Tural integrity on the T6SSSPI-6 gene cluster cloned onto R995 was verified by tilingPCR analysis so as to amplify ten fragments that cover the whole T6SS area (Figure S1). For competitive infections in chickens, the in vivo stability of plasmids R995 and R995+SPI-Materials and Solutions Bacteria and Development ConditionsThe bacterial strains applied in this perform are listed in Table 1. Bacteria have been routinely cultivated in LB broth (10 g/l tryptone, 5 g/l yeast extract, five g/l NaCl) at 37uC with aeration or on LB plates (15 g/l agar) supplemented with the appropriate antibiotic at the following concentrations: Ampicillin (Amp), 100 mg/ml; Kanamycin (Kan), 50 mg/ml; Chloramphenicol (Cam), 20 mg/ml; Trimethoprim (Tm), one hundred mg/ml; Spectinomycin (Sp), 250 mg/ml.DNA MethodsDNA manipulations have been performed utilizing standard protocols. Plasmid DNA was isolated from overnight cultures applying the QIAprep Spin Miniprep Kit (QIAGEN), as outlined by the makers directions. Genomic DNA was isolated from overnight cultures using the GenElute Bacterial Genomic DNA kit (Sigma) in line with the suppliers guidelines. PCR items had been purified working with the QIAquick PCR Purification Kit (QIAGEN). XbaI restriction enzyme (Fermentas) and T4 DNA ligase (New England Biolabs) had been used as per manufacturer guidelines. DNA samples had been routinely analyzed by electrophoresis in 1 agarose gels (1X Tris-acetate-EDTA buffer) and visualized below UV light following ethidium bromide staining.PLOS One | plosone.orgSPI-6 in Salmonella Infection in ChickensTable 1. Strains and plasmids utilised in this study.StrainsFeaturesSource of referenceEscherichia coliDH5a EC100D pir-116 EC100D pir-116/R995+SPI-6 EC100D pir-116/R995+SPI-19 DH5a/R995 DH5a/R995-VC6 F-W80lacZDM15D(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rk-, mk+) phoA supE44 thi-1 gyrA96 relA1 lF-mcrAD(mrr-hsdRMS-mcrBC)W 80dlacZDM15 DlacX74 recA1 endA1 araD139 D(ara, leu)7697 galU galK l- rpsL (StrR) nupG pir-116(DHFR) Strain with T6SSSPI-6 from S.1889290-53-2 In stock Typhimurium cloned in plasmid R995 Strain with T6SSSPI-19 from S. Gallinarum cloned in plasmid R995 Strain harboring an empty R995 vector Strain containing a derivative of plasmid R995 with a 1,209 bp DNA fragment of T6SSSPI-6 cloned from S. Typhimurium Laboratory collection Laboratory collection This study [42] This study This studySalmonella Typhimurium14028 s MTM753 MTM35 MTM2640 WT/R995 MTM35R MTM35R6 MTM35R19 Plasmids pKD46 pEKA30 pCLF2 pCLF4 pVEX1212 pVEX2212 R995 R995-VC6 R995+SPI-6 R995+SPI-19 doi:ten.1371/journal.pone.0063917.t001 bla PBAD bet exo pSC101 oriTs, AmpR IncQ plasmid that constitutively express Cre recombinase, AmpR Red-swap redesigned vector, CamR Red-swap redesigned vector, KanR Suicide vector harboring a loxP web site followed by a SpR cassette Suicide vector harboring a loxP internet site followed by a CamR cassette Self-transmissible broad-host range IncP vector A derivative of plasmid R995 with a 1,209 bp DNA fragment of T6SSSPI-6 cloned from S.Buy5-Azidopentan-1-amine Typhimurium T6SSSPI-6 cluster from S.PMID:23558135 Typhimurium 14028 s cloned in vector R995 T6SSSPI-19 cluster from S. Gallinarum 287/91 cloned in vector R995 [43] [44] [50] [50] [44] [44] [44] This study This study [42] Wild-type strain 14028 s DphoN 14028 s DSPI-6 T6SS 14028 s DclpV 14028 s containing an empty R995 vector MTM35 harboring R995 plasmid MTM35 complemented with plasmid R995+SPI-6 MTM35 complemented with plasmid R995+SPI-19 Laboratory collection This study This study This study This study This study This study This stu.
