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PLoS Pathog. 2014 Mar 6;10(3):e1003984. doi: 10.1371/journal.ppat.1003984. eCollection 2014 Mar.

ChIP-Seq and RNA-Seq reveal an AmrZ-mediated mechanism for cyclic di-GMP synthesis and biofilm development by Pseudomonas aeruginosa.

Author information

1
Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, Santa Cruz, California, United States of America; Department of Infection and Immunity and Center for Microbial Interface Biology, Ohio State University, Columbus, Ohio, United States of America.
2
Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America.
3
Department of Biology & Biochemistry, University of Bath, Claverton Down, Bath, United Kingdom.
4
Department of Microbiology, University of Washington, Seattle, Washington, United States of America.
5
Department of Microbiology, Ohio State University, Columbus, Ohio, United States of America.
6
Department of Infection and Immunity and Center for Microbial Interface Biology, Ohio State University, Columbus, Ohio, United States of America.
7
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
8
Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, Santa Cruz, California, United States of America; Department of Microbiology, Ohio State University, Columbus, Ohio, United States of America.

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.

PMID:
24603766
PMCID:
PMC3946381
DOI:
10.1371/journal.ppat.1003984
[Indexed for MEDLINE]
Free PMC Article

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