The broad objective of this investigation is to increase our understanding of the pathogenic mechanisms of Pseudomonas aeruginosa. The mucoid phenotype of P. aeruginosa plays a major role in the pathogenesis of pulmonary tract infections in cystic fibrosis patients. Mucoidy indicates the over- production of a capsule-like exopolysaccharide called alginate, which plays a role in colonization and persistence in the lung. Dr. Ohman has recently provided evidence that alginate production is part of a large regulon that is primarily under the control of AlgT. The AlgT primary sequence has homology to a number of alternate sigma factors, suggesting that it is directly involved in the activation of alginate gene promoters. He has also found that AlgT is under the control of adjacent genes with both negative and positive functions. Such sigma factor- regulator gene cluster are seen in other bacterial systems, but little is known about their regulation. The main objective of this proposal is to understand the role of AlgT in the activation of alginate production in P. aeruginosa. Both genetic and biochemical approaches will be employed. The investigator will characterize the mechanisms of action of AlgT,as either an alternate sigma factor or positive regulator, by developing an AlgT-dependent in vitro transcription system. AlgT regulates two simple promoters, its own and that of algR, which will be characterized. The role of AlgT will be evaluated in the transcriptional activation of algB and algK, encoding a sensory transducer and cognate sensor, as well as algR, also a sensory- regulator, and algD, the first gene in the alginate biosynthetic gene cluster. AlgT activity in the cell will be evaluated by characterizing its interaction with negative and positive regulatory proteins produced from genes adjacent to algT. Finally, there is evidence that AlgT is a global regulator, affecting a number of virulence systems, and this level of control will be evaluated. This research provides an excellent model for understanding multigenic control in bacteria and should result in new and important information on a significant mechanism of pathogenesis in a serious opportunist.
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