jejuni dba-dsbI genes, was used as a template for PCR-mediated mutagenesis. Point mutations M1R and L29stop (replacing a Leu codon with amber stop codon) were introduced using the respective pairs of primers: Cj18M1R – Cj18M1Rc and Cj18L29 – Cj18L29c. The resulting plasmids were introduced into E. coli cells by transformation and presence of desired mutations was verified by DNA sequencing. DNA fragments containing the C. jejuni dba-dsbI operon (with or without a point mutation) were then digested and inserted into the pRY107 shuttle vector. The resulting plasmids were named pUWM769

(containing wt dba-dsbI), pUWM811 (dba: M1R, wt dsbI) and pUWM812 (dba: L29stop, wt dsbI). These plasmids were subsequently introduced into C. jejuni 81-176 AL1 (dsbI::cat) and C. jejuni 81-176 AG6 (Δdba-dsbI::cat) knock-out cells by conjugation [28]. Construction of bacterial Thiazovivin datasheet mutant strains To inactivate dba and dsbI genes, three recombinant plasmids were constructed, based on pBluescript II KS (Stratagene) and pGEM-T Easy (Promega) vectors, which

are BAY 80-6946 suicide plasmids in C. jejuni Anlotinib manufacturer cells. A. van Vliet kindly furnished the fourth suicide plasmid, pAV80, which was previously used for C. jejuni NCTC11168 fur inactivation [25]. Correct construction of all the plasmids was confirmed by restriction analysis and sequencing. The plasmid for C. jejuni dba mutagenesis was generated by PCR-amplification of two C. jejuni 81-176 DNA fragments (600 bp and 580 bp long) that contained dba gene fragments with their adjacent regions GNAT2 with primer pairs: Cj19LX-2 – Cj18RM and Cj18LM – Cj17RM. Next they were cloned in native orientation in pBluescript II KS (Statagene). Using BamHI restrictase, the kanamycin resistance cassette (the 1.4 kb aphA-3 gene excised from pBF14) was inserted between the cloned dba arms in the same transcriptional orientation, generating the suicide plasmid pUWM622. To obtain the construct for C. jejuni dsbI mutagenesis the 1.5 kb DNA fragment containing the dsbI gene was PCR-amplified

from the C. jejuni 81-176 chromosome using primer pair: Cj17LSal – Cj17RBgl and was cloned into pGEM-T Easy (Promega). Subsequently, the internal 300 bp EcoRV-EcoRV region of dsbI was replaced by a SmaI-digested chloramphenicol resistance cassette (the 0.8 kb cat gene excised from pRY109) [27] inserted in the same transcriptional orientation as the dsbI gene, generating the suicide plasmid pUWM713. To obtain the construct for C. jejuni dba-dsbI mutagenesis, the 410 bp and 380 bp DNA fragments, containing dba upstream and dsbI downstream regions were PCR-amplified from the C. jejuni 81-176 chromosome using primer pairs: Cj19LX-2 – Cjj46mwR and Cjj43mwL – Cjj43Eco. These fragments were directly digested with BamHI restrictase, ligated in a native orientation and used as a template for a subsequent PCR reaction with the external primer pair: Cj19LX-2 – Cjj43Eco.