PMC

Genetic organization of the vanT DNA locus. Arrows indicate the direction of transcription.

Genetic organization of the cloned DNA fragments fused to the E. coli lacZ gene carried on pDM5-S5 and pDM5-S13. The horizontal arrows indicate the putative promoters and direction of transcription. The vertical arrow indicates the site of the plasmid insertion within the sat gene that created the polar mutation in the sat-vps73 DNA locus (strain DM70).

Quantification of bacteria in biofilms formed on PVC plastic by the wild-type (Wt) NB10, the vanT mutant, and the mutant carrying a polar mutation in the sat-vps73 DNA locus. Biofilms were allowed to form in a 96-well PVC microtiter dish. The microtiter dish was incubated for 6 h, 8 h, 10 h, 12 h, and 14 h at 24°C. The unattached bacteria were removed, and the biofilms were stained with crystal violet. The crystal violet was solubilized, and the absorbance, which correlated to the biofilm mass, was determined.

β-Galactosidase activity of vps73::lacZ and serA::lacZ transcriptional gene fusions. (A) vps73::lacZ was carried on pDM8-Vps73 and (C) serA::lacZ was carried on pDM8-SerA in the wild type (Wt) and vanT mutant. Overnight cultures were diluted into fresh TSB medium to an OD₆₀₀ of 0.05 and then incubated with shaking at 24°C. Samples were taken at various times and analyzed for growth (OD₆₀₀, open symbols) and β-galactosidase expression (Miller units, solid symbols). Miller units for the vector control were between 100 and 200 U. (B and D) Northern analysis. Total RNA was isolated from 30-h cultures of the wild type and vanT mutant grown at 24°C and hybridized to a DNA fragment complementary to either the vps73 gene (B) or the serA gene (D).

Measurement of pigment production and promoter activity of the hpdA gene. (A) For pigment production, overnight cultures of the wild type (Wt) and the vanT mutant containing pDM5-S13, which carries the hpdA gene and its promoter, were diluted to an OD₆₀₀ of 0.05 into TSB medium with (solid symbols) and without 5 mM l-tyrosine (open symbols) and incubated with shaking at 24°C. Samples were taken from the culture at various times, and the supernatant was filter sterilized to remove bacteria. Pigment production in the supernatant was estimated by measuring the absorbance at 400 nm. (B) For hpdA promoter activity, an hpdA-lacZ transcriptional gene fusion was made and contained on pDM8-HpdA in the wild type and the vanT mutant. Overnight cultures were diluted into fresh TSB medium to an OD₆₀₀ of 0.05 and then incubated with shaking at 24°C. Samples were taken at various times and analyzed for growth (OD₆₀₀, open symbols) and β-galactosidase expression (Miller units, solid symbols). Miller units for the vector control were between 100 and 200 U. (C) Northern analysis. Total RNA was isolated from 30-h cultures of the wild type and vanT mutant grown at 24°C and hybridized to a DNA fragment complementary to the hpdA gene.

Biofilm attachment to a glass surface for the wild type (wt), the vanT mutant, and the mutant carrying a polar mutation in the sat-vps73 DNA locus. (A) Typical view of the progression of biofilm formation for each strain at 4 h, 8 h, 12 h, and 16 h. Slides were stained with acridine orange and then viewed with a fluorescent microscope. Bar, 20 μm. (B) Biofilm formation was quantified by determining the percent area coverage on a glass slide. For each strain, five images were taken at each time point and saved as a computer image. These images were converted into black (background) and white (biofilm) pixels using the Image Tool Software, version 2, and the percentage of white pixels is given as the percentage of biofilm covering the glass surface.

Analyses of metalloprotease activity in the wild type (Wt) and the vanT mutant. (A) Growth curve and expression of empA::lacZ transcriptional gene fusion. Overnight cultures were diluted to an OD_{600 nm} of 0.05 and then incubated with shaking at 24°C. Samples were taken at various times and analyzed for growth (OD₆₀₀, open symbols) and β-galactosidase expression (Miller units, solid symbols). Miller units for the vector control were between 100 and 200 U. (B) Protease activity. Overnight cultures were diluted to an OD₆₀₀ of 0.05 and then incubated with shaking at 24°C. Samples were taken at various times and analyzed for growth (open symbols) and for azocasein degradation (OD₄₄₂, solid symbols). For protease activity, 100 μl of filtered-sterilized culture supernatant was mixed with 100 μl of azocasein solution and incubated at 30°C for 2 h. The reaction was stopped by the addition of trichloroacetic acid, the unreacted azocasein was removed by centrifugation, and the absorbance at 442 nm was determined. (C) Northern analysis. Total RNA was isolated from 30-h cultures of the wild type and vanT mutant grown at 24°C and hybridized to a DNA fragment complementary to the empA gene.