Discovery of a New Natural Product and a Deactivation of a Quorum Sensing System by Culturing a "Producer" Bacterium With a Heat-Killed "Inducer" Culture

Abstract

Herein we describe a modified bacterial culture methodology as a tool to discover new natural products via supplementing actinomycete fermentation media with autoclaved cultures of "inducer" microbes. Using seven actinomycetes and four inducer microbes, we detected 28 metabolites that were induced in UHPLC-HRESIMS-based analysis of bacterial fermentations. Metabolomic analysis indicated that each inducer elicited a unique response from the actinomycetes and that some chemical responses were specific to each inducer-producer combination. Among these 28 metabolites, hydrazidomycin D, a new hydrazide-containing natural product was isolated from the pair Streptomyces sp. RKBH-B178 and Mycobacterium smegmatis. This result validated the effectiveness of the strategy in discovering new natural products. From the same set of induced metabolites, an in-depth investigation of a fermentation of Streptomyces sp. RKBH-B178 and autoclaved Pseudomonas aeruginosa led to the discovery of a glucuronidated analog of the pseudomonas quinolone signal (PQS). We demonstrated that RKBH-B178 is able to biotransform the P. aeruginosa quorum sensing molecules, 2-heptyl-4-quinolone (HHQ), and PQS to form PQS-GlcA. Further, PQS-GlcA was shown to have poor binding affinity to PqsR, the innate receptor of HHQ and PQS.

Keywords: biotransformation; induction; metabolomics; natural product discovery; quorum sensing.

FIGURE 1

Inducer-producer culture strategy and metabolomic analysis. (A) Inducer-producer culture strategy overview. (B) UHPLC-HRESIMS based untargeted metabolomic analysis. 28 mass features are selected in the heatmap based on induction, upregulation, and peak area abundance. The vertical axis indicates actinomycete and inducer-producer pairings. Four inducer organisms include: PA, P. aeruginosa; BS, B. subtilis; MS, M. smegmatis; and AF, A. flavus. Preparation of the inducer organisms in ISP2 agar or broth is indicated by (A) or (B), respectively. The control experiments (CON) were prepared by adding twice-autoclaved ISP2 media to the mono-cultures of the producers. The two experimental replicates are indicated by 1 or 2. The horizontal axis indicates induced mass features (detected ions defined by a m/z and t_R window) from UHPLC-HRESIMS analysis. The mass features are presented in a m/z_t_R format and are clustered across different culture experiments. The white cells indicate peak areas <1E4, while peak areas from 1E4 to 1E8 were scaled from black (1E4) to red (1E8).

FIGURE 2

Structural characterization of hydrazidomycin D (1). (A) Hydrazidomycin D key COSY, ¹H-¹³C HMBC, and ¹H-¹⁵N HMBC (H→C) correlations. (B) Overall scheme of DMDS derivatization to determine the position of the double bond in the C18 alkyl chain.

FIGURE 3

Biotransformation of two quorum sensing molecules of P. aeruginosa. (A) Chemical structures of quinolones PQS-GlcA (6), HHQ, and PQS. Key COSY and HMBC (H→C) correlations for PQS-GlcA are shown. (B) Induction of the metabolite m/z 436.1966 [M+H]⁺, t_R 2.75 min from Streptomyces sp. RKBH-B178 treated with different concentrations of HHQ and PQS, experiments conducted in triplicate. The peak area was calculated from integration of UHPLC-HRESIMS data. Peak areas were standardized using an internal standard (20 μg/mL dioctyl phthalate) added post-extraction to each sample.

FIGURE 4

Biotransformation and its effects on PQS-PqsR quorum sensing. Molecular docking of PQS-GlcA (A) and PQS (B) with PqsR^CBD. Images on the left are ribbon representations and images on the right are hydrophobicity surface representations. Visualization was in Chimera (Pettersen et al., 2004). (C) Experimental comparison of quorum sensing activities of HHQ, PQS, and PQS-GlcA using the reporter strain Pseudomonas putida KT2440 [pBBR-pqsR-pqsA’-‘lacZ] (Müller and Fetzner, 2013). The results are expressed as mean ±SD (N = 5).

FIGURE 5

Microbial biotransformations of HHQ/PQS. (A) Oxidation of HHQ by P. aeruginosa (PqsH) (Gallagher et al., 2002), Rhodococcus sp. BG43 and Streptomyces sp. RKBH-B178. (B) Glucuronidation of PQS by Streptomyces sp. RKBH-B178. (C) Degradation of PQS to anthranilic acid (7) by Rhodococcus sp. BG43 (Muller et al., 2014). (D) Degradation of PQS to N-octanoylanthranilic acid (8) by Arthrobacter nitroguajacolicus strain Rü61a (Hod) (Pustelny et al., 2009). (E) Oxidation of PQS to HHQD (9) by Achromobacter xylosoxidans Q19 (Soh et al., 2015).