Article Figures & Data
Figures
- Fig. 1.
Genes present in the SR-11 Fad− 4.5-kbPstI DNA fragment that contains the chloramphenicol resistance gene (cam). Heavy lines below the genes denote regions in the fragment that were sequenced. Sequencing reactions were performed with the BigDye Terminator Cycle Sequencing Kit, and reactions were analyzed on the ABI PRISM 310 (PE Applied Biosystems, Forest City, Calif.). Nucleotide sequences were analyzed using Clone Manager and Align Plus 3 programs (Sci-Ed Software, Durham, N.C.). Arrows above the genes denote the direction of transcription. Arrows below the genes denote the positions of the forward and reverse primers used for PCR amplification of the wild-type and defectivecra genes. P, PstI.
- Fig. 2.
PCR amplification of the defective cra genes of serovar Typhimurium SR-11 Fad−, SR-11 Fad−AX-2, and UK-1 Fad− AX-1. Genomic DNA preparations were performed by standard techniques (13) or by following the manufacturer's instructions. Genomic DNA preparations of SR-11, SR-11 Fad−, SR-11 Fad− AX-2, UK-1, and UK-1 AX-1 were amplified by PCR using 5′-ATCGACTGCAGTGCGAAATCCGTGGTAACCCGG-3′ as the forward primer and 5′-TAGCTCTGCAGCCTGTTTAACGTGTGCGGTGCC-3′ as the reverse primer. PCR reaction mixtures contained 2.0 mM magnesium chloride and 5% dimethyl sulfoxide and were subjected to 25 cycles of 94°C for 30 s, followed by 65°C for 30 s, followed by 72°C for 2.5 min. Lane 1, kilobase ladder; lane 2, SR-11 Fad− AX-2; lane 3, SR-11 Fad−; lane 4, SR-11; lane 5, UK-1 Fad− AX-1; lane 6, UK-1; lane 7, no DNA added; lane 8, kilobase ladder.
Tables
- Table 1.
Bacterial strains and plasmids
Strain or plasmid Relevant genotype and/or phenotype Source or reference S. enterica serovar Typhimurium strains (plasmid) SR-11 gyr1816 4 SR-11 Fad− gyr1816 cra::Tn10 d cam 21 SR-11 Fad− AX-2 gyr1816 cra::Tn10 d cam This study SR-11 Fad−(pBR322) gyr1816 cra::Tn10 d cam, bla tet This study SR-11 Fad−(pJHA8) gyr1816 cra::Tn10 d cam, cra+tet This study LJ2443 fruR51::Tn10 3 UK-1 Virulent avian strain 5 UK-1 Fad− AX-1 cra::Tn10 d cam This study E. coli S-17 λpir Host for pLD55 and pMJN10 V. de Lorenzo Plasmids pBR322 bla tet 2 pJHA1 1.4-kb camgene from Tn10::d cam inBamHI site of pBR322 This study pJHA7 4.3-kbPstI fragment containingcra::Tn10 d cam in pBR322, cam tet This study pJHA8 1.4-kbcra + gene in PstI site of pBR322,tet This study pLD55 Suicide vector for allelic exchange, bla tetAR 15 pMJN10 4.3-kb PstI fragment containingcra::Tn10 d cam in pLD55, cat bla tetAR This study
