Induction of Antibacterial Metabolites by Co-Cultivation of Two Red-Sea-Sponge-Associated Actinomycetes Micromonospora sp. UR56 and Actinokinespora sp. EG49

Abstract

Liquid chromatography coupled with high resolution mass spectrometry (LC-HRESMS)-assisted metabolomic profiling of two sponge-associated actinomycetes, Micromonospora sp. UR56 and Actinokineospora sp. EG49, revealed that the co-culture of these two actinomycetes induced the accumulation of metabolites that were not traced in their axenic cultures. Dereplication suggested that phenazine-derived compounds were the main induced metabolites. Hence, following large-scale co-fermentation, the major induced metabolites were isolated and structurally characterized as the already known dimethyl phenazine-1,6-dicarboxylate (1), phenazine-1,6-dicarboxylic acid mono methyl ester (phencomycin; 2), phenazine-1-carboxylic acid (tubermycin; 3), N-(2-hydroxyphenyl)-acetamide (9), and p-anisamide (10). Subsequently, the antibacterial, antibiofilm, and cytotoxic properties of these metabolites (1-3, 9, and 10) were determined in vitro. All the tested compounds except 9 showed high to moderate antibacterial and antibiofilm activities, whereas their cytotoxic effects were modest. Testing against Staphylococcus DNA gyrase-B and pyruvate kinase as possible molecular targets together with binding mode studies showed that compounds 1-3 could exert their bacterial inhibitory activities through the inhibition of both enzymes. Moreover, their structural differences, particularly the substitution at C-1 and C-6, played a crucial role in the determination of their inhibitory spectra and potency. In conclusion, the present study highlighted that microbial co-cultivation is an efficient tool for the discovery of new antimicrobial candidates and indicated phenazines as potential lead compounds for further development as antibiotic scaffold.

Keywords: DNA gyrase; antibacterial; antibiofilm; co-cultivation; phenazine; pyruvate kinase; sponge-associated actinomycetes.

Figure 1

Outline of the procedure used in the present study.

Figure 2

Classes of metabolites produced from Micromonospora sp. UR56 and Actinokineospora sp. EG49 axenic and co-cultures.

Figure 3

Identified phenazine derivatives in the axenic Micromonospora sp. UR56 culture, and after its co-culture with Actinokineospora sp. EG49. 1: dimethyl phenazine-1,6-dicarboxylate, 2: phencomycin, 3: phenazine-1-carboxylic acid, 4: methyl saphenate, 5: 1-hydroxy methyl-6-carboxy phenazine, 6: griseolutic acid, 7: griseolutin A, 8: aestivophoenin C, 9: N-(2-hydroxyphenyl)-acetamide, and 10: p-anisamide.

Figure 4

Structure–activity relationship of the induced phenazine derivatives.

Figure 5

Docking of 1 (A,B), 2 (C,D) and 3 (E,F) within the active site of Staphylococcal Gyr-B. (G,H) The key binding interactions of Gyr-B co-crystallized ligand. The amino acid side chains were depicted in (A,C,E,G) for clarification.

Figure 6

Docking of 1 (A,B), 2 (C,D), and 3 (E,F) within the active site of Staphylococcus PK. (G,H) The key binding interactions of the Gyr-B co-crystallized ligand. The amino acid side chains are depicted in (A,C,E,G) for clarification.