One of the most intriguing examples of bacterial pathogenesis is the infection of the Cystic Fibrosis (CF) patients by Pseudomonas aeruginosa. The CF patients accumulate in their lungs a viscous, dehydrated mucus high in salts, which predisposes the patients to infection by P. aeruginosa. The initial infecting cells are typically nonmucoid, but on prolonged infection, they become highly mucoid due to the production of an exopolysaccharide called alginate, which encapsulates the infecting P. aeruginosa cells and is believed to protect them from phagocytosis and antibiotic treatment. In other infections such as eye, urinary tract or burn, they are seldom mucoid (i.e. alginate producer). We have recently demonstrated that many of the alginate structural genes are clustered at 34 min on the P. aeruginosa chromosome, and the expression of one of the terminally located genes, algD, is controlled by a promoter PalgD. This promoter must be activated by a set of regulatory proteins AlgR1, AlgR2 and AlgR3 under conditions of high osmolarity and ethanol-induced dehydration -- two conditions unique to the CF lung mucus environment. Supercoiling of the PalgD region by DNA gyrase is essential for transcription from PalgD. In addition, we have purified the positive regulatory protein AlgR1 and demonstrated its binding at two far upstream sites of the algD promoter. In this proposal, we want to determine how important the two binding sites are, and whether the orientation or the spacing of the binding sites may be important for algD promoter activation. We also want to determine if other alginate genes such as AlgA, Alg8, Alg44, etc. are also regulated. Recently, we have purified AlgR2 and demonstrated autophosphorylation of the AlgR2 protein with either ATP or GTP and subsequent transfer of the phosphate to algR1. It would be important to determine the mechanism of phosphorylation, the nature and extent of phosphorylated amino acids in the two proteins and the role phosphorylation plays in the activation of the algD promoter. AlgR1 is also phosphorylated in Escherichia coli by an AlgR2-analog protein which undergoes phosphorylation in presence of ATP or GTP. This raises interesting questions about the role of this intermediate phosphorylating agent in E. coli. In addition, another gene encoding a starvation induced protein SspA allows complementation of the algR2 mutation, raising the question of the role of AlgR2 as an RNA polymerase contacting protein.
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