PMC

Observation of the clumping phenotype (left panels) and phase-contrast images of B. melitensis (right panels) in exponential growth phase. (A) B. melitensis 16M; (B) strain CD100/pSB203 harboring vjbR(D82A).

Effect of polymixin B on the viability of B. melitensis 16M, CD100, CD100/pSB202, and CD100/pSB203. The data for the wt and CD100 strains are superimposed on the graph. The graph is representative of three distinct experiments.

Scanning electron micrographs of suspension cultures of Brucella strains. (A) wt B. melitensis strain 16M; (B and C) clumps formed by strain CD100; (D) clumps formed by strain CD100 harboring pSB203; (E) B. abortus 2308; (F) strain RMD100 harboring pSB203.

Identification of EPS(s) within aggregates formed by Brucella vjbR mutant strains. Phase-contrast images are shown in the left panels, and calcofluor white staining is shown in the right panels. (A) wt strain; (B) CD100 strain containing the vjbR(D82A) expression plasmid pSB203. Calcofluor white staining of the CD100/pSB202 strain gave similar results.

Interactions between FITC-labeled ConA lectin and aggregates formed by Brucella vjbR mutant visualized by confocal laser scanning microscopy. Culture samples of (A) B. melitensis strains 16M and (B) CD100 containing the vjbR(D82A) expression plasmid pSB203 were stained with ConA-FITC (green) and propidium iodide (red).

Omp31 is implicated in the clumping phenotype and EPS production or export in B. melitensis. (A) Clumping phenotypes of the wt strain, vjbR-defective strain CD100, and vjbR/omp31-defective strain SB200 in 96-well 2YT medium cultures. (B) ELLSA with ConA-peroxidase and supernatants of shaken stationary-phase cultures of the wt, CD100, SB200, and SB200/pSB305 strains. Peroxidase activity is represented by the OD₄₅₀-OD₆₅₀ value. Significant differences in relation to the wt strain are indicated by an asterisk (P < 0.05).

QS system of B. melitensis affects the production of several Omps. (A) Dot blot analysis of Omp amounts in the B. melitensis 16M wt strain cultivated with or without exogenous C₁₂-HSL. (B) Dot blot analysis of Omp amounts in the CD100 strain cultivated with or without exogenous C₁₂-HSL. (C) Densitometric quantification of Omps for dot blots in panels A and B. (D) Dot blot analysis of Omp amounts in the B. melitensis 16M wt strain and the CD100 strain complemented with pSB202. Cells were grown in 2YT medium and harvested in stationary phase. Whole-cell extracts were diluted as described in Materials and Methods, and dilutions were subjected to dot blot analysis using different Omp MAbs. ACN, negative control with the solvent (ACN) used for C₁₂-HSL dilution. The data are representative of three experiments.

Schematic representation of the VjbR mutated polypeptides. The pSB201-encoded wt VjbR polypeptide is shown at the top; the proposed autoinducer (AI) binding region is indicated by a solid bar, and the DNA binding region is indicated by a cross-hatched bar. Mutations in the VjbR polypeptide are indicated in the middle. The D82A substitution is indicated by an arrowhead, and conserved regions of deletants are represented. The relative levels of luciferase activity are indicated on the right. The values are expressed as percentages of the PvirB activity in the B. melitensis CD110 strain containing the pSB201 plasmid (top) grown without C₁₂-HSL. The average PvirB activity in the vjbR mutant was 40%. The values are the means of at least three experiments (the variation coefficients were between 1 and 8%).