Cytolytic hemolysin, a gene product of vvhA, is a putative virulence factor of the pathogenic bacterium Vibrio vulnificus. We have previously shown that hemolysin production is repressed by adding glucose to culture media and that production can be restored by adding cAMP. In this study, hemolysin activity and the level of vvh transcript were determined to reach a maximum in late exponential phase and were repressed when cells entered stationary phase. Northern blot and primer extension analyses revealed that vvhA is cotranscribed with a second gene, vvhB, located upstream of vvhA. Transcription of the vvhBA operon begins at a single site and is under the direction of a single promoter, P vvh . A crp null mutation decreased hemolysin production and the level of vvhBA transcript by reducing the activity of P vvh , indicating that the P vvh activity is under the positive control of cAMP receptor protein (CRP). A direct interaction between CRP and the regulatory region of the vvhBA operon was demonstrated by gel-mobility shift assays. The CRP binding site, centered at 59.5 bp upstream of the transcription start site, was mapped by deletion analysis of the vvhBA promoter region and confirmed by DNase I protection assays. These results demonstrate that the vvhBA expression is activated by CRP in a growth-dependent manner and that CRP exerts its effects by directly binding to DNA upstream of P vvh .

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.Change in the external osmolarity is one of the most common environmental stresses that bacteria routinely encounter (10). The main strategy that members of the family Enterobacteriaceae use to adapt to high osmolarity is the accumulation of organic solutes, known as compatible solutes or osmoprotectants (6,10,12,14). It has been well demonstrated that the osmolytes do not interfere with macromolecules and allow the maintenance of vital cellular functions. Hence, the osmolytes can be accumulated to high levels to prevent the loss of water caused by high external osmolarity and consequently contribute to the maintenance of the outwardly directed turgor pressure required for growth. Although a variety of novel organic osmolytes as osmoprotectants have been identified, glycine betaine, ectoine, proline, glutamate, and trehalose are probably the most widely used compatible solutes in bacteria (6, 10).Vibrio vulnificus is an opportunistic gram-negative bacterial pathogen that commonly contaminates raw oysters and is the causative agent of food-borne diseases, such as gastroenteritis and life-threatening septicemia. Mortality from septicemia is very high (Ͼ50%), and death may occur within 1 to 2 days after the first signs of illness (2,20). Like many other food-borne pathogenic bacteria, V. vulnificus occurs in various environments having different osmotic strengths; it naturally inhabits coastal seawaters, contaminates shellfish, survives current control practices (such as adding salt or sugar to suppress its growth), and colonizes in the human body. These facts indicate that V. vulnificus has to cope with ever-changing osmolarity in its growth environments...