GacA-Controlled Activation of Promoters for Small RNA Genes in Pseudomonas fluorescens

Abstract

The Gac/Rsm signal transduction pathway positively regulates secondary metabolism, production of extracellular enzymes, and biocontrol properties of Pseudomonas fluorescens CHA0 via the expression of three noncoding small RNAs, termed RsmX, RsmY, and RsmZ. The architecture and function of the rsmY and rsmZ promoters were studied in vivo. A conserved palindromic upstream activating sequence (UAS) was found to be necessary but not sufficient for rsmY and rsmZ expression and for activation by the response regulator GacA. A poorly conserved linker region located between the UAS and the ؊10 promoter sequence was also essential for GacA-dependent rsmY and rsmZ expression, suggesting a need for auxiliary transcription factors. One such factor involved in the activation of the rsmZ promoter was identified as the PsrA protein, previously recognized as an activator of the rpoS gene and a repressor of fatty acid degradation. Furthermore, the integration host factor (IHF) protein was found to bind with high affinity to the rsmZ promoter region in vitro, suggesting that DNA bending contributes to the regulated expression of rsmZ. In an rsmXYZ triple mutant, the expression of rsmY and rsmZ was elevated above that found in the wild type. This negative feedback loop appears to involve the translational regulators RsmA and RsmE, whose activity is antagonized by RsmXYZ, and several hypothetical DNA-binding proteins. This highly complex network controls the expression of the three small RNAs in response to cell physiology and cell population densities.

Regulation of Bacterial Virulence by Csr (Rsm) Systems

“…Integration host factor (IHF) binds to the promoter of the csrB gene of Salmonella enterica serovar Typhimurium and the rsmZ promoter from P. fluorescens (63,74). Deletion of ihfA in S. Typhimurium severely decreases csrB expression, similar to the case for a uvrY deletion, although it does not affect csrC expression.…”

“…UvrY directly binds to the csrB and csrC promoters in vitro (63,73), yet the details of its transcription activation mechanism have not been determined. Though Csr/Rsm-inhibitory sRNAs of various species may bear little sequence identity outside the CsrA/RsmA binding sequences, their expression in most cases is highly dependent on the UvrY ortholog (73)(74)(75)(76)(77)(78)(79). Whether UvrY and its various orthologs have additional regulatory roles remains to be determined, although the GacA protein of P. aeruginosa was proposed only to regulate Rsm sRNA transcription (80).…”

“…The GacS/GacA (BarA/ UvrY) TCS controls the expression of genes involved in the production/secretion of these factors, including the genes for 2,4-diacetylphloroglucinol biosynthesis, phlACBDE, the major exoprotease, aprA, and hydrogen cyanide synthase, hcnABC (119,160,(163)(164)(165). In the closely related pathogen P. aeruginosa, GacA activates the expression of RsmY and RsmZ sRNAs, and GacA from P. fluorescens follows a similar trend via RsmX, RsmY, and RsmZ (74,76). Therefore, some or all of the GacA effects on secreted biocontrol factors probably occur through activation of RsmX/Y/Z expression and, hence, inhibition of RsmA activity.…”

Low oxygen induces the type III secretion system in Pseudomonas aeruginosa via modulation of the small RNAs rsmZ and rsmY

“…RsmA activity is itself modulated by the two small, non-coding RNAs rsmY and rsmZ, which sequester and thus inactivate the protein (Kay et al, 2006). rsmY and rsmZ transcription is regulated by the response regulator GacA, and in the case of rsmZ may involve the DNA-binding protein integration host factor (IHF) (Humair et al, 2010).…”

Transcriptional and antagonistic responses of Pseudomonas fluorescens Pf0-1 to phylogenetically different bacterial competitors

“…Pseudomonas species, which are frequently isolated from complex environments, such as soil and rhizosphere, possess a range of two-component signal transduction proteins (for example, 212 in Pseudomonas fluorescens Pf0-1, 223 in P. fluorescens Pf-5, 179 in P. putida KT2440 and 203 in P. syringae DC3000 (http://genomics.ornl.gov/mist)), which are thought to permit rapid responses to environmental changes (Heeb and Haas, 2001;Humair et al, 2010). In these complex natural environments, Pseudomonas species coexist with many other bacterial species.…”