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.

FIG. 1.

Cell population density-dependent expression of transcriptional lacZ fusions to the rsmX, rsmY, and rsmZ promoters in P. fluorescens CHA0. Cultures of CHA0 carrying either pME7317 (rsmX-lacZ), pME6916 (rsmY-lacZ), or pME6091 (rsmZ-lacZ) were obtained either in full-strength NYB (open bars) or in 10-fold-diluted NYB (hatched bars), and β-galactosidase activities were determined in triplicate (mean ± standard deviation [SD]) at the OD₆₀₀ values indicated.

FIG. 2.

Alignment of rsmX, rsmY, and rsmZ promoter regions of P. fluorescens CHA0. The consensus UAS (28, 31) and the PsrA box (30) are shown above and below the promoter sequences, respectively. Two sequence elements showing resemblance to the IHF consensus recognition sequence WATCARN₄TTR (where W = A or T and R = A or G [51]) or AATCAAN₄TTG (35) are shown in bold. Conserved nucleotides are indicated by asterisks. The −10 promoter elements are underlined.

FIG. 3.

Expression of rsmZ in the wild-type strain CHA0 and in the psrA mutant CHA1063. Strains CHA0 (filled symbols) and CHA1063 (open symbols), carrying pME6091 (wild-type rsmZ-lacZ; squares) or pME7681 (PsrA box ΔTTT rsmZ-lacZ; circles) were grown in NYB, and β-galactosidase activities were determined in triplicate (mean ± SD). Plasmid pME7687 (PsrA box mutTTT rsmZ-lacZ) gave values that were indistinguishable from those for pME7681.

FIG. 4.

Electrophoretic mobility shift analysis of IHF binding to an rsmZ promoter fragment. Increasing concentrations of purified E. coli IHF protein were incubated with ³³P-labeled rsmZ promoter DNA, as specified in Materials and Methods, and separated by gel electrophoresis under nondenaturing conditions. F, free DNA fragment; C, IHF-DNA complex.

FIG. 5.

Northern blot revealing RsmY and RsmZ sRNAs. Total RNA was extracted from cells grown at 30°C in NYB to an OD₆₀₀ of ∼4.2. Experimental conditions are described in Materials and Methods. As a loading control, 5S rRNA was revealed in all samples by use of a specific probe.

FIG. 6.

Hypothetical model describing transcriptional regulation of the rsmY promoter. For explanation, see text in the Discussion. ⊥, negative control; arrow with plus sign, positive control; GacA∼P, phosphorylated GacA protein.