Secondary metabolites of Flustra foliacea and their influence on bacteria

Abstract

The North Sea bryozoan Flustra foliacea was investigated to determine its secondary metabolite content. Gas chromatography-mass spectrometry analysis of a dichloromethane extract of the bryozoan enabled 11 compounds to be identified. Preparative high-performance liquid chromatography of the extract resulted in the isolation of 10 brominated alkaloids (compounds 1 to 10) and one diterpene (compound 11). All of these compounds were tested to determine their activities in agar diffusion assays against bacteria derived from marine and terrestrial environments. Compounds 1, 3 to 7, 10, and 11 exhibited significant activities against one or more marine bacterial strains originally isolated from F. foliacea but only weak activities against all of the terrestrial bacteria. By using the biosensors Pseudomonas putida(pKR-C12), P. putida(pAS-C8), and Escherichia coli(pSB403) the antagonistic effect on N-acyl-homoserine lactone-dependent quorum-sensing systems was investigated. Compounds 8 and 10 caused reductions in the signal intensities in these bioassays ranging from 50 to 20% at a concentration of 20 micro g/ml.

FIG. 1.

Structures of secondary metabolites isolated from F. foliacea.

FIG. 2.

(A) GC-MS chromatogram showing separation of compounds 2 to 11 from the dichloromethane extract of F. foliacea. The GC parameters were as follows: an increase from 90°C (at time zero) to 160°C at a rate of 6°C/min and then an increase from 160 to 300°C at a rate of 10°C/min; sample concentration, 1 mg/ml; injection volume, 1 μl; split ratio, 1/5. (B) GC-MS chromatogram showing separation of compound 1 from the dichloromethane extract of F. foliacea. The GC parameters were as follows: an increase from 50°C (at zero time) to 230°C at a rate of 4°C/min and then an increase from 230 to 300°C at a rate of 10°C/min; sample concentration, 1 mg/ml; injection volume, 1 μl; split ratio, 1/5.

FIG. 3.

Interference of F. foliacea compounds 8 and 10 with AHL-dependent cell-cell communication. (A) Responses of different quorum-sensing reporter strains to the signal molecules and compounds 8 (C8) and 10 (C10). pKR-C12, P. putida harboring pKR-C12; pAS-C8, P. putida harboring pAS-C8; pSB403, E. coli harboring pSB403. The reporter strain signals (fluorescence and bioluminescence) in the absence of the compounds were defined as 100%. (B) Effects of compounds 8 and 10 on the growth of E. coli. The cultures were grown in the presence (20 or 100 μg/ml) or absence of compounds 8 and 10. Control cultures were supplemented with appropriate volumes of DMSO. OD_600nm, optical density at 600 nm. (C) Effects of compounds 8 and 10 on expression of QS-regulated exoproteases of P. aeruginosa PAO1. Cultures of PAO1 were grown in the absence or presence of 20 μg of compound 8 or 10 per ml. P. aeruginosa lasI rhlI mutant PAO1-JP2, an AHL-deficient mutant, was used as a negative control. The protease activities of spent culture supernatants were determined with azocasein as the substrate. The data are means of three independent experiments. The proteolytic activity of PAO1 in the absence of the compounds was defined as 100%. The error bars indicate the standard errors of the means.