Identification of a new LAP biosynthetic gene cluster

Klebsazolicin (KLB) from Klebsiella pneumoniae is a recently characterized LAP, which inhibits prokaryotic translation through the obstruction of the ribosome exit tunnel8. The BGC of this RiPP (klpACBDE) contains genes encoding the precursor peptide (klpA), the three enzymes involved in the formation of azole cycles (klpC, klpB, and klpD), as well as a transmembrane pump (klpE) exporting the modified peptide. We used the amino acid sequences of KlpB and KlpD proteins as queries to search the NCBI non-redundant protein database using the PSI-BLASTP algorithm11 followed by manual analysis of the genomic environment of the identified gene orthologues. Along with several previously described clusters of KLB homologs8 a distinct BGC phzEACBD was found in the genome of Rhizobium sp. Pop5. Two other related BGCs were found in the genomes of Rhizobium sp. PDO1-076 and Phyllobacterium myrsinacearum DSM 5893, also belonging to the order Rhizobiales. The alignment of amino acid sequences of the predicted core parts of precursor peptides revealed a similar pattern of Cys/Ser/Thr residues, which potentially could be cyclized in the mature LAP product (Fig. 1b, red). The putative PhzA precursor peptide has an amino acid sequence significantly different from that of the KLB precursor KlpA (Fig. 1a). As in the case with KlpA, the C-terminal part of the predicted PhzA precursor peptide is enriched in Ser, Thr, and Cys residues but also contains several positively charged residues absent in the core part of KlpA. Moreover, PhzA and its relatives lack the X-X-(S/T)P motif required for the formation of the N-terminal amidine cycle, which is strictly required for the activity of KLB12. Therefore, we hypothesized that the phzEACBD and similar BGCs from other Rhizobiales might encode closely related LAPs with structural and functional properties different from those of KLB or other known compounds of the family.

Fig. 1 Organization of the phazolicin (PHZ) biosynthetic gene cluster. a Comparison of klebsazolicin (KLB) biosynthetic gene cluster (klpACBDE) with the cluster found in the genome of Rhizobium sp. Pop5. Functions of the genes forming the BGCs are listed on the right. The numbers in the middle indicate the extent of identity between the amino acid sequences of the C, B, and D gene products. The sequence of the KLB precursor peptide is shown above its BGC with the leader and core parts indicated. Residues converted to azoles in the final product are highlighted in red. Residues involved in the formation of the N-terminal amidine cycle are highlighted in green. The sequence of the precursor peptide from the newly identified BGC is shown below it. Ser and Cys residues in the sequence of PhzA precursor involved in oxazole and thiazole formation, respectively, are shown in red. Positively charged residues of the predicted core part are highlighted in blue. b Alignment of the amino acid sequences of precursor peptides encoded in phzEACBD BGC and BGCs of PHZ homologs from other Rhizobiales (Rhizobium sp. PDO1-076 and Phyllobacterium myrsinacearum DSM 5893). Ser, Thr, and Cys residues in the predicted core parts of the peptides are shown in red. Positively charged residues are highlighted in blue. c Structure of the fully modified PHZ peptide after the cleavage of the leader. Cyclized residues are shown in red (Thz and Oxz). Positively charged amino acids are highlighted in blue Full size image

