Tropodithietic acid production in Phaeobacter gallaeciensis is regulated by N-acyl homoserine lactone-mediated quorum sensing

Abstract

The production of N-acyl homoserine lactones (AHLs) is widely distributed within the marine Roseobacter clade, and it was proposed that AHL-mediated quorum sensing (QS) is one of the most common cell-to-cell communication mechanisms in roseobacters. The traits regulated by AHL-mediated QS are yet not known for members of the Roseobacter clade, but production of the antibiotic tropodithietic acid (TDA) was supposed to be controlled by AHL-mediated QS in Phaeobacter spp. We describe here for the first time the functional role of luxR and luxI homologous genes of an organism of the Roseobacter clade, i.e., pgaR and pgaI in Phaeobacter gallaeciensis. Our results demonstrate that the AHL synthase gene pgaI is responsible for production of N-3-hydroxydecanoylhomoserine lactone (3OHC(10)-HSL). Insertion mutants of pgaI and pgaR are both deficient in TDA biosynthesis and the formation of a yellow-brown pigment when grown in liquid marine broth medium. This indicates that in P. gallaeciensis the production of both secondary metabolites is controlled by AHL-mediated QS. Quantitative real-time PCR showed that the transcription level of tdaA, which encodes an essential transcriptional regulator for TDA biosynthesis, decreased 28- and 51-fold in pgaI and pgaR genetic backgrounds, respectively. These results suggest that both the response regulator PgaR and the 3OHC(10)-HSL produced by PgaI induce expression of tdaA, which in turn positively regulates expression of the tda genes. Moreover, we confirmed that TDA can also act as autoinducer in P. gallaeciensis, as previously described for Silicibacter sp. strain TM1040, but only in the presence of the response regulator PgaR.

Fig. 1.

(A and B) Time course of TDA and pigment production of P. gallaeciensis grown on MB at 28°C under shaken (A) and stagnant (B) conditions. Bacterial growth (−) is shown as absorption at 600 nm as the average of three parallel cultures. TDA production (♦) of cell-free culture fluids was measured using agar diffusion assays with P. tunicata as target strain (n = 3). The TDA concentration was calculated by the use of an internal standard curve with pure TDA (r² = 0.98). Pigmentation (▾) of cell-free culture fluids was measured by spectroscopy at 398 nm (n = 3).

Fig. 2.

A. tumefaciens NTL4 well diffusion assay. (A) Relationship between the concentration of 3-N-hydroxy-decanoyl-l-homoserine lactone (3OHC₁₀-HSL) and TDA in well diffusion assays and resulting diameters of induced blue zones. Error bars indicate standard deviations (n = 3). (B) Well diffusion assays with sterile-filtered culture fluids from late-exponential-phase cultures of the wild-type strain DSM 17395, the tdaA mutant strain WP75, the pgaR mutant strain WP52, and the pgaI mutant strain WP38. The culture fluids were added to wells in agar containing A. tumefaciens NTL4 and X-Gal. Blue zones surrounding the wells indicate induced β-galactosidase activity.

Fig. 3.

(A to C) Total ion chromatograms and the characteristic ion traces m/z 102, 143, and 172 (slightly offset for improved visibility) of culture extracts of the P. gallaeciensis wild-type strain DSM 17395 (A), pgaR mutant strain WP52 (B), and pgaI mutant strain WP38 (C). (D) Mass spectrum of R-3OHC₁₀-HSL/peak at 42.6 min.

Fig. 4.

Growth, antimicrobial activity, and pigmentation of P. gallaeciensis DSM 17395 and derived mutants. The results demonstrate that the PgaI-PgaR QS system of P. gallaeciensis is essential for the TDA and pigment production. (A) Growth curves of P. gallaeciensis wild-type strain DSM 17395, pgaR mutant strain WP52, strain WP52 carrying pBB0808, pgaI mutant strain WP38, and strain WP38 in the presence of either added 100 nM R-3OHC₁₀-HSL or 10 μM TDA. The cultures were grown at 28°C in MB under shaken conditions. Growth was measured by OD₆₀₀ in triplicate cultures. The asterisks indicate points in time where culture fluids were taken for the analysis shown in panels B and C. (B) Zone of inhibition against P. tunicata determined in agar diffusion assays from cell-free culture fluids of different P. gallaeciensis strains at indicated points in time (n = 3). (C) Pigmentation of different P. gallaeciensis strains measured by spectroscopy at 398 nm (n = 3).

Fig. 5.

Effects of disrupting pgaR, pgaI, and tdaA genes on the expression of genes essential for TDA biosynthesis at the RNA level. (A) Relative transcription levels of tdaA, tdaB, tdaE, tdaF, paaZ2, paaG, and pgaI from wild-type P. gallaeciensis DSM 17395 (black bars), the pgaR mutant strain WP52, and the tdaA mutant strain WP75 grown in liquid MB and determined by using qRT-PCR analysis of reverse-transcribed RNA samples (see Materials and Methods for details). (B) Relative transcription levels of tda genes of the wild type and the pgaI mutant strain WP38 grown without or in the presence of either exogenously added R-3OHC₁₀-HSL or TDA. The transcription is shown as the relative expression of each target gene compared to rpoB. The error bars are derived from three independent cultures assayed each in triplicate.