Identification and characterization of mutations conferring resistance to D-amino acids in Bacillus subtilis.
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.


OBJECTIVE
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.
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
Sara A
SA
Leiman
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center in Shreveport, Shreveport, Louisiana, USA.
Charles
C
Richardson
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
Lucy
L
Foulston
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
Alexander K W
AK
Elsholz
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center in Shreveport, Shreveport, Louisiana, USA.
Eric A
EA
First
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA losick@mcb.harvard.edu.
Richard
R
Losick
D000596:Amino Acids / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D016680:Genome, Bacterial; D006131:Growth Inhibitors / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010457:Peptidoglycan / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014448:Tyrosine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism*
D000596:Amino Acids; D006131:Growth Inhibitors; D050505:Mutant Proteins; D010457:Peptidoglycan; D014448:Tyrosine-tRNA Ligase