Purification and characterization of a new LAP

Reverse-phase HPLC analysis of medium after cultivation of the Rhizobium sp. Pop5 led to the identification of several fractions absorbing at 254 nm, a wavelength previously used in the purification of other azole-containing compounds (Supplementary Fig. 1A)8,13. MALDI-TOF-MS analysis of the selected fractions showed the presence of compounds, which, based on the observed masses, appeared to be the products of the PhzA precursor peptide posttranslational modification (Supplementary Fig. 1B). The major compound with monoisotopic MH+ 2363.9 was named PHZ (Supplementary Fig. 1C). This mass matches the mass of the C-terminal part of the PhzA precursor peptide, if it was cleaved between the two alanine residues […Thr-Ala-↓-Ala-Thr-Cys…] and contained eight azole rings (loss of 20 Da per installation of each cycle corresponds to −18 Da for each dehydration/cyclization reaction and −2 Da for each azoline oxidation event) (Fig. 1c). Two other major compounds detected in the growth media (Supplementary Fig. 1B) also contained eight azole cycles but had an alternative site for the leader peptide cleavage situated one or two residues closer to the N-terminus of the precursor. These compounds are further referred as A-PHZ (MH+ = 2434.9) and TA-PHZ (MH+ = 2536.0). High-resolution MS measurements for PHZ, A-PHZ, and TA-PHZ resulted in MW values of 2362.8679, 2433.9052, and 2534.9524, which are all within 1.5 ppm of the corresponding masses calculated based on the brutto-formulas (Supplementary Fig. 2). Although due to extensive modification the fragmentation of the PHZ was relatively poor, MS/MS analysis using both ESI-MS/MS and MALDI-MS/MS techniques allowed predictions of the positions of azole cycles in the structure of PHZ (Supplementary Fig. 3). PHZ is a 27-residue long peptide with three thiazole and five oxazole cycles in its structure. The cycles are distributed evenly across the core part of the precursor peptide with every third residue being turned into an azole (Fig. 1c). In addition to the major fractions, several minor forms with a fewer (5–7) number of azole cycles were detected in the cultivation medium (Supplementary Fig. 1B). Compounds with less than eight cycles lacked the C-terminal cycles, consistent with the N- to C-terminus direction of modification of the PHZ precursor peptide.

PHZ demonstrates antimicrobial activity against Rhizobiales

When the purified PHZ was tested against Escherichia coli BW25113 grown on rich solid medium, small growth inhibition zones were observed only when the compound was applied in high concentrations (5–10 mM). Many known RiPPs demonstrate a narrow spectrum of antibacterial activity and are usually active against bacteria, which are evolutionary close to the producing strain7,14,15 serving as powerful weapons in the competition for the ecological niche between closely related species. Accordingly, we tested PHZ against a panel of microorganisms from the order Rhizobiales (class Alphaproteobacteria) grown in a rich liquid medium. The compound proved to be highly active against bacteria from genera Rhizobium, Sinorhizobium (Ensifer), and Azorhizobium with the minimal inhibitory concentrations (MICs) being around 1 μM (Table 1). PHZ is less active against two tested strains of Agrobacterium, while microorganisms from the genus Mesorhizobium appeared to be PHZ-resistant (Table 1). PHZ was also tested against a small set of plant pathogens, plant-associated and soil microorganisms from Gammaproteobacteria (Erwinia amylovora, Pantoea ananatis PA4, Pseudomonas putida KT2440, P. fluorescens Pf-5), Firmicutes (Bacillus subtilis 168, B. cereus ATCC 4342), and Actinobacteria (Arthrobacter sp. ATCC21022, Microtetraspora glauca NRRL B-3735). However, no activity was observed. Taken together, these observations demonstrate that, similarly to a number of other LAPs, PHZ is a narrow-spectrum antimicrobial compound specifically targeting several genera from Rhizobiales.

Table 1 Phazolicin MICs for various bacterial strains in rich YEB medium and Rhizobium medium (RM) Full size table

The resistance of producing strains to RiPPs with antimicrobial activity often results from the efficient export of the mature compound by a specific transporter encoded in the corresponding BGC16. However, there are examples of BGCs containing additional genes providing self-immunity through different mechanisms17,18. Expression of a plasmid-borne copy of the phzE gene, which was annotated as an exporter pump, in S. meliloti Sm1021 conferred resistance to external PHZ and increased the MIC more than 100-fold in comparison with the strain carrying an empty vector (λ). These data confirm the role of PhzE transporter in self-immunity of the producing strain to PHZ.

