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MOLECULAR AND CELLULAR PATHOGENESIS

Identification of the Perosamine Synthetase Gene ofBrucella melitensis 16M and Involvement of Lipopolysaccharide O Side Chain in BrucellaSurvival in Mice and in Macrophages

Fabrice Godfroid, Bernard Taminiau, Isabelle Danese, Philippe Denoel, Anne Tibor, Vincent Weynants, Axel Cloeckaert, Jacques Godfroid, Jean-Jacques Letesson
Fabrice Godfroid
Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d’Immunologie et de Microbiologie, Facultés Universitaires Notre Dame de la Paix, Namur, and
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Bernard Taminiau
Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d’Immunologie et de Microbiologie, Facultés Universitaires Notre Dame de la Paix, Namur, and
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Isabelle Danese
Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d’Immunologie et de Microbiologie, Facultés Universitaires Notre Dame de la Paix, Namur, and
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Philippe Denoel
Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d’Immunologie et de Microbiologie, Facultés Universitaires Notre Dame de la Paix, Namur, and
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Anne Tibor
Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d’Immunologie et de Microbiologie, Facultés Universitaires Notre Dame de la Paix, Namur, and
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Vincent Weynants
Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d’Immunologie et de Microbiologie, Facultés Universitaires Notre Dame de la Paix, Namur, and
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Axel Cloeckaert
Institut National de la Recherche Agronomique, Centre de Recherche de Tours, Nouzilly, France
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Jacques Godfroid
Centre d’Étude et de Recherche Vétérinaire et Agrochimique (CERVA), Brussels, Belgium, and
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Jean-Jacques Letesson
Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d’Immunologie et de Microbiologie, Facultés Universitaires Notre Dame de la Paix, Namur, and
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DOI: 10.1128/IAI.66.11.5485-5493.1998
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  • Fig. 1.
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    Fig. 1.

    Immunoblot analysis of Brucella whole-cell lysates probed with MAbs 12G12 and 2E11 (directed againstBrucella S-LPS) (a) and MAb A68/3F03/D05 (directed against R-LPS of Brucella) (b). Lanes: A, B. melitensis 16M (parental strain); B, B. melitensisB115; C, B. melitensis H38 rough mutant; D,B. melitensis B3B2 mutant.

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    Fig. 2.

    (a) pKSTn5R and primers used in this study. (b) Gene replacement strategy used to create B. melitensis 16M DR identical to the B3B2 insertion mutant. The construction of pKSTn5R and pSKoritTn5R is described in the text. Open regions, B. melitensischromosomal DNA; light-grey regions, SalI chromosomal DNA containing the mini-Tn5 in the B3B2 mutant; solid regions, kanamycin resistance gene and cat reporter gene of the mini-Tn5.

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    Fig. 3.

    The nucleotide sequence and the deduced amino acid sequence of the B. melitensis 16M perosamine synthetase gene. The putative ribosome binding site (RBS) is underlined. The asterisk denotes the termination codon. The arrow indicates the site of the mini-Tn5 insertion in strain B3B2.

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    Fig. 4.

    Simultaneous multiple alignment of perosamine synthetase amino acid sequences from B. melitensis 16M (1),V. cholerae O1 (2), and E. coli O157:H7 (3). The matching regions for the three sequences are outlined by boxes. Amino acids are numbered above the sequence. Shaded boxes indicate identities. Lowercase letters indicate unaligned residues.

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    Fig. 5.

    Growth of B. melitensis 16M (parental strain) and the rough insertion mutant B3B2 in bovine macrophages. The data presented are means ± standard deviations of quintuplicate plate counts and are representative of two experiments. p.i., postinfection.

Tables

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  • Table 1.

