Mutant construction and cloning The Δ chuT, see more Δ iroD, and Δ iucD mutants were generated in APEC E058 and UPEC U17

by allelic exchange. To enhance the numbers of recombinants, E058 and U17 were initially electroporated with pKD46 to express Red recombinase [50]. The genes were PCR amplified as described below and cloned into pMD18-T simple vector according to manufacturer’s instructions. The antibiotic resistance cassette was then inserted into the target gene. Each of the resultant constructs was then introduced into E058 or U17 by electroporation. All mutants were confirmed by PCR and verified by sequence analysis. The Δ chuT mutants, E058Δ chuT and U17Δ chuT, were Tozasertib cost constructed as follows: the chuT gene was amplified by PCR using the primers 5′-CTCGGATCCAGGATCATCACCAGGCCGTT-3′ and 5′-CTCAAGCTTTCAACGGTGATAATGCGCTG-3′. The products were cloned into pMD18-T simple vector to form pMD-chuT. To insert the kanamycin cassette into chuT, reverse PCR was adopted. The reverse PCR product was amplified from pMD-chuT using the primers 5′-CTCGAATTCGGTAATTACGCTATCCGG-3′ and 5′-CTCGAATTCCGTTACAGGTTCCTGAAC-3′. The kanamycin cassette was then introduced into

the chuT genes at the EcoRI site. The Δ iroD E058 and U17 mutants were constructed by amplifying and cloning the fragment into pMD18-T simple vector using the primers 5′-CTCGGATCCACCATGCGTAATCGTGAC-3′

and 5′-CTCAAGCTTTACTGACTGACTTCTGGCGCGA-3′. The cam cassette was introduced into this website the iroD genes at the internal EcoRV site. The aerobactin synthesis (iucD) mutants, E058Δ iucD and U17Δ iucD, were constructed by amplifying and cloning the iucD gene using the primers 5′- TCAGTCGACTCAGCATTGCTGCGTTGT-3′ and 5′-CGCGAATTCTACGT GCAGATCTCCATG −3′. The reverse PCR products were amplified from pMD-iucD using the primers 5′-GACGATATCTCATATGCTTCACACAGG-3′ Farnesyltransferase and 5′-CCTGCATG CCTGGAGGAAGATATTCGC−3′. The zeo cassette was introduced into the iucD genes at the EcoRV and SphI sites. To construct the triple knockout mutant, the Δ iroD Δ iucD double mutant was initially constructed by electroporating the disrupted iroD genes into the E058Δ iucD and U17Δ iucD competent cells. The disrupted chuT gene was then electroporated into the E058Δ iroD Δ iucD and U17Δ iroD Δ iucD double mutant competent cells to form triple mutants E058Δ chuT Δ iroD Δ iucD and U17Δ chuT Δ iroD Δ iucD. Complementation of the triple mutants using native iroD For complementation analysis, the native iroD gene was amplified using primers 5′-CTCGGATCCATGCTGAACATGCAACAA −3′and 5′-CTCGAATTCTCAACCCTGTAGTAAACC-3′ from E058 and U17. To determine whether the sequences were in-frame, the pGEM®-T Easy vector with the iroD insert was sequenced by Sangon Co. (Shanghai, China).