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 building and propagated aerobically in Luria-Bertani (LB-Lennox, RPI) broth at 37 °C. All experiments involving faecal communities had been carried out in an anaerobic chamber (70% N2, 25% CO2, 5% H2). Oligonucleotides (Sigma) and dsDNA gene blocks (IDT) utilized in plasmid building are listed in Supplementary Desk 4. Plasmid building steps and recombineering had been carried out utilizing enzymes obtained from NEB (NEBuilder HiFi DNA meeting grasp combine, T4 DNA ligase and DpnI) and lambda crimson (pKD46 and pKD3), respectively. Sequencing of all inserts was carried out utilizing Sanger sequencing. Plasmid sequencing of PR–lux revealed that the vector consists of two copies of the DNA-damage-responsive component (cI and PR). Development, reporter and lysis assays had been all carried out in M9 medium supplemented with 0.4% casamino acids (M9-CAS, High quality Organic) until in any other case specified. Antibiotics, inducers and indicators had 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), until in any other case specified.
Development 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 had been centrifuged (16,100g and 1 min) and the supernatant was handed by way of a 0.22-μm filter (Corning Spin-X). Development of non-colibactin-producing E. coli cultures was assayed in recent 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 had 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 PR–lux 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-primarily based reporter assay’).
For E. coli–E. coli competitors assays
In a single day cultures of lacZ + E. coli MG1655 (KIlacZ, Addgene: 52696) harbouring BAC-pks had been back-diluted 1:100 into recent 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 had 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 −) had 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, had been back-diluted 1:100 into recent M9-CAS. The monocultures had been incubated at 37 °C and the OD600 nm readings had been obtained after 24 h.
For E. coli–S. aureus competitors assays
S. aureus RN450 lysogenic for phi80α and S. aureus RN450 lysogenic for phi11 had been grown in a single day at 37 °C in recent mind coronary heart infusion (BHI) medium, and E. coli BW25113 harbouring BAC-pks or empty BAC had been grown in a single day at 37 °C in recent LB broth supplemented with chloramphenicol. The in a single day cultures had been back-diluted 1:100 into recent BHI medium and combined in a 1:1 ratio and incubated at 37 °C for twenty-four h. The cultures had been plated on LB agar supplemented with Cm for E. coli colony-forming models (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 recent LB and BHI media, respectively. The in a single day cultures had been back-diluted 1:100 into the identical respective recent medium and a twofold dilution collection of MMC was added to attain a remaining focus starting from 78 ng ml−1 to five,000 ng ml−1. Cultures had been subsequently incubated in a single day at 37 °C and OD600 nm readings had 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 recent LB and incubated at 37 °C. After reaching an OD600 nm of 0.4–0.5, MMC (500 ng ml−1 remaining focus) was added and the cultures had been returned to 37 °C for a further 3–5 h, over which period noticeable clearing occurred. After chloroform remedy and centrifugation (16,100g and 1 min), the clarified lysates had 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 had been back-diluted 1:100 into recent M9-CAS medium with acceptable antibiotics earlier than being distributed (200 μl) into white-walled 96-well plates (Corning 3610). For co-culture experiments, the 2 cultures had been combined 1:1 instantly after back-dilution. Monoculture controls for every pressure had 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) had been annealed in 10 mM aqueous Tris-HCl buffer, and the ensuing duplexes had been added to the wells on the indicated concentrations. Plates had been shaken at 37 °C and the OD600 nm and bioluminescence readings had been obtained after 24 h. Relative gentle models (RLU) had 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 accordance with the similar situations 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 had been carried out in M9-CAS, whereas co-cultures with phage-infected S. aureus and E. faecium had been carried out in BHI as the expansion medium. To organize phage lysates, cultures had 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 had been diluted logarithmically from 100 to 10−5, and 10 µl noticed on high agar (preparation beneath) containing the related indicator. For E. albertii 07-3866 phi12, 2-fold dilutions of the supernatants as a substitute of 10-fold had been used. Within the case of quantifying S. Typhimurium phages from faecal communities, tradition supernatants had 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 had 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 had been used for PCR amplification utilizing the Luna Common qPCR package (NEB) in a CFX96 real-time PCR detection system (Bio-Rad). Knowledge had been processed and analysed by evaluating the relative amplification inside samples of phage-specific primer pairs to host-specific primer pairs (Pfaffl methodology) utilizing the Gene Expression calculator within the CFX Supervisor software program (Bio-Rad).
