-tRNA deacylase.</jats:p><jats:p><jats:bold>IMPORTANCE</jats:bold>Proteins are composed of<jats:sc>l</jats:sc>-amino acids. Mischarging of tRNAs with<jats:sc>d</jats:sc>-amino acids or the misincorporation of<jats:sc>d</jats:sc>-amino acids into proteins causes toxicity. This work reports on mutations that confer resistance to<jats:sc>d</jats:sc>-amino acids and their mechanisms of action.</jats:p>