Abstract
L-Proline can be used by Bacillus subtilis as a sole source of carbon or nitrogen. We traced L-proline utilization genetically to the putBCP (ycgMNO) locus. The putBCP gene cluster encodes a high-affinity proline transporter (PutP) and two enzymes, the proline dehydrogenase PutB and the Δ(1)-pyrroline-5-carboxylate dehydrogenase PutC, which jointly catabolize L-proline to L-glutamate. Northern blotting, primer extension, and putB-treA reporter gene fusion analysis showed that the putBCP locus is transcribed as an L-proline-inducible operon. Its expression was mediated by a SigA-type promoter and was dependent on the proline-responsive PutR activator protein. Induction of putBCP expression was triggered by the presence of submillimolar concentrations of L-proline in the growth medium. However, the very large quantities of L-proline (up to several hundred millimolar) synthesized by B. subtilis as a stress protectant against high osmolarity did not induce putBCP transcription. Induction of putBCP transcription by external L-proline was not dependent on L-proline uptake via the substrate-inducible PutP or the osmotically inducible OpuE transporter. It was also not dependent on the chemoreceptor protein McpC required for chemotaxis toward L-proline. Our findings imply that B. subtilis can distinguish externally supplied L-proline from internal L-proline pools generated through de novo synthesis. The molecular basis of this regulatory phenomenon is not understood. However, it provides the B. subtilis cell with a means to avoid a futile cycle of de novo L-proline synthesis and consumption by not triggering the expression of the putBCP L-proline catabolic genes in response to the osmoadaptive production of the compatible solute L-proline.