Pseudomonas aeruginosa PA1006, Which Plays a Role in Molybdenum Homeostasis, Is Required for Nitrate Utilization, Biofilm Formation, and Virulence

Melanie J Filiatrault,Kendra Rumbaugh,Victoria E Wagner,Nadine Van Alst,Johanna Schwingel,Gregory Tombline,Pam Sokol,Barbara H Iglewski,Eric Cascales

doi:10.1371/journal.pone.0055594

Abstract

Pseudomonas aeruginosa (Pae) is a clinically important opportunistic pathogen. Herein, we demonstrate that the PA1006 protein is critical for all nitrate reductase activities, growth as a biofilm in a continuous flow system, as well as virulence in mouse burn and rat lung model systems. Microarray analysis revealed that ΔPA1006 cells displayed extensive alterations in gene expression including nitrate-responsive, quorum sensing (including PQS production), and iron-regulated genes, as well as molybdenum cofactor and Fe-S cluster biosynthesis factors, members of the TCA cycle, and Type VI Secretion System components. Phenotype Microarray™ profiles of ΔPA1006 aerobic cultures using Biolog plates also revealed a reduced ability to utilize a number of TCA cycle intermediates as well as a failure to utilize xanthine as a sole source of nitrogen. As a whole, these data indicate that the loss of PA1006 confers extensive changes in Pae metabolism. Based upon homology of PA1006 to the E. coli YhhP protein and data from the accompanying study, loss of PA1006 persulfuration and/or molybdenum homeostasis are likely the cause of extensive metabolic alterations that impact biofilm development and virulence in the ΔPA1006 mutant.

Highlights

P. aeruginosa (Pae) is a ubiquitous opportunistic nosocomial pathogen that infects individuals with pre-disposing conditions such as cancer, AIDS, burns, and importantly, Cystic Fibrosis (CF)
Aerobically grown overnight cultures of PAO1, the DPA1006 mutant and a complemented mutant (DPA1006(attB:PA1006)) containing a single copy of the wild-type (WT) PA1006 gene reintroduced onto the chromosome [26], were used to inoculate pre-reduced NY media supplemented with 100 mM KNO3 as the terminal electron acceptor
Since the most robust changes in gene expression in the DPA1006 mutant reflect a loss of nitrate responsiveness it is likely that a loss of signaling via denitrification pathway intermediates contributes significantly to the DPA1006 mutant phenotype

Summary

Introduction

P. aeruginosa (Pae) is a ubiquitous opportunistic nosocomial pathogen that infects individuals with pre-disposing conditions such as cancer, AIDS, burns, and importantly, Cystic Fibrosis (CF). Mounting evidence indicates that Pae experiences microaerobic as well as anaerobic environments during biofilm development and during infection in vivo [6,7,8,9,10,11,12,13,14]. Reduced oxygen tension does not appear to be a problem in infection, since biofilms can form under anaerobic conditions [8]. Compounding matters, biofilms and anaerobic growth appear to contribute to increased antibiotic resistance [6]. Proteins that enable biofilm development and anaerobic metabolism in vivo (in the host) are not well defined and likely contribute to virulence potential

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Results

Conclusion