The bacterial toxin colibactin triggers prophage induction


Bacterial strains, plasmids and routine cultivation

Bacterial strains and plasmids utilized in these research are listed in Supplementary Desk 2 and Supplementary Desk 3, respectively. Except in any other case famous, E. coli DH10β (NEB) was used for all pressure development and propagated aerobically in Luria-Bertani (LB-Lennox, RPI) broth at 37 °C. All experiments involving faecal communities have been carried out in an anaerobic chamber (70% N2, 25% CO2, 5% H2). Oligonucleotides (Sigma) and dsDNA gene blocks (IDT) utilized in plasmid development are listed in Supplementary Desk 4. Plasmid development steps and recombineering have been carried out utilizing enzymes obtained from NEB (NEBuilder HiFi DNA meeting grasp combine, T4 DNA ligase and DpnI) and lambda pink (pKD46 and pKD3), respectively. Sequencing of all inserts was carried out utilizing Sanger sequencing. Plasmid sequencing of PRlux revealed that the vector consists of two copies of the DNA-damage-responsive aspect (cI and PR). Progress, reporter and lysis assays have been all carried out in M9 medium supplemented with 0.4% casamino acids (M9-CAS, High quality Organic) except in any other case specified. Antibiotics, inducers and indicators have been used on the following concentrations: 100 μg ml−1 ampicillin (IBI Scientific), 50 μg ml−1 kanamycin (VWR), 25 μg ml−1 chloramphenicol (Sigma), 100 ng ml−1 MMC (Sigma), 40 μg ml−1 5-bromo-4-chloro-3-indolyl β-d-galactosidase (X-gal, Takara Bio), and 500 μM isopropyl β-d-1-thiogalactopyranoside (IPTG, Teknova), except in any other case specified.

Progress and competitors assays

For development inhibition by cell-free fluids

In a single day cultures of wild-type E. coli BW25113 harbouring both BAC-pks or the empty BAC have been centrifuged (16,100g and 1 min) and the supernatant was handed by way of a 0.22-μm filter (Corning Spin-X). Progress of non-colibactin-producing E. coli cultures was assayed in contemporary LB within the presence of various quantities of every supernatant (5%, 10%, 20%, 50% v/v). OD600 was measured at common intervals utilizing a BioTek Synergy HTX multi-mode plate-reader.

For testing ClbS safety from cell-free fluids

In a single day cultures of wild-type E. coli BW25113 harbouring both pTrc-clbS or pTrc-∆clbS have been centrifuged (16,100g and 1 min) earlier than addition at 10% v/v to co-cultures containing a 1:1 ratio of E. coli BW25113 harbouring the PRlux reporter and E. coli BW25113 harbouring both BAC-pks or the empty BAC. Bioluminescence was measured after 24 h and quantified in a plate-reader as outlined beneath (see ‘E. coli-based mostly reporter assay’).

For E. coliE. coli competitors assays

In a single day cultures of lacZ + E. coli MG1655 (KIlacZ, Addgene: 52696) harbouring BAC-pks have been back-diluted 1:100 into contemporary M9-CAS and combined in a 1:1 ratio with a equally back-diluted tradition of lacZ  E. coli MG1655 (delta-Z, Addgene: 52706) harbouring the empty BAC. The co-cultures have been incubated at 37 °C, and, at common intervals, an aliquot was taken for differential plating on LB supplemented with X-gal and IPTG. Each BAC combos (pks+ versus empty) and marker combos (lacZ + versus lacZ ) have been examined to rule out the affect of carrying the lacZ marker.

For assaying RecA-dependent development of pks
+clbS E. coli

In a single day cultures of wild-type E. coli BW25113 or wild-type E. coli DH10β, every individually harbouring BAC-pks or BAC-pks∆clbS, have been back-diluted 1:100 into contemporary M9-CAS. The monocultures have been incubated at 37 °C and the OD600 nm readings have been obtained after 24 h.

