Characterization of the Vibrio vulnificus putAP Operon, Encoding Proline Dehydrogenase and Proline Permease, and Its Differential Expression in Response to Osmotic Stress

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Abstract
The Vibrio vulnificus putAP genes encoding a proline dehydrogenase and a proline permease are transcribed in the same direction. Proline dehydrogenase activity and the level of putA transcript were determined to reach a maximum in exponential phase and were then repressed when growth slowed down. Northern blotting and primer extension analyses revealed that transcription of putAP genes results in two different transcripts, transcript A ( putA transcript) and transcript AP ( putAP transcript). Expression of putAP genes was directed by two promoters, promoter P putA and promoter P putAP . A crp null mutation decreased proline dehydrogenase activity and the level of the put transcripts, indicating that transcription of putAP is under the positive control of cyclic AMP receptor protein. Proline dehydrogenase and the level of both put transcripts were increased by proline but repressed by glutamate. In contrast, the level of transcript A, not transcript AP, increased when proline dehydrogenase was induced by NaCl. Since P putA activity, not P putAP activity, was increased by NaCl, it is apparent that transcript A and transcript AP are transcribed through P putA and P putAP , respectively. Cells challenged with NaCl and various hyperosmotic stresses accumulated higher levels of glutamate than control cells, indicating that glutamate is a compatible solute in V . vulnificus .