Redundant phenazine operons in Pseudomonas aeruginosa exhibit environment-dependent expression and differential roles in pathogenicity

Abstract

Evolutionary biologists have postulated that several fitness advantages may be conferred by the maintenance of duplicate genes, including environmental adaptation resulting from differential regulation. We examined the expression and physiological contributions of two redundant operons in the adaptable bacterium Pseudomonas aeruginosa PA14. These operons, phzA1-G1 (phz1) and phzA2-G2 (phz2), encode nearly identical sets of proteins that catalyze the synthesis of phenazine-1-carboxylic acid, the precursor for several phenazine derivatives. Phenazines perform diverse roles in P. aeruginosa physiology and act as virulence factors during opportunistic infections of plant and animal hosts. Although reports have indicated that phz1 is regulated by the Pseudomonas quinolone signal, factors controlling phz2 expression have not been identified, and the relative contributions of these redundant operons to phenazine biosynthesis have not been evaluated. We found that in liquid cultures, phz1 was expressed at higher levels than phz2, although phz2 showed a greater contribution to phenazine production. In colony biofilms, phz2 was expressed at high levels, whereas phz1 expression was not detectable, and phz2 was responsible for virtually all phenazine production. Analysis of mutants defective in quinolone signal synthesis revealed a critical role for 4-hydroxy-2-heptylquinoline in phz2 induction. Finally, deletion of phz2, but not of phz1, decreased lung colonization in a murine model of infection. These results suggest that differential regulation of the redundant phz operons allows P. aeruginosa to adapt to diverse environments. G ene duplications give rise to genetic redundancy, an unstable condition that would not be expected to persist over time (1). This property is exhibited by diverse genomes (2-4), however, and evolutionary theorists have proposed several mechanisms whereby duplicate genes might provide selective advantages (5); for example, redundancy may be favored when spatial or temporal differences in expression enable tissue-specific variation or survival under varying environmental conditions (6, 7). Products of duplicated genes are involved in crucial cellular processes, including signal transduction, development, and metabolism (8).The genome of the bacterium Pseudomonas aeruginosa, an opportunistic pathogen that thrives in both soil and host environments, contains two redundant seven-gene operons termed phzA1-G1 (phz1) and phzA2-G2 (phz2). These operons are nearly identical (∼98% similarity at the DNA level), and each encodes the biosynthetic enzymes for phenazine-1-carboxylic acid (PCA) (9). Downstream modifications derivatize this precursor, generating other phenazines (SI Appendix, Fig. S1A). Pseudomonad phenazines are toxic to many other organisms and cell types due to their inherent redox activity (10, 11). Studies conducted in various plant and animal models of infection have implicated phenazines in colonization and pathogenicity (12, 13). In addition, recent studies have elucidated beneficial roles...

The Pseudomonas aeruginosa efflux pump MexGHI-OpmD transports a natural phenazine that controls gene expression and biofilm development

“…The opportunistic pathogen P. aeruginosa produces phenazines, antibiotics that contribute to virulence not only by directly damaging host tissues but also by enhancing pseudomonad fitness during host colonization (18,21,22,46,55). In particular, P. aeruginosa is well known for production of the bright blue phenazine pyocyanin, which contributes to the coloration of sputum and pus associated with infections.…”

“…Deletion plasmids were generated using the yeast gap repair method as described previously (18,62). The same approach was used to complement the transposon insertion mutant mexH:: tn, with the modification that plasmid pLD1574, instead of pMQ30, was used to replace the disrupted allele with the wild-type sequence.…”

“…Markerless deletions in specific loci were generated by homologous recombination as described previously (18). Deletion plasmids were generated using the yeast gap repair method as described previously (18,62).…”

“…P. aeruginosa is an opportunistic pathogen that causes biofilmbased infections with devastating effects (16). This Gram-negative bacterium synthesizes phenazines, redox-cycling antibiotics that affect redox homeostasis and gene expression in both the producer and hosts (17)(18)(19)(20)(21)(22). Several years ago, our group showed that pyocyanin (5-N-methyl-1-hydroxyphenazine), the most wellknown P. aeruginosa phenazine (23), activates the redox-active transcription factor SoxR and thereby induces expression of a small regulon that includes the RND (resistance-nodulation-division) efflux pump-encoding operon mexGHI-opmD (hereafter referred to as "mex") (24,25).…”

Pseudomonas Aeruginosa AmpR Transcriptional Regulatory Network

“…P. aeruginosa PAO1 and PA14 have two redundant operons phzA1-G1 (PA4210-PA4216; phz1 operon) and phzA2-G2 (PA1899-PA1905; phz2 operon) that are involved in biosynthesis of the phenazine precursor, phenazine-1-carboxylic acid (27). This precursor is then sequentially modified by a methyltransferase (PhzM, PA4209) and a monooxygenase The phz1 operon is known to be QS regulated while the regulators of the phz2 operon are not known (388,541). Downregulation of the phz2 operon in PAO∆ampR suggests a potential positive regulatory role for AmpR of this operon and is a novel finding.…”