A Silent Operon of Photorhabdus luminescens Encodes a Prodrug Mimic of GTP

Abstract

With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups of bacteria. Teixobactin, the first antibiotic without detectable resistance that binds lipid II, comes from an uncultured Eleftheria terra, a betaproteobacterium; odilorhabdins, from Xenorhabdus, are broad-spectrum inhibitors of protein synthesis, and darobactins from Photorhabdus target BamA, the essential chaperone of the outer membrane of Gram-negative bacteria. Xenorhabdus and Photorhabdus are symbionts of the nematode gut microbiome and attractive producers of secondary metabolites. Only small portions of their biosynthetic gene clusters (BGC) are expressed in vitro. To access their silent operons, we first separated extracts from a small library of isolates into fractions, resulting in 200-fold concentrated material, and then screened them for antimicrobial activity. This resulted in a hit with selective activity against Escherichia coli, which we identified as a novel natural product antibiotic, 3'-amino 3'-deoxyguanosine (ADG). Mutants resistant to ADG mapped to gsk and gmk, kinases of guanosine. Biochemical analysis shows that ADG is a prodrug that is converted into an active ADG triphosphate (ADG-TP), a mimic of GTP. ADG incorporates into a growing RNA chain, interrupting transcription, and inhibits cell division, apparently by interfering with the GTPase activity of FtsZ. Gsk of the purine salvage pathway, which is the first kinase in the sequential phosphorylation of ADG, is restricted to E. coli and closely related species, explaining the selectivity of the compound. There are probably numerous targets of ADG-TP among GTP-dependent proteins. The discovery of ADG expands our knowledge of prodrugs, which are rare among natural compounds. IMPORTANCE Drug-resistant Gram-negative bacteria have become the major problem driving the antimicrobial resistance crisis. Searching outside the overmined actinomycetes, we focused on Photorhabdus, gut symbionts of enthomopathogenic nematodes that carry up to 40 biosynthetic gene clusters coding for secondary metabolites. Most of these are silent and do not express in vitro. To gain access to silent operons, we first fractionated supernatant from Photorhabdus and then tested 200-fold concentrated material for activity. This resulted in the isolation of a novel antimicrobial, 3'-amino 3'-deoxyguanosine (ADG), active against E. coli. ADG is an analog of guanosine and is converted into an active ADG-TP in the cell. ADG-TP inhibits transcription and probably numerous other GTP-dependent targets, such as FtsZ. Natural product prodrugs have been uncommon; discovery of ADG broadens our knowledge of this type of antibiotic.

Keywords: antibiotic resistance; natural product; nucleoside analog.

FIG 1

ADG structure and biosynthetic gene cluster (BGC). (a) Structures of ADG, guanosine, and puromycin. (b) Comparison between the puromycin BGC from S. alboniger and the ADG BGC from P. luminescens. The color-coded genes highlighted are homologs between the two BGCs. (c) Heterologous expression of ADG. Extracted ion chromatogram (EIC; m/z 283.11 to 283.19) on the left and inhibitory activity on E. coli lawn on the right of ADG standard (60 ng/mL ADG in H₂O) (line A), partially purified extract of E. coli Bap1+pNS-ADG (line B), partially purified extract of E. coli Bap1+pRSFduett-1 (negative control) (line C), and EIC (m/z 283.11 to 283.19) of coinjection of lines A and B (line D).

FIG 2

Chromosomal mutations that confer resistance to ADG in E. coli. (a) Mutations in gsk conferring resistance to ADG. (b) Mutations in E. coli MG1655 gsk++ conferring resistance to ADG.

FIG 3

Phosphorylation of ADG in vitro and in vivo. (a) Consecutive phosphorylation of ADG. (Left) In vitro phosphorylation of ADG using recombinant Gsk, Gmk, and Ndk. (Right) Schematic cascade of phosphorylation of ADG. Gsk, guanosine/inosine kinase; Gmk, GMP kinase; Ndk, nucleoside diphosphate kinase; PK, pyruvate kinase; PEP, phosphoenolpyruvate; ADG-MP, ADG-DP, and ADG-TP, ADG monophosphate, diphosphate, and triphosphate, respectively. (b) Targeted mass spectrometry analysis of E. coli metabolome extracts. Presence of ADG-MP, ADG-DP, and ADG-TP mass candidates in cell extract metabolome 1 h after treatment with ADG. Data are means from three biological replicates. Error bars are standard deviations from the means. The mass of ADG and its nucleotide derivatives were not detected in the nontreated samples.

FIG 4

Elucidation of ADG mode of action. (a) Impact of ADG on macromolecular biosynthesis of E. coli. Incorporation of [¹⁴C]thymidine (DNA), [¹⁴C]uridine (RNA), [¹⁴C]l-amino acid mixture (protein), and [¹⁴C]acetic acid (fatty acid) was determined in cells treated with ADG at 2× MIC. Ciprofloxacin (2× MIC), rifampicin (2× MIC), chloramphenicol (2× MIC), and triclosan (2× MIC) were used as controls. Means from three biological replicates are shown. Error bars are standard deviations from the means. (b) In vitro inhibition of RNA synthesis by ADG-TP. The effect of 2-fold dilutions of ADG-TP in an in vitro RNA synthesis reaction mixture was evaluated by agarose gel electrophoresis. This experiment was repeated three times. This gel is a representative of three experiments. (c) Diagram showing the similarity between termination of DNA synthesis by ddNTP (left) and termination of RNA synthesis by ADG-TP (right). (d) Anion-exchange traces of in vitro transcription reactions. Standard traces for starting-material nucleotides are included on the top. Peak positions of major oligonucleotide products (with 5′-triphosphate) are indicated by arrows. Identity of AAG, AA(ADG), and AAGA were confirmed using mass spectrometry (Fig. S5c).

FIG 5

Predictive modeling based on compounds with known modes of action. (a) CoHEC model decision graph for ADG representing MOA predictions. Prediction paths where each terminal colored node depicts an MOA, each internal gray node represents a submodel decision point, the solid line edge width corresponds to the probability according to the model for the respective path, and the dotted opaque path represents standard errors along the decision path. (b) Unsupervised hierarchical clustering of held-out test set prediction probabilities for each compound unobserved by the model. (c) Standard error profiles for each of the submodel predictions for the held-out test compounds. ADG and darobactin are flagged with novel activity by their high standard error profiles and uncertainty with respect to the model.

FIG 6

FtsZ ring localization and cell division is inhibited by ADG. ADG and ciprofloxacin inhibit localization of FtsZ at the division site. E. coli MG1655 FtsZ-GFP was grown and treated with either ADG (128 μg/mL), ciprofloxacin (1 μg/mL), cephalexin (8 μg/mL), or no drug for 1 h at 37°C. Cells were spotted onto a 1.5% low-melting-point agarose pad and observed with a fluorescence microscope. This experiment was repeated three times. This image is a representative of three independent experiments.