Small RNA-dependent expression of secondary metabolism is controlled by Krebs cycle function in Pseudomonas fluorescens

Abstract

Pseudomonas fluorescens CHA0, an antagonist of phytopathogenic fungi in the rhizosphere of crop plants, elaborates and excretes several secondary metabolites with antibiotic properties. Their synthesis depends on three small RNAs (RsmX, RsmY, and RsmZ), whose expression is positively controlled by the GacS-GacA two-component system at high cell population densities. To find regulatory links between primary and secondary metabolism in P. fluorescens and in the related species Pseudomonas aeruginosa, we searched for null mutations that affected central carbon metabolism as well as the expression of rsmY-gfp and rsmZ-gfp reporter constructs but without slowing down the growth rate in rich media. Mutation in the pycAB genes (for pyruvate carboxylase) led to down-regulation of rsmXYZ and secondary metabolism, whereas mutation in fumA (for a fumarase isoenzyme) resulted in up-regulation of the three small RNAs and secondary metabolism in the absence of detectable nutrient limitation. These effects required the GacS sensor kinase but not the accessory sensors RetS and LadS. An analysis of intracellular metabolites in P. fluorescens revealed a strong positive correlation between small RNA expression and the pools of 2-oxoglutarate, succinate, and fumarate. We conclude that Krebs cycle intermediates (already known to control GacA-dependent virulence factors in P. aeruginosa) exert a critical trigger function in secondary metabolism via the expression of GacA-dependent small RNAs.

FIGURE 1.

Physical map of the pycA, pycB, and fumA genes in P. fluorescens CHA0 and construction of strains CHA1321 (pycA) and CHA1322 (fumA). ▾, Tn5 insertion in the pycB gene of strain CHA1201; Ω, region replaced by the ΩSp/Sm cassette in strain CHA1321; ▵, region deleted in the fumA of strain CHA1322. The open boxes below indicate the regions used for complementation, either in mini-Tn7 (via pME9951 and pME9955) or in the pME6031 derivatives pME9952 and pME9956, as described under “Experimental Procedures.” PFL gene numbers refer to the genome of P. fluorescens Pf-5 (15).

FIGURE 2.

Effect of pycB and fumA mutations on the transcriptional expression of the rsmX, rsmY, and rsmZ genes. The expression of a chromosomal rsmX-lacZ fusion (A and D), that of a chromosomal rsmY-lacZ fusion (B and E), and that of a plasmid-borne rsmZ-lacZ fusion on pME6091 (C and F) were determined in P. fluorescens CHA0 (wild type, open squares), CHA1201 (pycB⁻, open triangles), CHA1201C/CHA1201Cp (pycB⁺, open circles), CHA1322 (fumA⁻, closed triangles), and CHA1322C/CHA1322Cp (fumA⁺, closed circles). The growth of each strain in the same medium (NYB) was monitored in parallel (G and H). Incubation was carried out in Erlenmeyer flasks as described under “Experimental Procedures.” Measurements were carried out in triplicate. The symbols indicate averages, and the error bars indicate S.D. values. OD_{600 nm}, optical density at 600 nm.

FIGURE 3.

Detection of RsmZ RNA in P. fluorescens CHA0, CHA1201 (pycB⁻) and CHA1322 (fumA⁻) by Northern blot. Total RNA was extracted from CHA0 (wild type), CHA1201, CHA1322, CHA1201C (pycB⁺), and CHA1322C (fumA⁺) at the end of the exponential phase (A_{600 nm} ≈ 3). As a loading control, 5 S rRNA was revealed in all samples. RsmZ expression levels were quantitated using ImageQuant software, with an estimated error of ±20%.

FIGURE 4.

Expression of a phlA′-′lacZ fusion. This reporter carried by pME6702 was measured, in triplicate, in P. fluorescens CHA0 (wild type, open squares), CHA1201 (pycB⁻, open triangles), and CHA1322 (fumA⁻, closed triangles). The strains were grown in Erlenmeyer flasks containing NYB. The symbols indicate averages, and the error bars indicate S.D. values. OD_{600 nm}, optical density at 600 nm.

FIGURE 5.

Effects of pycB and fumA mutations on antibiotic activity toward B. subtilis. Antibiotic activities of P. fluorescens strains grown on GCM were evaluated by the size of growth inhibition zone of B. subtilis. Antibiotic activities of P. fluorescens CHA0 (wild type), CHA1201 (pycB⁻), CHA1201C (pycB⁺), and CHA19 (gacS⁻) are compared in A. Antibiotic activities of P. fluorescens CHA0 (wild type), CHA1322 (fumA⁻), CHA1322C (fumA⁺), and CHA19 (gacS⁻) are compared in B.

FIGURE 6.

Expression of an aprA′-′lacZ fusion. This reporter carried by pME6060 was measured, in triplicate, in P. fluorescens CHA0 (wild type, open squares), CHA1201 (pycB⁻, open triangles), and CHA1322 (fumA⁻, closed triangles). The strains were grown in Erlenmeyer flasks containing NYB. The symbols indicate averages, and the error bars indicate S.D. values. OD_{600 nm}, optical density at 600 nm.

FIGURE 7.

Effect of Krebs cycle intermediates and other carbon sources on rsmZ-gfp expression. Strain CHA0/pME7402 was streaked on solid minimal media containing each compound as the sole carbon source. Concentrations were as follows: citrate, 10 mm; cis-aconitate, 10 mm; isocitrate, 10 mm; 2-oxoglutarate, 12 mm; succinate, 15 mm; fumarate, 15 mm; malate, 15 mm; glycerol, 20 mm; glucose, 10 mm; pyruvate, 20 mm; acetate, 30 mm. The pH was adjusted to pH 6.8 with NaOH, and agarose was added at a concentration of 1.0%. Two days after inoculation, colonies on each plate were scraped and suspended in 0.9% NaCl. The fluorescence (excitation at 480 nm and emission at 520 nm) and the optical density at 600 nm were measured with a Fluostar fluorescence microplate reader (BMG Lab Technologies).