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

Author(s)

Christopher J. Jones, The Ohio State University
David Newsom, Nationwide Children's Hospital
Benjamin Kelly, Nationwide Children's Hospital
Yasuhiko Irie, University of DaytonFollow
Laura K. Jennings, University of Washington - Seattle Campus
Binjie Xu, The Ohio State University
Dominique H. Limoli, The Ohio State University
Joe J. Harrison, University of CalgaryFollow
Matthew R. Parsek, University of Washington - Seattle Campus
Peter White, Nationwide Children's Hospital
Daniel J. Wozniak, The Ohio State University

Document Type

Article

Publication Date

3-2014

Publication Source

PLOS Pathogens

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.

Inclusive pages

1-14

ISBN/ISSN

1553-7366

Document Version

Published Version

Comments

This document is provided for download in compliance with the publisher's policy on self-archiving. Permission documentation is on file.

Copyright

Copyright © 2014, Jones et al.

Publisher

PLOS

Volume

10

Peer Reviewed

yes

Issue

3

eCommons Citation

Jones, Christopher J.; Newsom, David; Kelly, Benjamin; Irie, Yasuhiko; Jennings, Laura K.; Xu, Binjie; Limoli, Dominique H.; Harrison, Joe J.; Parsek, Matthew R.; White, Peter; and Wozniak, Daniel J., "ChIP-Seq and RNA-Seq Reveal an AmrZ-Mediated Mechanism for Cyclic di-GMP Synthesis and Biofilm Development by Pseudomonas aeruginosa" (2014). Biology Faculty Publications. 209.
https://ecommons.udayton.edu/bio_fac_pub/209