Research Article: ChIP-Seq and RNA-Seq Reveal an AmrZ-Mediated Mechanism for Cyclic di-GMP Synthesis and Biofilm Development by Pseudomonas aeruginosa

Date Published: March 6, 2014

Publisher: Public Library of Science

Author(s): Christopher J. Jones, David Newsom, Benjamin Kelly, Yasuhiko Irie, Laura K. Jennings, Binjie Xu, Dominique H. Limoli, Joe J. Harrison, Matthew R. Parsek, Peter White, Daniel J. Wozniak, Urs Jenal.

http://doi.org/10.1371/journal.ppat.1003984

Abstract

The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ΔamrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ΔamrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ΔamrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon, and characterized AmrZ as an activator or repressor at each binding site. We identified an AmrZ-repressed DGC-encoding gene (PA4843) from this cohort, which we named AmrZ dependent cyclase A (adcA). PAO1 overexpressing adcA accumulates 29-fold more c-di-GMP than the wild type strain, confirming the cyclase activity of AdcA. In biofilm reactors, a ΔamrZ ΔadcA double mutant formed smaller microcolonies than the single ΔamrZ mutant, indicating adcA is responsible for the hyper biofilm phenotype of the ΔamrZ mutant. This study combined the techniques of ChIP-Seq and RNA-Seq to define the comprehensive regulon of a bifunctional transcriptional regulator. Moreover, we identified a c-di-GMP mediated mechanism for AmrZ regulation of biofilm formation and chronicity.

Partial Text

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a major burden on the health care industry. Up to 10% of all nosocomial infections are attributed to P. aeruginosa, with mortality rates approaching 40% in patients with bacteremia [1], [2]. This bacterium is often a causative agent of sepsis, as well as acute and chronic infections of the airway, burn wounds, skin, and medical devices such as catheters [1], [3].

Understanding how bacteria respond to varying conditions in the environment and during infection is clearly of importance. Here, we present a comprehensive analysis of a bacterial transcription factor regulon obtained by combining ChIP-Seq and RNA-Seq. The power of these techniques stems from the unbiased and genome-wide production of the entire regulon, but also the activity of the transcription factor at these binding sites. These techniques have been established in eukaryotes [62], [63], however they have recently been adapted as powerful tools to investigate the activity of bacterial transcription factors [27], [28], [58], [64]–[67]. We were able to identify 398 regions bound by AmrZ in the P. aeruginosa genome. Additionally, we developed a transcriptional profile of both the ΔamrZ mutant and its complemented strain. This allowed us to combine the results of ChIP-Seq and RNA-Seq and divide loci into several categories, either activated, repressed, or unaffected by AmrZ. Each of these groups were then further categorized into directly or indirectly regulated.

 

Source:

http://doi.org/10.1371/journal.ppat.1003984

 

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