Thiamine-auxotrophic mutants of Pseudomonas fluorescens CHA0 are defective in cell-cell signaling and biocontrol factor expression

Abstract

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 x 10(-8) M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.

FIG. 1.

Plasmid pME7402 contains a transcriptional rsmZ-gfp fusion in the vector pPROBE-TT′ and was constructed as described in Materials and Methods. Tc^r, tetracycline resistance; prsmZ, promoter of rsmZ; gfp, gene for green fluorescent protein; rep, replication origin; mob, mobility function; RBS, ribosome binding site; GacA-box, upstream activating sequence in the rsmZ promoter required for activation by GacA (38); lollipop symbol, transcription terminator T1.

FIG. 2.

Gfp expression in P. fluorescens CHA0/pME7402 (triangles), CHA50/pME7402 (thiC) (circles), and CHA52/pME7402 (gacS) (diamonds) and complemented mutant CHA52.1/pME7402 (gacS⁺) (squares). The strains were grown in microplate wells containing GCM without added thiamine. P. fluorescens CHA0/pME7402 and CHA50/pME7402 were cultivated in the presence (solid symbols) and absence (open symbols) of 1 mM thiamine. Gfp expression and cell densities were measured with a FluoStar fluorimeter. Each value is the average from eight different cultures; the error bars indicate standard deviations. OD₆₀₀, optical density at 600 nm.

FIG. 3.

Expression of the small regulatory RNAs RsmX, RsmY, and RsmZ in P. fluorescens CHA0 (diamonds) and CHA50 (thiC) (squares) grown in GCM amended with 5 × 10⁻⁸ M thiamine. (A) Expression of rsmX-lacZ on pME7317. (B) Expression of rsmY-lacZ on pME6916. (C) Expression of rsmZ-lacZ on pME6091. Measurements were carried out in triplicate. Experiments were done three times. The symbols indicate averages, and the error bars indicate standard deviations. OD₆₀₀, optical density at 600 nm.

FIG. 4.

Expression of AprA exoprotease and DAPG in P. fluorescens CHA0 (diamonds) and CHA50 (thiC) (squares) grown in GCM amended with 5 × 10⁻⁸ M thiamine. (A) Expression of phlA′-′lacZ on pME6259. (B) Expression of aprA′-′lacZ on pME6060. Measurements were carried out in triplicate. Experiments were done three times. The symbols indicate averages, and the error bars indicate standard deviations. OD₆₀₀, optical density at 600 nm.

FIG. 5.

Growth inhibition of P. ultimum. P. fluorescens strains CHA0, CHA19 (gacS), and CHA50 (thiC), as well as P. ultimum, were grown on minimal GCM plates not supplemented with thiamine (because agar contains traces of thiamine) (A) or supplemented with 10⁻³ M thiamine (B). wt, wild type.

FIG. 6.

Protection of cress against P. ultimum provided by P. fluorescens CHA0, CHA19 (gacS), and CHA50 (thiC). Cress plants were grown on minimal GCM plates not supplemented with thiamine (open bars) or supplemented with 10⁻³ M thiamine (solid bars). The relative stem length (100% = 14 cm) was calculated by determining the sum for 15 cress stems after 5 days of incubation at room temperature. Different letters above columns indicate significant differences at a P value of 0.05, as determined by a Student-Newman-Keuls test. Ctrl, control without added bacteria; Pu, Pythium. The data are averages ± standard deviations for six experiments.