Characterization of Rhamnolipids Produced by an Arctic Marine Bacterium from the Pseudomonas fluorescence Group

Abstract

The marine environment is a rich source of biodiversity, including microorganisms that have proven to be prolific producers of bioactive secondary metabolites. Arctic seas are less explored than warmer, more accessible areas, providing a promising starting point to search for novel bioactive compounds. In the present work, an Arctic marine Pseudomonas sp. belonging to the Pseudomonas (P.) fluorescence group was cultivated in four different media in an attempt to activate biosynthetic pathways leading to the production of antibacterial and anticancer compounds. Culture extracts were pre-fractionated and screened for antibacterial and anticancer activities. One fraction from three of the four growth conditions showed inhibitory activity towards bacteria and cancer cells. The active fractions were dereplicated using molecular networking based on MS/MS fragmentation data, indicating the presence of a cluster of related rhamnolipids. Six compounds were isolated using HPLC and mass-guided fractionation, and by interpreting data from NMR and high-resolution MS/MS analysis; the structures of the compounds were determined to be five mono-rhamnolipids and the lipid moiety of one of the rhamnolipids. Molecular networking proved to be a valuable tool for dereplication of these related compounds, and for the first time, five mono-rhamnolipids from a bacterium within the P. fluorescence group were characterized, including one new mono-rhamnolipid.

Keywords: OSMAC (one strain, many compounds); arctic bacteria; bioactive; molecular networking; rhamnolipids.

Figure 1

Structures of compounds 1–6 isolated from Pseudomonas sp.

Figure 2

Selected 2D-NMR correlations for compound 3 and 6. HMBC and H2BC revealed the position of unsaturation in the lipid chain, and the full lipid spin systems were identified in HSQC-TOCSY. HMBC and ROESY correlations confirmed the rhamnose moiety structure, while ROESY as well as homo- and heteronuclear coupling constants determined the olefinic protons to be in cis configuration.

Figure 3

Growth inhibition assay of 1–6 tested at three concentrations against the Gram-positive bacteria E. faecalis, S. aureus and S. agalactiae. Bacteria and medium (50:50) were used as negative growth controls. Values are means of two replicates, error bars indicate standard deviation.

Figure 4

Biofilm formation inhibition assay performed on S. epidermidis. Values are mean of three replicates, ± standard error.

Figure 5

A cell viability MTS assay was used to evaluate the cytotoxicity of compounds 1–6: (A) Human melanoma cells (A2058); (B) non-malignant cells (MRC5). Values are mean of three replicates, ± standard error.