Taken together, these results show that B. melitensis exopolysaccharide is a new mannose-rich polymeric structure. Besides exopolysaccharide, extracellular matrices often contain DNA, which may contribute to the structural integrity of biofilms (Whitchurch et al., 2002; Steinberger & Holden, 2005). To test whether Brucella’s clumps include DNA, culture samples were incubated in
the presence of DNAseI and the enzyme effect was observed under a microscope. Two hours after DNAseI incubation (Fig. 5b), clumps appeared to be digested by the nuclease while culture samples incubated with the enzyme buffer did not (Fig. 5a). This effect was increased after 24 h of incubation (Fig. 5c). Brucella melitensis wild-type strain or bearing a control vector (MG200 strain), used as negative aggregation controls, showed no effect of DNAseI treatment. These results Metformin research buy demonstrate that DNA is a component of the
extracellular matrix of B. melitensis aggregates Proteasome inhibitor review and contributes significantly to their structure. Because a recent study showed that OMVs are classical components of biofilm matrices (Schooling & Beveridge, 2006), we wondered whether our MG210 clumping strain could overproduce OMVs. We tested this hypothesis using transmission electron microscopy (TEM). We analyzed the abundance of OMVs’ structure in culture samples from MG210 and the wild-type strain collected in the stationary growth phase. Compared with the wild-type strain, we observed that the production of OMV-like structures was strongly increased in the clumping strain (Fig. 6a and b). Moreover, we took a set of minimum 20 TEM pictures for each strain on which we counted both the number of OMVs-like structures and the amount of bacteria to obtain quantitative data. Counting was performed in triplicate for each strain. As shown in Fig. 6c, we counted a mean of 73 OMVs per 100 bacteria in the
aggregative strain, but only four OMVs per 100 bacteria in the wild-type strain. These data indicate that OMVs could be a component of the matrix of the clumps formed by B. melitensis as described for other biofilm matrices. To confirm this hypothesis, we compared Amino acid the abundance of two major OMPs of the OMVs formed by Brucella (Omp25 and Omp31) (Gamazo & Moriyon, 1987; Boigegrain et al., 2004) in B. melitensis wild-type and MG210 strains by dot-blot analysis using specific MAbs (Cloeckaert et al., 1990). Omp16 (PAL lipoprotein) was used as an internal loading control. Dot blotting was carried out with B. melitensis culture supernatants (containing the OMVs fraction) (Fig. 7) from stationary-phase cultures. OD600 nm were used to normalize all samples. As shown in Fig. 7, the abundance of both tested OMPs of B. melitensis’ OMVs is strongly increased in MG210 supernatants compared with the control strain. Omp16 presented almost the same relative abundance in the two strains tested.
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