High-throughput platform for the discovery of elicitors of silent bacterial gene clusters

Abstract

Over the past decade, bacterial genome sequences have revealed an immense reservoir of biosynthetic gene clusters, sets of contiguous genes that have the potential to produce drugs or drug-like molecules. However, the majority of these gene clusters appear to be inactive for unknown reasons prompting terms such as "cryptic" or "silent" to describe them. Because natural products have been a major source of therapeutic molecules, methods that rationally activate these silent clusters would have a profound impact on drug discovery. Herein, a new strategy is outlined for awakening silent gene clusters using small molecule elicitors. In this method, a genetic reporter construct affords a facile read-out for activation of the silent cluster of interest, while high-throughput screening of small molecule libraries provides potential inducers. This approach was applied to two cryptic gene clusters in the pathogenic model Burkholderia thailandensis. The results not only demonstrate a prominent activation of these two clusters, but also reveal that the majority of elicitors are themselves antibiotics, most in common clinical use. Antibiotics, which kill B. thailandensis at high concentrations, act as inducers of secondary metabolism at low concentrations. One of these antibiotics, trimethoprim, served as a global activator of secondary metabolism by inducing at least five biosynthetic pathways. Further application of this strategy promises to uncover the regulatory networks that activate silent gene clusters while at the same time providing access to the vast array of cryptic molecules found in bacteria.

Keywords: cryptic metabolites; malleilactone; natural products discovery.

Fig. 1.

Cryptic biosynthetic gene clusters and their products modulated in this study. (A) Gene organization in the silent mal cluster (Upper). Gene transcripts that were monitored by RT-qPCR in this study are shown in aqua. A translational lacZ reporter fusion to malL was used as a read-out for malleilactone production. Structures of malleilactone A (1), determined previously (19, 20), and malleilactone B (2), elicited and solved in this study, are shown (Lower). (B) The bhc cluster (Upper); a lacZ reporter fusion to bhcF was used in this study. The structure of burkholdac A (3), the product of the bhc cluster is shown (Lower) (24). (C) Structures of two secondary metabolites produced by B. thailandensis and elicited in this study, HMNQ drawn as its quionolone tautomer (4) and thailandamide A (5), produced by the hmq and tha gene clusters, respectively (–27).

Fig. 2.

High-throughput screening as a strategy to awaken silent gene clusters. Two library plates (Upper and Lower) containing a total of 640 compounds were screened in duplicates against malL-lacZ. Each assay plate contained positive (btaK-lacZ) and negative (malL-lacZ with no elicitors) controls, which are shown in green bars. The averaged luminescence as a result of induced LacZ activity is shown for each compound and normalized to the positive control. The data are presented in clusters of 16 compounds. Horizontal blue lines correspond to a Z score of 10 (top line) and the averaged luminescence of the negative control (bottom line). Hits were defined as those with Z scores greater than 6. No hits were obtained on the second library plate (Lower). A total of nine hits were obtained on the first plate (Upper). The structures for the four best hits (orange and red bars), with Z scores greater than 10, are shown. These correspond to piperacillin (6), trimethoprim (7), ceftazidime (8), and cefotaxime (9). Five additional hits belonging to the family of fluoroquinolone antibiotics (tosufloxacin, sarafloxacin, ofloxacin, gatifloxacin, and ciprofloxacin) are shown in blue bars (SI Appendix, Tables S1 and S2).

Fig. 3.

Elicitors of cryptic metabolite production. (A) Hit validation by dose–response analysis for the ability of 6 (red dots) and 7 (blue dots) to turn on the silent mal cluster. Each data point is the average of two independent end-point luminescence measurements using malL-lacZ and varying concentrations of 6 or 7. Black lines describe fits to Eq. S3 (SI Appendix) and yield EC₅₀ values of 1.4 μM (6) and 18 μM (7). (B) RT-qPCR analysis of mal genes in wt E264 (control, black bars) and as a function of 6 (red bars) or 7 (blue bars). The level of each gene is normalized to the E264 control and expressed as fold-change. Each bar is the average of two independent measurements, each determined in triplicates. (C) HPLC-MS analysis of E264 extracts containing no elicitor (black trace), 8.6 μM 6 (red trace), or 15.3 μM 7 (blue trace), which correspond to elicitor concentrations used in the screen. (Inset) Effect of saturating concentrations of 7 (30.6 μM) on malleilactone production. The starred peaks correspond to 1. (D) HPLC-MS analysis of E264 extracts containing no elicitor (black trace) or β-lapachone (green trace). Elution of the mass ion of 3 (starred peak) is shown as a function of retention time. The traces in C and D are normalized for OD_600 nm of the corresponding cultures and vertically offset for clarity.

Fig. 4.

Trimethoprim activates multiple gene clusters in B. thailandensis. (A) Analysis of secondary metabolites in E264 extracts treated with DMSO (control, black trace) or with saturating concentrations of trimethoprim (30.6 μM, blue trace). Peaks corresponding to malleilactone (1), which does not absorb appreciably at 280 nm, HMNQ (4), the thailandamide isomers (5), and a set of new compounds elicited by trimethoprim (starred peaks) are indicated. (Inset) Characteristic absorption spectrum of thailandamide A (5). (B) RT-qPCR analysis of two genes within the thailandamide cluster (tha) in E264 cultures supplemented with DMSO (control, black bars) or with 30.6 μM trimethoprim (blue bars). The level of each gene is normalized to the E264 control and expressed as fold-change. Each bar is the average of two independent measurements, each determined in triplicates. (C) Induction of 3 by trimethoprim (30.6 μM, blue trace) compared with untreated samples (black trace). The traces in A and C are normalized for OD_600 nm of the corresponding cultures and vertically offset for clarity.