For E. coliS. aureus competitors assays

S. aureus RN450 lysogenic for phi80α and S. aureus RN450 lysogenic for phi11 have been grown in a single day at 37 °C in contemporary mind coronary heart infusion (BHI) medium, and E. coli BW25113 harbouring BAC-pks or empty BAC have been grown in a single day at 37 °C in contemporary LB broth supplemented with chloramphenicol. The in a single day cultures have been back-diluted 1:100 into contemporary BHI medium and combined in a 1:1 ratio and incubated at 37 °C for twenty-four h. The cultures have been plated on LB agar supplemented with Cm for E. coli colony-forming items (CFUs), and mannitol salt phenol-red agar (Sigma) for S. aureus CFUs.

For differential MMC susceptibility of phage-free S. aureus and E. coli

lacZ  E. coli MG1655 (delta-Z, Addgene: 52706) and S. aureus RN450were grown in a single day at 37 °C in contemporary LB and BHI media, respectively. The in a single day cultures have been back-diluted 1:100 into the identical respective contemporary medium and a twofold dilution sequence of MMC was added to realize a last focus starting from 78 ng ml−1 to five,000 ng ml−1. Cultures have been subsequently incubated in a single day at 37 °C and OD600 nm readings have been obtained after 24 h. Normalized OD600 nm was calculated because the OD600 nm at a given MMC focus relative to the OD600 nm of the identical pressure to which no MMC was added (outlined as 100%).

Manufacturing and isolation of phage lambda by MMC induction

An in a single day tradition of the lambda lysogen was back-diluted 1:100 into contemporary LB and incubated at 37 °C. After reaching an OD600 nm of 0.4–0.5, MMC (500 ng ml−1 last focus) was added and the cultures have been returned to 37 °C for a further 3–5 h, over which period noticeable clearing occurred. After chloroform therapy and centrifugation (16,100g and 1 min), the clarified lysates have been filter-sterilized and saved at 4 °C earlier than use.

Quantification of phage induction by colibactin

E. coli-based reporter assay

In a single day cultures have been back-diluted 1:100 into contemporary M9-CAS medium with acceptable antibiotics earlier than being disbursed (200 μl) into white-walled 96-well plates (Corning 3610). For co-culture experiments, the 2 cultures have been combined 1:1 instantly after back-dilution. Monoculture controls for every pressure have been ready by including 100 μl of the back-diluted cultures to an equal quantity of M9-CAS. For DNA interference experiments, herring sperm DNA (Promega) was used. To check DNA with various AT richness, complementary oligonucleotide pairs (JWO-1046 and JWO-1047) and (JWO-1044 and JWO-1045) have been annealed in 10 mM aqueous Tris-HCl buffer, and the ensuing duplexes have been added to the wells on the indicated concentrations. Plates have been shaken at 37 °C and the OD600 nm and bioluminescence readings have been obtained after 24 h. Relative gentle items (RLU) have been calculated by dividing the bioluminescence by the OD600 nm.

Phage quantification for phages of E. coli, S. Typhimurium, S. aureus, E. albertii 07-3866 and E. faecium E1007

