Regulation of an auxiliary, antibiotic-resistant tryptophanyl-tRNA synthetase gene via ribosome-mediated transcriptional attenuation

Abstract

cis-Acting RNA elements in the leaders of bacterial mRNA often regulate gene transcription, especially in the context of amino acid metabolism. We determined that the transcription of the auxiliary, antibiotic-resistant tryptophanyl-tRNA synthetase gene (trpRS1) in Streptomyces coelicolor is regulated by a ribosome-mediated attenuator in the 5' leader of its mRNA region. This regulatory element controls gene transcription in response to the physiological effects of indolmycin and chuangxinmycin, two antibiotics that inhibit bacterial tryptophanyl-tRNA synthetases. By mining streptomycete genome sequences, we found several orthologs of trpRS1 that share this regulatory element; we predict that they are regulated in a similar fashion. The validity of this prediction was established through the analysis of a trpRS1 ortholog (SAV4725) in Streptomyces avermitilis. We conclude that the trpRS1 locus is a widely distributed and self-regulating antibiotic resistance cassette. This study provides insights into how auxiliary aminoacyl-tRNA synthetase genes are regulated in bacteria.

FIG. 1.

Schematic representation of the mutually exclusive secondary structures formed by the 157-nucleotide leader of the trpRS1 transcript. The start codon (GUG), tryptophan codon (UGG), and stop codon (UGA) of the trpRS1 leader peptide ORF are in boldface. (A) The proposed terminator structure of the wild-type trpRS1 leader peptide attenuator. An S. coelicolor strain harboring the reporter construct with the wild-type promoter/leader fails to grow on medium containing only kanamycin (growth is indolmycin dependent). (B) The proposed antiterminator structure of the trpRS1 leader peptide attenuator. An S. coelicolor strain harboring the wild-type reporter construct grows on medium containing kanamycin and indolmycin.

FIG. 2.

Sequence alignments of the regions upstream of trpRS1 and its orthologs in various Streptomyces species and of the cognate leader peptides. (A) The regions upstream of the trpRS1 orthologs are highly homologous to the attenuator elements of the trpRS1 leader. The sequences of the dyads of symmetry are shaded gray. The start codon (GUG), tryptophan codon (UGG), and stop codon (UGA) of the leader peptide ORFs are in boldface. (B) Alignment of the predicted peptide encoded by the small ORF in the trpRS1 leader with those of its orthologs. The peptides are grouped based on their tryptophan content. The tryptophan residues are shaded gray. (C) Comparison of the leader peptide sequences of S. avermitilis (SAV4725), S. coelicolor (trpRS1), and E. coli (trpL).

FIG. 3.

Schematic representation of the engineered trpRS1 leaders defective with respect to transcription termination. (A) Schematic representation of the trpRS1 reporter construct in which the 3:4 hairpin is replaced by the native trpRS1 ribosome-binding site. The strain harboring this construct grows in an indolmycin-independent fashion on media supplemented with kanamycin. (B) Schematic representation of the trpRS1 reporter construct in which the terminal four nucleotides of the A+U-rich tract are replaced by the native trpRS1 ribosome-binding site. The strain harboring this construct grows in an indolmycin-independent fashion on media supplemented with kanamycin. The start codon (GUG), tryptophan codon (UGG), and stop codon (UGA) of the leader peptide ORF are in boldface. The start codon (ATG) of the neo open reading frame at position +1 also is in boldface.