    Bacterial strains and plasmids

    Strain or plasmidGenotype, serotype, or descriptionReference or source
    E. coli strains
     XL1-BluerecA endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac (F′ proAB lacIq ZΔM15Tn10[Tetr])Stratagene
     S17-1thi pro hsdR hsdM+recA::RP4-2−Tc::Mu-Km::Tn752
    Brucella strains
     B. melitensis 16MaWild typeATCC 23456
     B. ovis Reo 198aRough, CO2independentBCCN R22 1
     B. melitensis 16M NalraSpontaneous nalidixic acid-resistant mutant60
     B. melitensis B115aRough mutant, O side chain production in the cytoplasmBCCN R19 1, 16
     B. melitensisH38RaRough mutant, no O side chain productionBCCN V3r 6, 14
     B. melitensis B3B2Rough mini-Tn5 insertion mutantThis study
     B. melitensis DR 1 to 4Rough double recombinantsThis study
    Plasmids
     pUTmini-Tn5KmcatSuicide plasmid inBrucella spp. containing mini-Tn5Kmcat20
     pBluescript KS(−)Phagemid cloning vector, AmprStratagene
     pBluescript SK(−) oriTMobilizable phagemid cloning vector, Ampr,oriT59
     pKSTn5RpBluescript KS derivative containing a 6.5-kb SalI chromosomal DNA fragment from the B3B2 insertion mutant; this fragment contains mini-Tn5This study
     pSKoritTn5RpBluescript SK(−)oriT derivative containing a 6.5-kb SalI chromosomal DNA fragment from the B3B2 insertion mutant; this fragment contains mini-Tn5This study
     pCAT19pUC19 containing a chloramphenicol acetyltransferase-encoding cassette28
    • ↵a This strain was received from J.-M. Verger (Laboratoire de Pathologie Infectieuse et d’Immunologie, Institut National de Recherche Agronomique, Nouzilly, France).

  • Table 2.

    ELISA binding of antibodies to whole cells and whole-cell lysates of three Brucella strains

    MAbSpecificityOD490 after subtraction of blank value
    Whole cellsWhole-cell lysates
    B. melitensis 16MaB. melitensis B3B2bB. ovisReo 198cB. melitensis16MaB. melitensisB3B2bB. ovis Reo 198c
    12G12S-LPS1.3280.0060.0031.1980.0050.039
    2E11S-LPS0.9530.0020.0001.3870.0010.042
    A68/3F03/D05R-LPS0.1240.2120.2810.4431.1971.117
    3D6PG0.0110.0040.0000.3500.1940.376
    • ↵a Parental strain.

    • ↵b Insertion mutant.

    • ↵c Rough strain.

  • Table 3.

    Bacterial counts in mouse spleens examined 1 and 4 weeks after i.p. infection

    StrainMedian no. of CFU/spleen at indicated weeka
    14
    Wild-type B. melitensis 16M6.2 × 105 (3.9 × 105– 9.5 × 105)1.8 × 103 (4.6 × 102– 1.8 × 103)
    Rough B3B2 mutant5.0 × 102 (0–3 × 103)0 (0)
    • ↵a Lowest and highest values are presented in parentheses.

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Identification of the Perosamine Synthetase Gene ofBrucella melitensis 16M and Involvement of Lipopolysaccharide O Side Chain in BrucellaSurvival in Mice and in Macrophages
Fabrice Godfroid, Bernard Taminiau, Isabelle Danese, Philippe Denoel, Anne Tibor, Vincent Weynants, Axel Cloeckaert, Jacques Godfroid, Jean-Jacques Letesson
Infection and Immunity Nov 1998, 66 (11) 5485-5493; DOI: 10.1128/IAI.66.11.5485-5493.1998

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Identification of the Perosamine Synthetase Gene ofBrucella melitensis 16M and Involvement of Lipopolysaccharide O Side Chain in BrucellaSurvival in Mice and in Macrophages
Fabrice Godfroid, Bernard Taminiau, Isabelle Danese, Philippe Denoel, Anne Tibor, Vincent Weynants, Axel Cloeckaert, Jacques Godfroid, Jean-Jacques Letesson
Infection and Immunity Nov 1998, 66 (11) 5485-5493; DOI: 10.1128/IAI.66.11.5485-5493.1998
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KEYWORDS

Brucella melitensis
Carbohydrate Epimerases
Lipopolysaccharides
Macrophages, Peritoneal
Transaminases

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