Making ready and measuring viral titres from co-cultures with phage-infected isolates was carried out in keeping with the an identical circumstances used for the reporter assays with the exception that the reporter pressure was substituted for the related lysogen. Co-cultures with phage-infected E. coli, S. Typhimurium and E. albertii have been carried out in M9-CAS, whereas co-cultures with phage-infected S. aureus and E. faecium have been carried out in BHI as the expansion medium. To organize phage lysates, cultures have been transferred after 24 h co-culture to microcentrifuge tubes and centrifuged at 16,100g for 1 min. The supernatant was eliminated and handed by way of a 0.22-µm filter. For phage quantification by plaque assays, supernatants have been diluted logarithmically from 100 to 10−5, and 10 µl noticed on prime agar (preparation beneath) containing the related indicator. For E. albertii 07-3866 phi12, 2-fold dilutions of the supernatants as an alternative of 10-fold have been used. Within the case of quantifying S. Typhimurium phages from faecal communities, tradition supernatants have been concentrated roughly 40-fold from their beginning quantity in protein concentrators (Pierce, 100 kDa MWCO, spin columns) earlier than use in plaque assays. For phage quantification by qPCR, supernatants have been diluted 100-fold, handled with DNase (Promega) to take away residual DNA, then boiled to launch encapsidated phage DNA. Host (JWO-1120 and JWO-1121) and phage (JWO-1116 and JWO-1117) particular primer pairs have been used for PCR amplification utilizing the Luna Common qPCR equipment (NEB) in a CFX96 real-time PCR detection system (Bio-Rad). Knowledge have been processed and analysed by evaluating the relative amplification inside samples of phage-specific primer pairs to host-specific primer pairs (Pfaffl technique) utilizing the Gene Expression calculator within the CFX Supervisor software program (Bio-Rad).

Preparation of prime agar

The symptoms used to assay every phage-bacteria system have been as follows: for lambda-E. coli and phi12-E. albertii (wild-type E. coli BW25113 or the lambda-resistant lamB::kan mutant); for P22-S. Typhimurium (S. Typhimurium D23580ΔΦ); for BTP1-S. Typhimurium (S. Typhimurium SNW22 D23580 ΔBTP1); for Gifsy-1-S. Typhimurium (S. Typhimurium D23580 ΔΦ ΔwaaG::aph); for phi80α and phi11 (S. aureus RN450). In every case, in a single day cultures of the related indicator strains have been back-diluted 1:100 into LB (for E. coli and S. Typhimurium) or BHI (for S. aureus) and incubated at 37 °C. At an OD600 nm of 0.3–0.5, E. coli and S. Typhimurium cultures have been diluted 1:10 into molten LB-agar (0.6%) supplemented with 10 mM MgSO4 and 0.2% maltose and poured onto a LB-agar (1.5%) plate. For S. aureus, cultures have been back-diluted 1:10 into molten tryptic soy agar (0.6%) supplemented with 10 mM CaCl2 and poured onto a denser layer (1.5%) of the identical agar.

ELISA for Stx2dact detection

Detection of Stx2dact from each cardio co-cultures and faecal communities was carried out utilizing the Premier EHEC take a look at equipment, which particularly detects Shiga toxins I and II (Meridian Biosciences), following the producer’s directions with the next modifications: for cardio co-cultures, in a single day monocultures of C. rodentium harbouring stx2dact have been back-diluted 1:100 and co-cultured in M9-CAS at a 1:1 ratio with E. coli BW25113 harbouring both BAC-pks or the empty BAC. For faecal group experiments, anaerobic monocultures of C. rodentium harbouring stx2dact have been combined with faecal communities in BHI as described within the related part beneath. To confirm toxin manufacturing in response to a recognized DNA-damaging agent below these circumstances, MMC (1.5 µg ml−1 last focus) was added to cardio cultures of exponentially rising stx2dact-harbouring C. rodentium in M9-CAS. In all instances, samples collected after 24-h incubations (precise volumes detailed beneath) have been diluted in 200 µl of diluent buffer offered by the producer earlier than addition to Stx-specific antibody-coated microwells. The usage of kit-provided constructive and damaging controls in addition to all wash and substrate addition steps have been carried out precisely in keeping with the manufacturer-supplied protocol. The cease reagent was added roughly 2–5 min after including the ultimate substrate to every effectively, at which period the pictures utilized in Fig. 3c have been taken. Absorbance at 450 nm (Abs450 nm) was measured utilizing a plate-reader. In keeping with the producer, Abs450 nm values ≥ 0.180 are thought of a constructive take a look at end result. For cardio experiments together with MMC controls, 2 µl of tradition supernatants have been used because the pattern enter. For faecal group experiments, tradition supernatants have been first concentrated roughly 20-fold from the preliminary quantity in protein concentrators (Pierce, 30 kDa MWCO, spin columns). Forty microlitres of the concentrated retentate was used because the pattern enter.