PHZ inhibits bacterial protein translation in vivo and in vitro

To determine the mode of action of PHZ we tested its activity in vivo using an E. coli-based double-reporter system that allows one to identify antibiotics inducing ribosome stalling or inhibiting DNA replication19. In this system, pDualrep2 plasmid harbors two genes encoding fluorescent proteins under different regulation: the red fluorescent protein (RFP) gene is transcribed in the presence of sublethal concentrations of SOS-response inducing agents (e.g., fluoroquinolones or microcin B17), while the gene for far-red protein Katushka2S is transcribed only when translation inhibitors (e.g., macrolides) are present in the medium in sublethal concentrations. Thus, when a drop containing antimicrobial agent is deposited onto the lawn of cells carrying pDualrep2, the compound diffuses into the media and usually kills cells adjacent to the deposition spot. However, further away from the deposition spot at the outer edge of a growth inhibition zone the compound is present in a sublethal concentration and can induce production of either the RFP or Katushka2S fluorescent protein depending on whether the compound targets DNA replication or translation, respectively. Similar to KLB and erythromycin (ERY), PHZ induces expression of Katushka2S but not RFP, indicating that PHZ is an inhibitor of translation (Fig. 2a, left panel). In this assay, we observed a difference in the size of PHZ-induced inhibition zones between the E. coli strain with the deleted tolC gene and the wild-type strain (Fig. 2a, right panel). TolC is a major outer membrane multidrug efflux porin that is responsible for the export of various compounds from the cell including siderophores (e.g., enterobactin20), macrolide antibiotics21, and some RiPPs (e.g., microcin J2522). In contrast to the situation observed with PHZ, the KLB-induced inhibition zones were comparable in size between the ΔtolC and the wild-type E. coli strains, suggesting that PHZ but not KLB is subject to TolC-dependent efflux.

Fig. 2 Phazolicin is an inhibitor of protein synthesis both in vivo and in vitro. a In vivo testing of PHZ activity using BW25113 ΔtolC pDualRep2 and BW25113 pDualRep2 (WT) reporter strains. The induction of RFP expression (green halo around the inhibition zone, pseudocolors) is triggered by DNA-damage while the induction of Katushka2S protein (red halo) occurs in response to ribosome stalling. Erythromycin (ERY), levofloxacin (LEV), and klebsazolicin (KLB) are used as controls. b Kinetic curves showing inhibition of protein synthesis by increasing concentrations of PHZ in the in vitro cell-free translation in E. coli S30 extract. AU arbitrary units Full size image

To further confirm that PHZ is a protein synthesis inhibitor, we tested its ability to decrease the translation of the luciferase mRNA in vitro using an E. coli S30 extract. Concentration-dependent inhibition of in vitro translation by PHZ was observed (Fig. 2b) with 1 μM of PHZ decreasing the luciferase signal by approximately three-fold (Fig. 2b, blue curve), while at 5 μM PHZ inhibited translation by ~95% (Fig. 2b, red curve).

PHZ obstructs the nascent peptide exit tunnel (NPET) of the ribosome

Protein synthesis inhibitors can interfere with the activity of the ribosome or any of the various other enzymes associated with protein production (translation factors, aminoacyl-tRNA synthetases, etc.). Following the analogy with KLB, we proposed that PHZ acts upon the bacterial ribosome. To determine the exact mechanism of translation inhibition by PHZ we attempted to crystallize the 70S ribosome from Thermus thermophilus (Tth) in complex with PHZ, mRNA, and tRNAs, as previously done with KLB8, and several other translation inhibitors23,24,25,26,27,28,29,30. Despite numerous attempts, we were not able to observe any positive electron density peaks corresponding to the ribosome-bound PHZ anywhere within the Tth 70S ribosome. Moreover, in vitro translation inhibition experiments using E. coli (Eco) 70S or hybrid 70S (Eco 30S subunit + Tth 50S subunit) demonstrated that PHZ does not target Tth ribosome (Supplementary Fig. 4). However, the same data also suggested that PHZ does target the large ribosomal subunit of the E. coli ribosome.

Since the crystallization-based approach was unsuccessful, we switched to cryo-EM to determine the structure of PHZ bound to the sensitive Eco 70S ribosome. The obtained charge density map, characterized by the overall resolution of 2.87 Å according to the “gold-standard” Fourier shell correlation (FSC) method (Supplementary Fig. 5A), revealed PHZ bound to the ribosome (Fig. 3a). The high resolution and excellent quality of the map in the PHZ-binding site (Supplementary Fig. 5B), allowed us not only to fit its atomic model (residues 2–23; Fig. 3b) but also to confirm the positions of seven azoles in the structure of the modified peptide proposed according to the MS–MS analysis. While the results of tandem mass spectrometry indicate another oxazole present at the position 24, we left Ser23 unmodified in the model since Ser24 was not visible in the map due to its poor quality in that region (Fig. 3a).

