The spent culture supernatant of Pseudomonas syringae contains azelaic acid

Abstract

Background: Pseudomonas syringae pv. actinidiae (PSA) is an emerging kiwifruit bacterial pathogen which since 2008 has caused considerable losses. No quorum sensing (QS) signaling molecule has yet been reported from PSA and the aim of this study was to identify possible intercellular signals produced by PSA.

Results: A secreted metabolome analysis resulted in the identification of 83 putative compounds, one of them was the nine carbon saturated dicarboxylic acid called azelaic acid. Azelaic acid, which is a nine-carbon (C9) saturated dicarboxylic acid, has been reported in plants as a mobile signal that primes systemic defenses. In addition, its structure,(which is associated with fatty acid biosynthesis) is similar to other known bacterial QS signals like the Diffusible Signal Facor (DSF). For these reason it could be acting as s signal molecule. Analytical and structural studies by NMR spectroscopy confirmed that in PSA spent supernatants azelaic acid was present. Quantification studies further revealed that 20 μg/L of were present and was also found in the spent supernatants of several other P. syringae pathovars. The RNAseq transcriptome study however did not determine whether azelaic acid could behave as a QS molecule.

Conclusions: This study reports of the possible natural biosynthesis of azelaic acid by bacteria. The production of azelaic acid by P. syringae pathovars can be associated with plant-bacteria signaling.

Keywords: Azelaic acid; Metabolomics; Pseudomonas syringae pv. actinidiae; Quorum sensing.

Fig. 1

Representative HPLC and GC-MS chromatograms of standard azelaic acid and azelaic acid isolated from PSA. The presence of azelaic acid in the PSA extracellular metabolome was determined in comparison with standard azelaic acid. Using HPLC on C₁₈ reverse phase column compounds were analyzed and they collectively retained at similar retention time (RT) 13 min (a). GC-MS analysis of HPLC collected fraction at RT 13, showing similar mass fragmentation pattern with standard azelaic acid, confirms the presence of azelaic acid (b)

Fig. 2

¹H-NMR studies of Azelaic acid. Spectra of a solution of standard azelaic acid (a) and of the azelaic acid extracted and purified from PSA (b) recorded at 25 °C in CD₃Cl

Fig. 3

Depicts COSY 2-D spectrum of standard and azelaic acid isolated from PSA. The relevant cross-peaks, connected with dashed lines, are identical in the standard sample and in the extracted sample (a and b)

Fig. 4

Represents PSA azelaic acid production efficiency in different sugars. Production of azelaic acid by PSA grown in M9 minimal medium with respective sugars as the sole carbon source. Azelaic acid from spent medium was extracted and quantified at 70 h. Bar indicates the means and standard deviation of experiment performed in three biological replicates. Asterisks indicate significant difference between the azelaic acid production in glucose and other carbon sources (P < 0.05)

Fig. 5

Production of azelaic acid from different P. syringae pathovars. Strains belonging to different P. syringae pathovars were cultured in M9 sucrose medium for 42 h and azelaic acid from spent medium was extracted and quantified. Bar indicates the mean and standard deviation of experiment performed in three biological replicates. Asterisks indicate significant difference between the azelaic acid production by PSA and different P. syringae pathovars (P < 0.05)