Omics Technologies to Understand Activation of a Biosynthetic Gene Cluster in Micromonospora sp. WMMB235: Deciphering Keyicin Biosynthesis

Abstract

DNA sequencing of a large collection of bacterial genomes reveals a wealth of orphan biosynthetic gene clusters (BGCs) with no identifiable products. BGC silencing, for those orphan clusters that are truly silent, rather than those whose products have simply evaded detection and cluster correlation, is postulated to result from transcriptional inactivation of these clusters under standard laboratory conditions. Here, we employ a multi-omics approach to demonstrate how interspecies interactions modulate the keyicin producing kyc cluster at the transcriptome level in cocultures of kyc-bearing Micromonospora sp. and a Rhodococcus sp. We further correlate coculture dependent changes in keyicin production to changes in transcriptomic and proteomic profiles and show that these changes are attributable to small molecule signaling consistent with a quorum sensing pathway. In piecing together the various elements underlying keyicin production in coculture, this study highlights how omics technologies can expedite future efforts to understand and exploit silent BGCs.

Figure 1

Structure of coculture-dependent polyketide keyicin 1 (a) and differential gene expression from WMMB235 genome in coculture with Rhodococcus sp. WMMA185 (b). Genes from the kyc gene cluster are indicated as red spheres within the circled (dashed lines) region.

Figure 2

AHL inducers of keyicin. (a) Six out of 96 AHLs screened for kyc cluster activation and subsequent keyicin production: 2 and 3 are natural AHLs, whereas 4–7 are synthetic. (b) Increase in keyicin production shown as positive log fold change in the absorbance at 470 nm on treatment with AHLs compared to untreated monoculture; absorbance at 470 nm also enables detection of aglycone-containing precursors to 1. Coculture with WMMA185 shown as positive control (red bar). AHLs 2 and 3 are the native LuxR signals in P. aeruginosa and V. fischeri, respectively.

Figure 3

Summary of quantitative proteomics studies of WMMB235 fermented in WMMA185 supernatant (Rhodococcus cell free) for 8 d. FC, Fold change as compared to WMMB235 monoculture conditions. N = 3, P < 0.05.

Figure 4

GNPS and Cytoscape visualization of keyicin analog masses (from LC-MS/MS of cocultured WMMB235) reflect varying extents of glycosylation over time (days 2, 5, 8, and 14). Continuous color mapping for each node in the network represents the relative concentrations of the species for which MS data is shown. Color intensities correlate to concentrations of each species for which MS data is acquired. The m/z signal for keyicin (805.347) is indicated at each time point with a thick diagonal arrow.

Figure 5

Summary of kyc cluster orf expression profiles in WMMB235/WMMA185 coculture compared to those generated in WMMB235 monoculture. Out of 49 orfs within the kyc cluster, only 6 undergo less than a 4-fold increase in expression and two (kyc30, 31 in red) appear to be suppressed in coculture. That these orfs appear to be dispersed at 3–4 different groupings within the kyc cluster suggests that kyc cluster regulation calls for more than just one global regulator. Beyond the earlier stated orf-to-function projections, a comprehensive listing of kyc genes and their putative roles in keyicin biosynthesis is provided in Table S3 of Supporting Information. FC, fold change. False Discovery Rate (q-value) for each gene expression change ≪ 0.01.

Figure 6

Global changes in BGC expression profiles in cocultured WMMB235 shown as logarithm of the fold change (FC) with base 2. The RPKMO over all the ORFs annotated by PRISM for each cluster were used to calculate the overall FCs. BGC numbers correlating to Table 1 are above each relevant bar, and expression profiles were obtained following 2 day (blue) or 5 day (purple) fermentations. N = 3.

Figure 7

Early schematics of BGC2–10 (Table 1, Figure 6) from WMMB235 as annotated using both PRISM and AntiSmash. Blue ORFs indicate core biosynthetic operons. Transporter operons in BGC2 (yellow), luxR operons found in BGC3 and 8 (green), and AHBA synthase genes (red) in BGC8 are all highlighted.

Figure 8

Summary of KEGG mapping for WMMB235 monoculture versus coculture with WMMA185. Lane contents are shown by combination of bracketing and lane coding below the categories listing. Coculturing and duration of fermentations both impact gene expression within WMMB235. Categories of function not abundant enough to depict graphically involved cell communication, cell motility, and signal molecules and interaction. All other categories are depicted in one or more of lanes 1–4.