Fig. 3 The structure of PHZ in complex with the bacterial ribosome. a Cryo-EM map of PHZ in complex with the E. coli 70S ribosome (green mesh). The fitted model of the compound is displayed in its respective charge density viewed from two different perspectives. The map is contoured at 2.5σ. Carbon atoms are colored yellow, nitrogen atoms are blue, oxygen atoms are red, and sulfur atoms are green. b Schematic diagram showing that only residues 2–23 of the ribosome-bound PHZ molecule are visible in the cryo-EM map. c, d Overview of the PHZ-binding site (yellow) on the E. coli large ribosomal subunit (light blue) viewed from two different perspectives. In c, the 50S subunit is viewed from the inter-subunit interface (30S subunit is removed for clarity) as indicated by the inset. The view in d is from the cytoplasm onto the A site. e, f Close-up views of the PHZ-binding site in the ribosome exit tunnel. E. coli numbering of the nucleotides in the 23S rRNA is used. Potential H-bond interactions are indicated with dotted lines Full size image

Similar to KLB, linear modified PHZ peptide forms a compact globule that binds in the upper part of the NPET of the 50S ribosomal subunit where it extensively interacts with the 23S rRNA and ribosomal proteins (Fig. 3c, d; Supplementary Movie 1). In comparison to antibiotics of smaller molecular weight, such as ERY, which binds at the same location but only partially occludes the NPET (Supplementary Fig. 6A, B), binding of both KLB and PHZ results in nearly complete obstruction of the NPET (Supplementary Fig. 6C, D). A prominent feature of the ribosome-bound PHZ molecule is its complex intramolecular interactions that involves both face-to-face and edge-to-face π–π stacking of Thz12, Oxz21, Thz6, and Oxz18, along with the nucleobase U2609 from the 23S rRNA (Fig. 3f; Supplementary Fig. 7D). Moreover, we observed two major types of interactions between the PHZ and the nucleotides of the 23S rRNA. First, three azoles (Thz3, Oxz15, and Oxz18) are involved in π–π stacking with nucleotides A751, C2611, and U2609, respectively (Fig. 3e, f). Second, two positively charged side chains of Arg5 and Arg11, as well as hydrophilic side chains of Asp7 and Ser8, form additional stabilizing hydrogen bonds (H-bonds) with nucleobases and backbone phosphate groups of the 23S RNA (Fig. 3e, f; Supplementary Fig. 7A–C). The presence of intramolecular π–π stacking and positively charged side chains, as well as the absence of the N-terminal amidine cycle, make the mode of PHZ binding significantly different from that of KLB. Unlike KLB, the N-terminal part of PHZ is not critical for binding to the ribosome, which is further supported by the observation that naturally occurring longer forms with an alternative leader cleavage site (A-PHZ and TA-PHZ) demonstrate the same level of in vitro translation inhibition activity as PHZ (Supplementary Fig. 8).

Interestingly, the PHZ-binding site overlaps with those of several other classes of antibiotics, such as macrolides and type B streptogramins. Comparison of the structures of ribosome-bound PHZ and KLB reveals that PHZ binds further away than KLB from the peptidyl transferase center (PTC) of the ribosome. We superimposed our new structure of the ribosome-bound PHZ (Fig. 4a) or the previously published KLB (Fig. 4b) with the cryo-EM structure of the 70S ribosome containing ErmBL nascent peptide chain connected to the P-site tRNA31. Apparently, there is more space between the ribosome-bound drug and the PTC in the case of PHZ in comparison to KLB. This suggests that PHZ should, in principle, allow for a few more amino acids to be incorporated into the nascent polypeptide chain before it encounters the NPET-bound PHZ molecule and translation stalls. To test this hypothesis, we performed a toe-printing (primer-extension inhibition) assay, which allows identification of drug-induced ribosome stalling on a given mRNA template with a single-nucleotide precision32. Because PHZ causes strong induction (stalling of the ribosomes) on Dualrep2 reporter used in our in vivo bioactivity test (Fig. 2a), we have chosen the corresponding mRNA (trpL-2Ala) as a template for our toe-printing assay. Addition of PHZ to the PURExpress cell-free transcription–translation system programmed with trpL-2Ala mRNA resulted in dose-dependent ribosome stalling at the seventh codon of mRNA (Fig. 4c, lanes 4–7), while the addition of KLB induced major stalling at the third and the fifth codons (Fig. 4c, lane 3) corroborating our hypothesis. Moreover, these data indicate that PHZ is an inhibitor of the elongation step during protein synthesis, as is the KLB. In the absence of any inhibitors in our toe-printing reactions, ribosomes stall at codons 9–10 due to a specific secondary structure in the mRNA (Fig. 4c, lane 8).

