To construct plasmid pYA4463 (Figure 1 panel A), a XbaI-HincII fragment containing the tetA promoter and 568 bp of the 5′ end of tetA, was excised from pACYC184 and ligated into XbaI-EcoRV digested pACYC184. To generate plasmid pYA4590 (Figure 1 panel A), the 5′ end of tetA gene together with its

promoter was amplified from pACYC184 with primers P1 and P2, which contain engineered XbaI and KpnI restriction sites, respectively. The resulting PCR fragment was digested with XbaI and KpnI. The kan gene was amplified from plasmid p15A-PB2-kan, a pACYC184 derivative carrying a influenza virus PB2 gene and a kan cassette, with primers P3 and P4, which were engineered to contain KpnI and BamHI sites, respectively. The resulting PCR fragment was digested with KpnI and BamHI. The two digested PCR fragments were ligated into pACYC184

BIBW2992 manufacturer digested with XbaI and BamHI. The resulting see more plasmid, pYA4590, contains the tetA promoter and 891 bp of the 5′ end of tetA, a 1041-bp fragment encoding kan and its promoter followed by 902 bp of the 3′end of tetA. To construct plasmid pYA4464 (Figure 1 panel B), plasmid pACYC184 was digested with XbaI and EcoRV to remove the 5′ 102 bp of the tetA gene and the tetA promoter. The cohesive ends were filled using the Klenow large fragment of DNA polymerase and the linear plasmid was self-ligated to yield plasmid pYA4464. To construct plasmid pYA4465 (Figure 1 panel B), the 5′ 853 bp of tetA together with its promoter was amplified from pACYC184 using primers P5 and P6, which were engineered with SmaI and BglII sites, respectively. The resulting PCR fragment was digested with SmaI and BglII, and ligated to EcoRV and BglII digested pBAD-HisA. Creation of rec deletions The recA62 deletion, which deletes 1062 bp, encompassing the entire recA open reading frame, introduced into the bacterial chromosome using either λ Red recombinase-mediated recombination [54], or conjugation with E. coli strain χ7213(pYA4680) followed by selection/counterselection

with chloramphenicol and PLX4032 ic50 sucrose, respectively Sitaxentan [55]. The cat-sacB cassette was amplified from plasmid pYA4373 by PCR with primers P7 and P8 to add flanking sequence. The PCR product was further amplified with primer P9 and P10 to extend the flanking sequence. Those two steps of amplification resulted in the cat-sacB cassette flanked by 100 bp of recA flanking sequences at both ends. The PCR product was purified with QIAquick Gel Extraction Kit (QIAGEN) and electroporated into Salmonella strains carrying plasmid pKD46 to facilitate replacement of the recA gene with the cat-sacB cassette. Electroporants containing the cat-sacB cassette were selected on LB plates containing 12.5 μg chloramphenicol ml-1. From S. Typhimurium chromosome, a 500-bp sequence upstream recA gene was amplified with primers P11 and primer P12 and a 500-bp sequence downstream recA gene was amplified with primers P13 and P14. Primers P12 and P13 were engineered with a KpnI site.