Restriction enzymes

were purchased from Fermentas, and primers were purchased from Sigma-Aldrich. DNA fragments were amplified by PCR from B. abortus 2308 genomic DNA extracted as previously described [26]. High-fidelity PCR was performed using Vent polymerase (New England Biolabs), and standard PCR was performed using Taq (Qiagen). PCR products were purified using BIIB057 mw GenElute™ PCR Clean-Up (Sigma). Amplified products were cloned in pGEM®-T Easy (Promega) or pJET1.2 (Fermentas) depending on the polymerase used. The DNA sequence of the final plasmids was determined to rule out mutations introduced by PCR. Gateway cloning was made according to the manufacturer instructions (Invitrogen). The oligonucleotides Selleck A1155463 used are listed in Table 1. Construction of an aphT resistance cassette Plasmid pFJS235 carrying the aminoglycoside 3′-phosphotransferase gene (which encodes for kanamycin resistance) devoid of its transcription terminator (aphT) was constructed as follows. Primer aphT.F, derived from pUC4K [27] and located 5′ from

the aph gene, and primer aphT.R, derived from the aph sequence [28], were used to amplify a 1,005 bp DNA fragment from plasmid pUC4K. The amplified fragment was digested with PstI and cloned into pUC4K/PstI, yielding plasmid

pFJS235. The aphT gene can be retrieved from Sclareol pFJS235 by using PstI, HincII, SalI, or EcoRI. Construction of mutants and complementation plasmids To construct a polar ΔureT mutant (ΔureTp) from B. abortus strain 2308, ureT was replaced by aph. DNA fragments both upstream and downstream of ureT were amplified with the following set of primers: U_BMEI0642_XbaI.F and U_BMEI0642_BamHI.R were used to amplify a region of 578 bp upstream of ureT (U_ureT) and D_BMEI0642_BglII.F and D_BMEI0642_PstI.R were used to amplify a region of 589 downstream of ureT (D_ureT). PCR fragments of the expected size were ALK inhibitor gel-purified and cloned into pGEM®-T Easy resulting in plasmids pFJS225 and pFJS226 respectively. pFJS225 was linearized with BamHI and pFJS226 with BglII, and ligated to a 1.2 kb BamHI fragment from pUC4K, containing aph with its transcription terminator. An XbaI &PstI fragment of 1.4 kb was obtained directly from the partially digested ligation mixture, and cloned into pDS132 digested with PstI and partially with XbaI, to obtain pFJS227b, that was used to construct the corresponding ΔureTp mutants in Brucella, as described below. For the construction of a non-polar ΔureT mutant from B.