GacA-Controlled Activation of Promoters for Small RNA Genes in <i>Pseudomonas fluorescens</i>

<jats:title>ABSTRACT</jats:title> <jats:p> The Gac/Rsm signal transduction pathway positively regulates secondary metabolism, production of extracellular enzymes, and biocontrol properties of <jats:italic>Pseudomonas fluorescens</jats:italic> CHA0 via the expression of three noncoding small RNAs, termed RsmX, RsmY, and RsmZ. The architecture and function of the <jats:italic>rsmY</jats:italic> and <jats:italic>rsmZ</jats:italic> promoters were studied <jats:italic>in vivo</jats:italic> . A conserved palindromic upstream activating sequence (UAS) was found to be necessary but not sufficient for <jats:italic>rsmY</jats:italic> and <jats:italic>rsmZ</jats:italic> 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 <jats:italic>rsmY</jats:italic> and <jats:italic>rsmZ</jats:italic> expression, suggesting a need for auxiliary transcription factors. One such factor involved in the activation of the <jats:italic>rsmZ</jats:italic> promoter was identified as the PsrA protein, previously recognized as an activator of the <jats:italic>rpoS</jats:italic> gene and a repressor of fatty acid degradation. Furthermore, the integration host factor (IHF) protein was found to bind with high affinity to the <jats:italic>rsmZ</jats:italic> promoter region <jats:italic>in vitro</jats:italic> , suggesting that DNA bending contributes to the regulated expression of <jats:italic>rsmZ</jats:italic> . In an <jats:italic>rsmXYZ</jats:italic> triple mutant, the expression of <jats:italic>rsmY</jats:italic> and <jats:italic>rsmZ</jats:italic> 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. </jats:p>