Fig. 4 PHZ inhibits the elongation step during protein synthesis. a, b In silico modeling of the nascent polypeptide chain in the ribosome exit tunnel in the presence of klebsazolicin (a, PDB entry 5W4K8) or phazolicin (b, current structure) using the cryo-EM structure of the 70S ribosome with ErmBL peptide bound to the P-site tRNA (PDB entry 5JTE31). c Ribosome stalling by PHZ on trpL-2Ala mRNA in comparison with other translation inhibitors (thiostrepton, THS; erythromycin, ERY; klebsazolicin, KLB), as revealed by reverse-transcription primer-extension inhibition (toe-printing) assay in a cell-free translation system. trpL-2Ala mRNA nucleotide sequence and the corresponding amino acid sequence are shown on the left. Colored triangles show ribosome stalling at 1st, 3rd, 5th, and 7th codons. Note that owing to the large size of the ribosome, the reverse transcriptase stops at the nucleotide + 16 relative to the codon located in the P-site Full size image

Species-specificity of PHZ action

The PHZ-binding site is adjacent to the NPET constriction point—the most narrow part of the NPET formed by the extended loops of the ribosomal proteins uL4 and uL22 (Fig. 5a, b). The N-terminal and C-terminal parts of the PHZ molecule are located between the uL4 and uL22 loops (Fig. 5c) and occupy more space in this part of the NPET than the C-terminal part of KLB (Fig. 5d). The amino acid sequences of uL4 and uL22 proteins including their loop regions vary between distinct bacteria (Fig. 5e). Superpositioning of our Eco 70S ribosome structure with bound PHZ with the structure of Tth 70S reveals that residues His69 of the Tth protein uL4 sterically clashes with the PHZ molecule (Fig. 5c) rationalizing why PHZ does not bind to or act upon the Tth ribosome. Because the same residue in Eco ribosome is represented by a less bulky Gly64 (uL4), we hypothesized that the differences in the uL4 loop sequence are responsible for the inability of PHZ to bind in the NPET and, hence, to inhibit the translation by Tth ribosome. To test our hypothesis, we expressed a plasmid-borne copy of the S. meliloti rplD gene encoding for the uL4 protein carrying the Tth-like G68H substitution (rplDG68H) and showed that the loop structure of Tth uL4 confers resistance to PHZ and allows growth of otherwise sensitive strain on the medium with 8xMIC of the antibiotic (Fig. 5f). The expression of rplDK65A mutant, which carries an alanine substitution of the Eco Arg61-equivalent (Fig. 5e) that forms H-bond with the PHZ molecule in our structure (Fig. 5c), does not confer resistance to PHZ. This is likely due to multiple contacts, which the PHZ molecule forms with the ribosome, and the loss of one H-bond does not affect the overall binding. The same superpositioning of the Eco and Tth ribosome structures also revealed that residue Arg90 of the Tth protein uL22 sterically clashes with the PHZ molecule (Fig. 5c), while the comparable in size-equivalent residue Lys90 in the Eco protein uL22 does not clash with the PHZ and is repositioned to the side relative to the ribosome-bound PHZ to avoid a similar clash. Unlike His69Tth, which is located in a confined pocket and cannot re-adjust its position in response to PHZ binding, residues Arg90Tth and Lys90Eco have more space around and should be able to easily readjust their positions when PHZ is present to avoid a possible clash. Indeed, the substitution of Lys90 in the loop of protein uL22 with a larger Arg residue (rplVK90R) does not render any resistance to PHZ (Fig. 5f). Thus, the fine structure of the PHZ-binding site (formed in part by the loop regions of ribosomal proteins uL4 and uL22) together with the repertoire of peptide transporters involved in the uptake contribute to the specificity of the antibiotic action.