Bacillus subtilis possesses interlinked routes for the synthesis of
proline. The ProJ-ProA-ProH route is responsible for the production of
proline as an osmoprotectant, and the ProB-ProA-ProI route provides
proline for
protein synthesis. We show here that the transcription of the anabolic proBA and proI genes is controlled in response to
proline limitation via a T-box-mediated termination/antitermination regulatory mechanism, a
tRNA-responsive
riboswitch. Primer extension analysis revealed
mRNA leader transcripts of 270 and 269 nt for the proBA and proI genes, respectively, both of which are synthesized from
SigA-type promoters. These leader transcripts are predicted to fold into two mutually exclusive secondary
mRNA structures, forming either a terminator or an antiterminator configuration. Northern blot analysis allowed the detection of both the leader and the full-length proBA and proI transcripts. Assessment of the level of the proBA transcripts revealed that the amount of the full-length
mRNA species strongly increased in
proline-starved cultures. Genetic studies with a proB-treA operon fusion reporter strain demonstrated that proBA transcription is sensitively tied to
proline availability and is derepressed as soon as cellular
starvation for
proline sets in. Both the proBA and the proI leader sequences contain a CCU
proline-specific specifier
codon prone to interact with the corresponding uncharged
proline-specific tRNA. By replacing the CCU
proline specifier
codon in the proBA T-box leader with UUC, a
codon recognized by a Phe-specific
tRNA, we were able to synthetically re-engineer the
proline-specific control of proBA transcription to a control that was responsive to
starvation for
phenylalanine.