Title: The Roles and Regulation of the Redundant Phenazine Biosynthetic Operons in Pseudomonas aeruginosa PA14
Description: The opportunistic pathogen Pseudomonas aeruginosa has been well studied for its ability to cause nosocomial infections in immunocompromised patients. However, its pathogenicity is only one aspect of the biology that makes this bacterium one of the most versatile of its genus. Since its first description in 1885, P. aeruginosa has been known to produce colorful, small molecules called phenazines. These redox-active compounds were originally thought of as mere secondary metabolites or virulence factors that allow P. aeruginosa to infect plant and animal hosts. However, recently we have gained an appreciation for their diverse functions that directly benefit their producer: phenazines act as signaling molecules, regulate intracellular redox homeostasis and are implicated in iron uptake. As a result, phenazines also have dramatic effects on the structural development of multicellular communities of P. aeruginosa, generally referred to as biofilms. How phenazine production is regulated in response to environmental cues to allow for this functional diversity is still poorly understood.

Pseudomonas aeruginosa produces at least five different phenazines, each of which have distinct chemical properties. The genes encoding the core phenazine biosynthetic enzymes are found in two redundant 7-gene operons. These operons, phzA1-G1 (phz1) and phzA2-G2 (phz2), encode two sets of proteins that catalyze the synthesis of phenazine-1-carboxylic acid (PCA), the precursor for all other phenazine derivatives. Although the phz1 and phz2 operons are nearly identical (~98% similarity), they are differentially regulated. phz1 is regulated by quorum sensing (QS), while the factors controlling phz2 expression have not yet been identified. Furthermore, the contribution of phz2 to phenazine production is not fully understood. The phz2 operon is conserved among all P. aeruginosa species and we hypothesize that it may be vital to their ability to adapt to diverse environments. In this work, we have investigated the regulation of the phz2 operon and its contribution to colony biofilm development in P. aeruginosa PA14 (Chapter 2).

We found that (1) phenazine production in biofilms is mediated exclusively through the phz2 operon, (2) phz2 expression is required for biofilm development and host colonization and (3) phz2 is regulated by quinolones, which are prominent signaling molecules in P. aeruginosa's QS system. We then investigated the roles of individual phenazines in colony development (Chapter 3) and the specificity of SoxR activation by redox active molecules (Chapter 4). We found that the effects of individual phenazines are not redundant and may be used in combination to modulate colony development. SoxR is a transcription factor that is activated by redox-active molecules including phenazines. Investigations into SoxR specificity showed that SoxR activation in non-enteric bacteria is tuned to specific redox potentials. Together, the findings presented in this thesis have expanded our knowledge about the role of phenazine production in biofilms and pathogenicity.