BIOFILM
The observation that Stewart's wilt disease development depends on the cell density-regulated expression of the EPS virulence factor suggested that the esaI, AHL-deficient mutant strain, ESN51, is locked into a low-cell density mode and the esaR mutant strains ES∆R and ES∆IR into a high cell density mode with respect of EPS synthesis. We postulated that the premature, constitutive synthesis of EPS must interference with one or several key steps of sessile bacterial community or biofilm development (1-4).
Adhesion: In an in vitro adhesion assay, the wild type strain, DC283, grown in microtiter dish wells, adheres at low but significant levels to the plastic surface (35 OD570units), while the QS/EPS repressed strain, ESN51, adheres with high efficiency (320 OD570units), as are mutants of the cps locus. For example, strain DM220, which is EPS deficient due to an insertion in a key EPS biosynthetic enzyme, I highly adherent (430 OD570units). Strain DM223 is an EPS biosynthetic mutant with an intermediate EPS and adhesion phenotype (110 OD570units). Most significantly, the hypermucoid strain ES∆IR is virtually adhesion deficient (4 OD570units). We conclude from these data that EPS synthesis interferes with P. stewartii bacterial adhesion (5).
In vitro biofilms examined by confocal fluorescence microscopy: GFP-tagged wild type and mutant strains were grown on glass cover slips and analyzed by confocal microscopy.
Panel
A shows the wild type strain beginning to colonize a site in the xylem. The
bacteria attach at relatively few sites on the xylem wall and form towering
structures that appear to grow toward the center of the lumen where they
coalesce. These structures continue to develop into a heavily matrix-encased
biofilm (Panel B). Panel C shows the AHL-deficient mutant strain
ESN51, which is greatly limited in its ability to spread within the xylem.
The mutant bacteria cover the xylem wall and form compact biofilms lacking
an EPS matrix. Panel D shows a heavily matrix-encased amorphous
and detached biofilm produced by the hypermucoid mutant ES∆IR,
which generally collapses during cryo treatment for EM studies (5).