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MOLECULAR AND CELLULAR PATHOGENESIS

A Functional cra Gene Is Required forSalmonella enterica Serovar Typhimurium Virulence in BALB/c Mice

James H. Allen, Maryjane Utley, Han van den Bosch, Piet Nuijten, Maarten Witvliet, Beth A. McCormick, Karen A. Krogfelt, Tine Rask Licht, Derek Brown, Michael Mauel, Mary P. Leatham, David C. Laux, Paul S. Cohen
James H. Allen
Department of Biochemistry, Microbiology, and Molecular Genetics, University of Rhode Island, Kingston, Rhode Island 02881;
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Maryjane Utley
Department of Biochemistry, Microbiology, and Molecular Genetics, University of Rhode Island, Kingston, Rhode Island 02881;
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Han van den Bosch
Bacteriological and Immunological Research and Development, Intervet International, 5830 AA Boxmeer, The Netherlands;
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Piet Nuijten
Bacteriological and Immunological Research and Development, Intervet International, 5830 AA Boxmeer, The Netherlands;
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Maarten Witvliet
Bacteriological and Immunological Research and Development, Intervet International, 5830 AA Boxmeer, The Netherlands;
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Beth A. McCormick
Combined Program in Pediatric Gastroenterology and Nutrition, Harvard Medical School, Massachusetts General Hospital, Charlestown, Massachusetts 02120;
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Karen A. Krogfelt
Department of Gastrointestinal Infections, Statens Seruminstitut, 2300 Copenhagen, and
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Tine Rask Licht
Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark; and
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Derek Brown
Scottish Salmonella Reference Laboratory, Stobhill NHS Trust, Glasgow G21 3UW, Scotland
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Michael Mauel
Department of Biochemistry, Microbiology, and Molecular Genetics, University of Rhode Island, Kingston, Rhode Island 02881;
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Mary P. Leatham
Department of Biochemistry, Microbiology, and Molecular Genetics, University of Rhode Island, Kingston, Rhode Island 02881;
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David C. Laux
Department of Biochemistry, Microbiology, and Molecular Genetics, University of Rhode Island, Kingston, Rhode Island 02881;
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Paul S. Cohen
Department of Biochemistry, Microbiology, and Molecular Genetics, University of Rhode Island, Kingston, Rhode Island 02881;
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DOI: 10.1128/IAI.68.6.3772-3775.2000
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    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.

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    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.

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  • Table 1.

    Bacterial strains and plasmids

    Strain or plasmidRelevant genotype and/or phenotypeSource or reference
    S. enterica serovar Typhimurium strains (plasmid)
     SR-11gyr18164
     SR-11 Fad−gyr1816 cra::Tn10 d cam21
     SR-11 Fad− AX-2gyr1816 cra::Tn10 d camThis study
     SR-11 Fad−(pBR322)gyr1816 cra::Tn10 d cam, bla tetThis study
     SR-11 Fad−(pJHA8)gyr1816 cra::Tn10 d cam, cra+tetThis study
     LJ2443fruR51::Tn103
     UK-1Virulent avian strain5
     UK-1 Fad− AX-1cra::Tn10 d camThis study
    E. coli S-17 λpirHost for pLD55 and pMJN10V. de Lorenzo
    Plasmids
     pBR322bla tet2
     pJHA11.4-kb camgene from Tn10::d cam inBamHI site of pBR322This study
     pJHA74.3-kbPstI fragment containingcra::Tn10 d cam in pBR322, cam tetThis study
     pJHA81.4-kbcra+ gene in PstI site of pBR322,tetThis study
     pLD55Suicide vector for allelic exchange, bla tetAR15
     pMJN104.3-kb PstI fragment containingcra::Tn10 d cam in pLD55, cat bla tetARThis study
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A Functional cra Gene Is Required forSalmonella enterica Serovar Typhimurium Virulence in BALB/c Mice
James H. Allen, Maryjane Utley, Han van den Bosch, Piet Nuijten, Maarten Witvliet, Beth A. McCormick, Karen A. Krogfelt, Tine Rask Licht, Derek Brown, Michael Mauel, Mary P. Leatham, David C. Laux, Paul S. Cohen
Infection and Immunity Jun 2000, 68 (6) 3772-3775; DOI: 10.1128/IAI.68.6.3772-3775.2000

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A Functional cra Gene Is Required forSalmonella enterica Serovar Typhimurium Virulence in BALB/c Mice
James H. Allen, Maryjane Utley, Han van den Bosch, Piet Nuijten, Maarten Witvliet, Beth A. McCormick, Karen A. Krogfelt, Tine Rask Licht, Derek Brown, Michael Mauel, Mary P. Leatham, David C. Laux, Paul S. Cohen
Infection and Immunity Jun 2000, 68 (6) 3772-3775; DOI: 10.1128/IAI.68.6.3772-3775.2000
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KEYWORDS

Bacterial Proteins
Genes, Bacterial
Repressor Proteins
Salmonella Typhimurium

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