Faecal pattern processing

Faecal pellets from C57BL/6J mice from the Jackson Laboratory (which lack Enterobacteriaceae and don’t comprise colibactin-producing organisms) have been suspended in pre-reduced PBS supplemented with 0.1% l-cysteine (5% w/v), then left to face to permit insoluble particles to settle. The supernatant was fastidiously eliminated and combined with an equal quantity of 40% glycerol. Aliquots (50 µl) of this suspension have been saved at –80 °C till required.

Ex vivo tradition with faecal communities

An aliquot of the faecal suspension ready above was thawed and inoculated into BHI (1:100) and incubated at 37 °C in an anaerobic chamber alongside the related human-associated phage-containing micro organism and E. coli BW25113 harbouring both BAC-pks or the empty BAC. After 24 h incubation, the in a single day cultures have been back-diluted (1:1,000) and combined in equal proportions in contemporary BHI, then incubated for an additional 24 h. Phages and toxin produced from faecal communities have been measured in the identical assays utilized in two-way cultures, involving plaque assays for S. Typhimurium (BTP1 and Gifsy-1) and S. aureus (phi80α and phi11), qPCR for E. faecium (phi1), and Stx ELISA for C. rodentium (stx2dact).

Assaying safety by clbS-like open studying frames

For reporter assays, every of the clbS-like open studying frames (ORFs) (or the pTrc-∆clbS assemble) have been reworked into E. coli BW25113 harbouring the PRlux reporter. After in a single day development of every pressure in monoculture, strains have been back-diluted 1:100 and co-cultured in M9-CAS at a 1:1 ratio with E. coli BW25113 harbouring BAC-pks. Bioluminescence was measured after 24 h incubation at 37 °C in a plate-reader as detailed for all different E. coli-based reporter assays above. For measuring safety by the clbS-like ORFs from phage induction, the identical clbS-like ORF-encoding constructs from the reporter assay have been individually reworked right into a E. coli BW25113 lambda lysogen. An an identical dilution and co-culture process to that of E. coli BW25113 harbouring BAC-pks was used, after which phage manufacturing was measured by plaque assay as described within the related part above. To measure safety offered by a chromosomal copy of E. albertii-encoded clbS (clbSalbertii), the locus surrounding clbSalbertii from the E. albertii 07-3866 genome was PCR-amplified and transferred utilizing lambda-red recombineering into wild-type E. coli BW25113 (JSO-1966–1973; Supplementary Desk 4). The PRlux reporter plasmid was launched into the ensuing pressure, E. coli::clbSalbertii, and measured for its capability to be induced by colibactin utilizing the an identical co-culture process as that used for E. coli-based reporter assays, as famous within the related part above.

Quantification of N-myristoyl-d-asparagine prodrug manufacturing by pks
+
E. coli

For tradition circumstances and pattern preparation

In a single day cultures of E. coli BW25113 harbouring both BAC-pks or the empty BAC have been back-diluted 1:100 and co-cultured in M9-CAS at a 1:1 ratio with phage-free E. coli BW25113 or lambda-infected BW25113. Cultures (1 ml) have been disbursed into deep-well plates (VWR) and incubated with shaking at 37 °C. After 24 h, 10 µl deuterated (d27) N-myristoyl-d-asparagine (10 µM in DMSO inventory resolution) was added to every pattern. Samples have been flash-frozen in liquid nitrogen, lyophilized for 48 h, then reconstituted in methanol (1 ml) and vortexed for 1 min. 300 microlitres of the combination was filtered by way of a 0.22 µm filter (Pall) earlier than mass spectrometry evaluation.

