Characterization of a New Key Regulator for Phenazine Production in Pseudomonads

描述（由申请人提供）：在了解已知毒力因子非那嗪的生产如何在致病性细菌铜绿假单胞菌中进行转录调节方面存在根本差距。这一差距的持续存在是一个重要的问题，因为在填补这一差距之前，了解铜绿假单胞菌和其他假单胞菌如何以及为什么产生非那嗪在很大程度上是无法理解的。为了解决这个问题，我们的长期目标是确定PA2449在整个生理中的功能

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how the production of phenazines, known virulence factors, is transcriptionally regulated in the pathogenic bacterium, Pseudomonas aeruginosa. Continued existence of this gap represents an important problem because, until it is filled, understanding how and why phenazines are produced by P. aeruginosa and other pseudomonads will be largely incomprehensible. To address this, our long-term goal is to define the function(s) of PA2449 in the global physiology of P. aeruginosa. Specifically, it is imperative to know what genes and operons are regulated by PA2449, and what signals PA2449 responds to in order to activate transcription. The overall objective of this application will be to define the regulon of PA2449 in the medically relevant bacterial strain, P. aeruginosa, and determine its roles in the production of the phenazines virulence factors. The central hypothesis is that PA2449 encodes an enhancer binding protein (EBP) transcription factor that regulates the production of phenazines in P. aeruginosa. The hypothesis is based on our own preliminary findings, which were generated by comparing physiological and transcriptomic data for wild type P. aeruginosa PAO1 and a P. aeruginosa PA2449 transposon deletion strain. The rationale for the proposed research is that knowledge of the genetic network regulated by PA2449 will generate new strategies to prevent the expression of these virulence factors and thereby restrict the ability of P. aeruginosa to cause infection, particularly in the cystic fibrosis lung. Guided by strong preliminary data, the hypothesis will be tested by pursuing two specific aims: 1) Define the regulon of PA2449 and 2) Define the metabolic profile for PA2449. Under the first aim, techniques, which have been established as feasible in the applicant's hands, will be used; A) to perform microarray transcriptomic studies to define the regulon of PA2449; and B) to perform chromatin-immunoprecipitation-exonuclease (ChIP-exo) experiments to define the DNA binding sites of PA2449 throughout the genome of P. aeruginosa. Under the second aim, the investigators will use common HPLC and LC-MS-MS methodologies to assess the amino acid utilization of the PA2449 strain. The approach is innovative because it identifies and characterizes, for the first time, an EBP that specifically regulates the production of phenazines virulence factors in P. aeruginosa. The proposed research is significant, because it is expected to vertically advance and expand understanding of how and why phenazines are produced by P. aeruginosa. Ultimately, such knowledge has the potential to identify new targets to interfere with virulence factor production in P. aeruginosa.