Article Figures & Data
Figures
- 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.
- 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.
- 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.
- 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.
- 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
- Table 1.
Bacterial strains and plasmids
Strain or plasmid Genotype, serotype, or description Reference or source E. coli strains XL1-Blue recA endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac (F′ proAB lacIq ZΔM15Tn10[Tetr]) Stratagene S17-1 thi pro hsdR hsdM+recA::RP4-2−Tc::Mu-Km::Tn7 52 Brucella strains B. melitensis 16Ma Wild type ATCC 23456 B. ovis Reo 198a Rough, CO2independent BCCN R22 1 B. melitensis 16M Nalra Spontaneous nalidixic acid-resistant mutant 60 B. melitensis B115a Rough mutant, O side chain production in the cytoplasm BCCN R19 1, 16 B. melitensisH38Ra Rough mutant, no O side chain production BCCN V3r 6, 14 B. melitensis B3B2 Rough mini-Tn5 insertion mutant This study B. melitensis DR 1 to 4 Rough double recombinants This study Plasmids pUTmini-Tn5Kmcat Suicide plasmid inBrucella spp. containing mini-Tn5Kmcat 20 pBluescript KS(−) Phagemid cloning vector, Ampr Stratagene pBluescript SK(−) oriT Mobilizable phagemid cloning vector, Ampr,oriT 59 pKSTn5R pBluescript KS derivative containing a 6.5-kb SalI chromosomal DNA fragment from the B3B2 insertion mutant; this fragment contains mini-Tn5 This study pSKoritTn5R pBluescript SK(−)oriT derivative containing a 6.5-kb SalI chromosomal DNA fragment from the B3B2 insertion mutant; this fragment contains mini-Tn5 This study pCAT19 pUC19 containing a chloramphenicol acetyltransferase-encoding cassette 28 ↵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
MAb Specificity OD490 after subtraction of blank value Whole cells Whole-cell lysates B. melitensis 16Ma B. melitensis B3B2b B. ovisReo 198c B. melitensis16Ma B. melitensisB3B2b B. ovis Reo 198c 12G12 S-LPS 1.328 0.006 0.003 1.198 0.005 0.039 2E11 S-LPS 0.953 0.002 0.000 1.387 0.001 0.042 A68/3F03/D05 R-LPS 0.124 0.212 0.281 0.443 1.197 1.117 3D6 PG 0.011 0.004 0.000 0.350 0.194 0.376 - Table 3.
Bacterial counts in mouse spleens examined 1 and 4 weeks after i.p. infection
Strain Median no. of CFU/spleen at indicated weeka 1 4 Wild-type B. melitensis 16M 6.2 × 105 (3.9 × 105– 9.5 × 105) 1.8 × 103 (4.6 × 102– 1.8 × 103) Rough B3B2 mutant 5.0 × 102 (0–3 × 103) 0 (0) ↵a Lowest and highest values are presented in parentheses.