For prodrug quantification

Evaluation of the N-myristoyl-d-asparagine prodrug in samples was carried out utilizing an ultra-high efficiency liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) system mannequin Xevo TQ-S (Waters). The mass spectrometer system consists of a triple quadrupole outfitted with a dual-spray electrospray ionization (ESI) supply. Samples have been analysed utilizing an Agilent Poroshell 120 EC-C18 column (2.7 mm, 4.6 mm × 50 mm) with the next elution circumstances: isocratic maintain at 90% solvent A in solvent B for 0.5 min: linear gradient from 90% to five% solvent A in solvent B from 0.5–2 min; isocratic maintain at 5% solvent A from 2–3 min, gradient from 5% to 98% solvent A in solvent B from 3–3.5 min; isocratic maintain at 98% solvent A in solvent B from 3.5–4 min (solvent A: 95% water + 5% methanol + 0.03% ammonium hydroxide; solvent B: 80% isopropanol + 15% methanol + 5% water; move fee = 0.75 ml min−1; injection quantity = 5 µl). The mass spectrometer was run in negative-mode MRM with a Cone voltage of fifty V, monitoring transitions of m/z 341 -> m/z 114 (retention time (rt) = 2.2 min, collision power (CE) = 24 V) for the prodrug scaffold and m/z 368 -> m/z 114 (rt = 2.2 min, CE = 28 V) for the deuterated inside customary (d27-N-myristoyl-d-asparagine). For all samples, peak areas for the m/z 341 -> m/z 114 have been normalized to the m/z 368 -> m/z 114 transition for a similar pattern, after which normalized values in comparison with a regular curve of unlabelled N-myristoyl-d-asparagine containing 100 nM d27- N-myristoyl-d-asparagine, which was run in triplicate.

Bioinformatic analyses

NCBI tBLASTn (nr/nt database, anticipate threshold = 0.05, phrase dimension = 6, BLOSUM62 matrix) was used to determine clbS genes that match E. coli ClbS (WP_000290498) however which are discovered outdoors of pks clusters. The extra distantly associated ClbS-like proteins examined on this research (Fig. 3d) have been compiled from BLASTp outcomes utilizing E. coli ClbS because the question (nr protein sequences database, anticipate threshold = 0.05, phrase dimension = 6, BLOSUM62 matrix, 5,000 entries). After excluding entries that happen in genomes with pks clusters, the isolation supply of the remaining hits was thought of in figuring out bee intestine and human-associated isolates. Different members within the consultant panel chosen for cloning and heterologous expression have been chosen heuristically and to cowl the vary in per cent identities returned by the BLAST search (spanning Mixta theicola having 80% pairwise identification and Bifidobacterium longum with 26.8% pairwise identification to E. coli ClbS). The genomes encoding clbS-like genes within the consultant panel have been submitted to PHASTER for identification of prophage areas. Genes encoded by predicted intact prophages (rating larger than 90) have been additional analysed by area evaluation (InterPro) for options matching the lambda repressor (DNA-binding and peptidase domains), as talked about in the primary textual content and Supplementary Dialogue, and proven in Prolonged Knowledge Fig. 4d.

Quantification and statistical evaluation

Software program used to gather and analyse knowledge generated on this research consisted of: GraphPad Prism 9 for evaluation of growth- and reporter-based experiments; Gen5 v.3 for assortment of growth- and reporter-based experiments; Bio-Rad CFX Supervisor 3.0 for quantification and evaluation of qPCR knowledge; ImageJ 1.53c for colony counting in competitors experiments; and Geneious Prime 2020 for evaluation of publicly out there knowledge and primer design. Knowledge are introduced as imply ± s.d. except in any other case indicated within the determine legends. The variety of unbiased organic replicates for every experiment is indicated for every experiment and included within the legend.

Reporting abstract

Additional info on analysis design is on the market within the Nature Analysis Reporting Abstract linked to this paper.

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