Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0

Abstract

In Pseudomonas fluorescens CHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA. This protein was purified from P. fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus, rsmZ. This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3' end. Expression of rsmZ depended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth. This signal appeared to be unrelated to N-acyl-homoserine lactones. A conserved upstream element in the rsmZ promoter, but not the stress sigma factor RpoS, was involved in rsmZ expression. Overexpression of rsmZ effectively suppressed the negative effect of gacS and gacA mutations on target genes, i.e., hcnA (for hydrogen cyanide synthase) and aprA (for the major exoprotease). Mutational inactivation of rsmZ resulted in reduced expression of these target genes in the presence of added signal. Overexpression of rsmA had a similar, albeit stronger negative effect. These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal. By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs.

FIG. 1.

lysC-rsmA region in strain CHA0. ΩKm^r, insertion in rsmA mutants CHA807, CHA808, and CHA809; T1 and T2, potential rho-independent terminators.

FIG. 2.

Regulation of aprA"-"lacZ and hcnA"-"lacZ fusions in strains overexpressing the translational repressor RsmA from pME6073 or harboring the vector pME6001 alone in NYB. □, ▪, CHA207 (hcnA"-"lacZ)/pME6001; ◊, ⧫, CHA805 (aprA"-"lacZ)/pME6001; ○, •, CHA207/pME6073; ▵, ▴, CHA805/pME6073. Open symbols, β-galactosidase activity; solid symbols, OD₆₀₀. Each value is the average from three different cultures ± standard deviation.

FIG. 3.

(A) The 4.1-kb region of P. fluorescens CHA0 with rpoS, rsmZ, and fdxA. The HindIII sites introduced to produce the rsmZ deletion mutant are indicated by Δ. The published prrB sequence of P. fluorescens F113 and its −35, −10, and +1 sites (1) are aligned above the rsmZ sequence. Rho-independent terminators T1, T2, and T3, predicted by the method of Brendel and Trifonov (10), are indicated by arrows beneath the sequence. The nucleotides deleted in the pME6093 reporter plasmid are shown in a box. (B) Predicted secondary structure of RsmZ using the Mfold program (63).

FIG. 4.

(A) Northern blot of RsmZ. Total RNA was extracted from strains CHA0 (lanes 1 to 4), CHA810 (rsmZ) (lane 5), and CHA89 (gacA) (lane 6) grown in NYB and hybridized with a DIG-labeled probe covering the full-length rsmZ transcript. In each lane, 3 μg of RNA was loaded. OD₆₀₀ values at the time of harvesting were, in lanes 1 to 6, 0.5, 1.0, 1.5, 3.4, 2.4, and 2.6, respectively. (B) Northern blot of total RNA extracted from strain CHA0 at an OD₆₀₀ of 3.4 was hybridized with the same probe as in panel A (lane 1), with the DIG-labeled oligonucleotide DIGRSMZ1, specific for the 5" end of the transcript (lane 2), or with the DIGRSMZ2 probe, specific for the 3" end (lane 3). X-ray film exposure for chemiluminescent detection was 2 min for lane 1 and 40 min for lanes 2 and 3. Std, RNA standards; sizes are shown in bases.

FIG. 5.

Suppression of gacS and gacA mutations by overexpressed RsmZ. (A) Expression of a chromosomal aprA"-"lacZ translational fusion and growth were determined in a wild-type context (◊, ⧫, CHA805/pME6032), in a gacS mutant (○, •, CHA806/pME6032), and in a gacS mutant overexpressing rsmZ (□, ▪, CHA806/pME6359). (B) Expression of a chromosomal hcnA"-"lacZ translational fusion and growth in a wild-type context (◊, ⧫, CHA207/pME6032), in a gacA mutant (○, •, CHA89.207/pME6032), and in a gacA mutant overexpressing rsmZ (□, ▪, CHA89.207/pME6359). Open symbols, β-galactosidase; solid symbols, OD₆₀₀. Each value is the average from three different cultures ± standard deviation.

FIG. 6.

Regulation of rsmZ expression. β-Galactosidase activities of transcriptional rsmZ-lacZ fusions containing the full promoter (pME6091), the promoter with the conserved upstream sequence (black box) deleted (pME6093), or no promoter (pME6095) in strains CHA0 (wild type), CHA89 (gacA), and CHA815 (rpoS). As a background control, the reporter vector pME6016 carrying the lacZ gene with its ribosome-binding site (dashed box) was used. ND, not determined. Each value is the average ± standard deviation from three different overnight cultures grown to an OD₆₀₀ of ≈3.0

FIG. 7.

Regulation of rsmZ expression and stimulation by a signal from spent medium. β-Galactosidase activities of a transcriptional rsmZ-lacZ fusion carried by pME6091 were determined in strain CHA0 (◊, ⧫), in strain CHA0 amended with extract from spent medium (□, ▪), and in the gacA mutant CHA89 (○, •). Open symbols, β-galactosidase activity; solid symbols, OD₆₀₀. Each value is the average from three different cultures ± standard deviation.

FIG. 8.

Effect of an rsmZ deletion on expression of the aprA and hcnA genes. (A) β-Galactosidase activities of a chromosomal aprA"-"lacZ translational fusion and growth were measured in the rsmZ⁺ strain CHA805 without (◊, ⧫) and with extract (○, •) and in the rsmZ mutant CHA812 without (□, ▪) and with extract (▵, ▴). (B) β-Galactosidase activities of a chromosomal hcnA"-"lacZ translational fusion and growth were determined in the rsmZ⁺ strain CHA207 without (◊, ⧫) and with extract (○, •) and in the rsmZ mutant CHA811 without (□, ▪) and with extract (▵, ▴). Open symbols, β-galactosidase; solid symbols, OD₆₀₀. Each value is the average from three different cultures ± standard deviation.