Preparation of high agar
The indications used to assay every phage-bacteria system had 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 had 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 had 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 had 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 check package, 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 had 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 neighborhood experiments, anaerobic monocultures of C. rodentium harbouring stx2dact had 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 situations, MMC (1.5 µg ml−1 remaining 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 (actual volumes detailed beneath) had been diluted in 200 µl of diluent buffer supplied by the producer earlier than addition to Stx-specific antibody-coated microwells. The usage of kit-provided optimistic and destructive controls in addition to all wash and substrate addition steps had been carried out precisely in accordance 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 had been taken. Absorbance at 450 nm (Abs450 nm) was measured utilizing a plate-reader. In accordance with the producer, Abs450 nm values ≥ 0.180 are thought-about a optimistic check outcome. For cardio experiments together with MMC controls, 2 µl of tradition supernatants had been used because the pattern enter. For faecal neighborhood experiments, tradition supernatants had 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) had 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 had 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 had been back-diluted (1:1,000) and combined in equal proportions in recent BHI, then incubated for an extra 24 h. Phages and toxin produced from faecal communities had 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) had been reworked into E. coli BW25113 harbouring the PR–lux reporter. After in a single day development of every pressure in monoculture, strains had 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 had been individually reworked right into a E. coli BW25113 lambda lysogen. An similar 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 supplied 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 PR–lux reporter plasmid was launched into the ensuing pressure, E. coli::clbSalbertii, and measured for its potential to be induced by colibactin utilizing the similar 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
For tradition situations and pattern preparation
In a single day cultures of E. coli BW25113 harbouring both BAC-pks or the empty BAC had 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) had been distributed 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 had 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 geared up with a dual-spray electrospray ionization (ESI) supply. Samples had been analysed utilizing an Agilent Poroshell 120 EC-C18 column (2.7 mm, 4.6 mm × 50 mm) with the next elution situations: 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; movement price = 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 vitality (CE) = 24 V) for the prodrug scaffold and m/z 368 -> m/z 114 (rt = 2.2 min, CE = 28 V) for the deuterated inner customary (d27-N-myristoyl-d-asparagine). For all samples, peak areas for the m/z 341 -> m/z 114 had been normalized to the m/z 368 -> m/z 114 transition for a similar pattern, after which normalized values in comparison with a normal curve of unlabelled N-myristoyl-d-asparagine containing 100 nM d27- N-myristoyl-d-asparagine, which was run in triplicate.
NCBI tBLASTn (nr/nt database, anticipate threshold = 0.05, phrase measurement = 6, BLOSUM62 matrix) was used to determine clbS genes that match E. coli ClbS (WP_000290498) however which might be discovered outdoors of pks clusters. The extra distantly associated ClbS-like proteins examined on this research (Fig. 3d) had been compiled from BLASTp outcomes utilizing E. coli ClbS because the question (nr protein sequences database, anticipate threshold = 0.05, phrase measurement = 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-about in figuring out bee intestine and human-associated isolates. Different members within the consultant panel chosen for cloning and heterologous expression had 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 had been submitted to PHASTER for identification of prophage areas. Genes encoded by predicted intact prophages (rating larger than 90) had 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 information 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 information; ImageJ 1.53c for colony counting in competitors experiments; and Geneious Prime 2020 for evaluation of publicly out there information and primer design. Knowledge are introduced as imply ± s.d. until in any other case indicated within the determine legends. The variety of impartial organic replicates for every experiment is indicated for every experiment and included within the legend.
Additional data on analysis design is on the market within the Nature Analysis Reporting Abstract linked to this paper.