The Systematic Investigation of the Quorum Sensing System of the Biocontrol Strain Pseudomonas chlororaphis subsp. aurantiaca PB-St2 Unveils aurI to Be a Biosynthetic Origin for 3-Oxo-Homoserine Lactones

Abstract

The shoot endophytic biocontrol strain Pseudomonas chlororaphis subsp. aurantiaca PB-St2 produces a wide range of exoproducts, including enzymes and antibiotics. The production of exoproducts is commonly tightly regulated. In order to get a deeper insight into the regulatory network of PB-St2, the strain was systematically investigated regarding its quorum sensing systems, both on the genetic and metabolic level. The genome analysis of PB-St2 revealed the presence of four putative acyl homoserine lactone (AHL) biosynthesis genes: phzI, csaI, aurI, and hdtS. LC-MS/MS analyses of the crude supernatant extracts demonstrated that PB-St2 produces eight AHLs. In addition, the concentration of all AHL derivatives was quantified time-resolved in parallel over a period of 42 h during the growth of P. aurantiaca PB-St2, resulting in production curves, which showed differences regarding the maximum levels of the AHLs (14.6 nM- 1.75 μM) and the production period. Cloning and heterologous overexpression of all identified AHL synthase genes in Escherichia coli proved the functionality of the resulting synthases PhzI, CsaI, and AurI. A clear AHL production pattern was assigned to each of these three AHL synthases, while the HdtS synthase did not lead to any AHL production. Furthermore, the heterologous expression study demonstrated unequivocally and for the first time that AurI directs the synthesis of two 3-oxo-AHLs.

Fig 1. Structural formulas and MS/MS fragmentation of AHLs.

(A) Structural formulas of AHLs produced by P. aurantiaca PB-St2. (B) Fragmentation of AHLs into the [M+H]⁺ ion of the lactone moiety in the collision cell.

Fig 2. Identification of the produced AHLs using LC-MS/MS.

Extracted ion chromatograms (LC-MS/MS, precursor ion scan, positive ionization mode) of [M+H]⁺ ions of AHLs present in (A) P. aurantiaca PB-St2 crude extract after 22 h cultivation, (B) E. coli XL1-Blue/pBluescript SK(-)ΔlacZ crude extract, and extracts of E. coli XL1-Blue expressing (C) phzI, (D) csaI, (E) aurI, and (F) hdtS. C4-HSL (black, m/z 172–173), 3-OH-C6-HSL (red, m/z 216–217), 3-oxo-C6-HSL (blue, m/z 214–215), C6-HSL (pink, m/z 200–201), 3-OH-C8-HSL (green, m/z 244–245), 3-oxo-C8-HSL (cyan, m/z 242–243), 3-OH-C10-HSL (orange, m/z 272–273), C8-HSL (purple, m/z 228–229).

Fig 3. Growth curve of P. aurantiaca PB-St2 and production curve of phenazines.

The growth curve of P. aurantiaca PB-St2 was measured as OD₆₀₀ (black) and the production curve of phenazines as A₃₆₅ (red). Data represent means with corresponding standard deviation of three independent replicates.

Fig 4. Production of AHLs in PB-St2; quantified using LC-MS/MS precursor ion scans and calibration curves.

(A) 3-OH-C6-HSL (red, quantification was possible between 5 and 42 h) and 3-oxo-C6-HSL (blue, quantification was possible between 5 and 42 h). (B) C4-HSL (black, quantification was possible between 5 and 42 h), C6-HSL (pink, quantification was possible between 9 and 42 h), and 3-OH-C8-HSL (green, quantification was possible between 11 and 42 h). (C) C8-HSL (purple, quantification was possible between 11 and 42 h), 3-oxo-C8-HSL (cyan, quantification was possible between 9 and 27 h), and 3-OH-C10-HSL (orange, quantification was possible between 11 and 42 h). Data represent means with corresponding standard deviation of three independent replicates. When the amount was lower than the limit of quantification (see S2 Table) the production was considered as zero.