Two more pieces of the colibactin genotoxin puzzle from Escherichia coli show incorporation of an unusual 1-aminocyclopropanecarboxylic acid moiety

Biosynthetic pathway intermediates related to genotoxin colibactin formation: a linear compound 3 bearing a rare 7-methyl-4-azaspiro[2.4]hept-6-en-5-one residue.


Gene inactivation by replacement of an antibiotic resistance marker in the chromosome of E. coli Nissle 1917 using Red/ET recombineering
The target genes were replaced by an antibiotic selection marker (chloramphenicol resistance gene Cm R , kanamycin resistance gene Km R , or spectinomycin resistance gene Spect R ) using Red/ET recombineering.
The detailed procedure of Red/ET recombineering is provided in previous publications. [1][2][3][4] The antibiotic resistance genes flanked with homology arms (~50 bp) were generated by polymerase chain reaction (PCR) amplification using Phusion high-fidelity polymerases (Thermo-Scientific) according to the manufacture's manual, and the templates for Cm R , Km R and Spect R were plasmids Pirate-cm-new (GeneBridges), Pirate-km (GeneBridges), and pR6K-Spect-BAC (GeneBridges), respectively. For Red/ET recombineering, purified PCR products of resistance genes flanked by different homology arms, were transformed into E. coli Nissle 1917 containing the recombinase expression plasmid pSC101-BAD-gbaA-tet by electroporation, respectively. 1;5 Recombinants were selected on LB plates containing 20 µg/mL chloramphenicol, 20 µg/mL kanamycin, or 40 µg/mL spectinomycin, respectively. To generate the double mutants, the antibiotic resistance gene was used to replace the target gene by Red/ET recombineering in E. coli Nissle 1917 ΔclbP containing pSC101-BAD-gbaA-tet. Correct clones were verified by colony PCR (Table S1). The recombinase expression plasmid pSC101-BAD-gbaA-tet was removed by culture temperature shift to 42 °C. A list of mutants generated in this study is provided as Table S1. Oligonucleotides used for gene deletions are listed in Table S2. An exemplarily example of a diagram for gene inactivation (clbH) by replacement of an antibiotic resistance marker in the chromosome of E. coli Nissle 1917 using Red/ET recombineering is shown in Fig. S1.

General methods for cultivation and culture extraction for LC-MS analysis
The E. coli strains Nissle 1917 wildtype and verified mutants were incubated in 25 mL Luria broth in 100 mL glass flasks amended with suitable antibiotics at 30℃ (200 rpm) for 2 days. The culture was extracted by 25 mL EtOAc. Twenty mL organic phase was evaporated to dryness. The resulting residue was dissolved in 200 µL of methanol for LC-MS analysis. Standard analysis of crude extracts was performed on a Dionex Ultimate 3000 LC system using a Waters Acquity BEH C-18, 50 x 2 mm, 1.7 μm dp column.
Separation of a 2 μL sample was achieved by a linear gradient with (A) H 2 O + 0.1 % formic acid (FA) to (B) acetonitrile (ACN) + 0.1 % FA at a flow rate of 600 μL/min and at 45 °C. The gradient was initiated by a 0.5 min isocratic step at 5 % B, followed by an increase to 95 % B in 9 min to end up with a 1.5 min step at 95 % B before re-equilibration employing initial conditions. UV spectra were recorded by a DAD The gradient was initiated by a 1 min isocratic step at 5 % B, followed by an increase to 95 % B in 6 min to end up with a 1.5 min step at 95 % B before reequilibration with initial conditions. UV spectra were recorded by a DAD in the range from 200 to 600 nm. The LC flow was split to 75 µL/min before entering the maXis4Ghr-ToF mass spectrometer (BrukerDaltonics, Bremen, Germany) using the standard ESI source. Mass spectra were acquired in centroid mode ranging from 150 -2000 m/z at 2 Hz scan speed.

LC-MS Analysis of L/D-FDLA Derivatives
Approximately 0.25 mg of compound was hydrolyzed with 6 N HCl (0. of FA, using a gradient from 5-95% of ACN over 9 min.