Table 1.
Strains and plasmids used in this study.
Figure 1.
Hierarchical clustering of the transcriptional response of B. bronchiseptica strain RB50 throughout biofilm development identified by comparing cDNA from planktonic cells to biofilm cells at 6, 12, 24, 36, and 48 hours of growth.
A) Expression profiles representing global transcriptional changes B) Expression profiles of annotated B. bronchiseptica transcription factors. C) Expression profiles of annotated B. bronchiseptica transcription factors maximally expressed under biofilm growth conditions at 6, 12, 24, 36, and 48 hours. Data are mean centered for each array element and averaged from three biological replicates. All expression profiles of genes are in row and are represented using the color scale at top. Yellow, indicates increased expression in biofilm cells; blue, decreased gene expression in biofilm cells; black, no significant change in gene expression.
Figure 2.
Hierarchical clustering of the transcriptional response of BvgAS-regulated genes throughout biofilm development identified by comparing cDNA from planktonic cells to biofilm cells at 6, 12, 24, 36, and 48 hours of growth.
Expression profiles representing transcriptional changes of genes regulated by BvgAS, along with 16S. Data are mean centered for each array element and averaged from three biological replicates. All expression profiles of genes are in rows and are represented using the color scale at top with gray indicating missing data. Yellow, indicates increased expression in biofilm cells; blue, decreased gene expression in biofilm cells; black, no significant change in gene expression.
Figure 3.
Hierarchical clustering of the transcriptional response of genes located within the motility locus throughout biofilm development identified by comparing cDNA from planktonic cells to biofilm cells at 6, 12, 24, 36, and 48 hours of growth.
Expression profiles representing transcriptional changes of genes located within the motility locus, along with 16S. Data are mean centered for each array element and averaged from three biological replicates. All expression profiles of genes are in row and are represented using the color scale at top. Yellow, indicates increased expression in biofilm cells; blue, decreased gene expression in biofilm cells; black, no significant change in gene expression. The flagella structural gene flaA is indicated.
Figure 4.
Differential expression of flaA during biofilm development.
Gene expression of the flagella structural gene flaA as determined by quantitative RT-PCR in attached, planktonic, or cells grown in shaking culture conditions at 2, 8, and 48 hours of growth. Gene expression standardized to rpoD is plotted along the y-axis and attached, planktonic, and shaking culture conditions are shown along the x-axis. Data shown are averages obtained from triplicate cultures. The error bars represent +/− the standard deviation. Asterisks represent a p value less than 0.005.
Figure 5.
Flagella are necessary for initial surface contact but deleterious for biofilm maturation.
Kinetics of biofilm development for the different strains was analyzed by the microtiter assay. Optical densities (OD540) of solubilized crystal violet from surface associated cells are plotted along the y-axis and time in hours, is shown along the x-axis. Data shown are averages obtained from at least 6 wells each time from 2 independent experiments. The error bars represent +/− the standard error. Asterisks represent a p value less than 0.05.
Figure 6.
Flagella are deleterious for biofilm maturation.
Kinetics of biofilm development for different strains was analyzed by the microtiter assay. Optical densities (OD540) are plotted along the y-axis and time in hours, is shown along the x-axis. Data shown are averages obtained from at least 6 wells each time from 2 independent experiments. The error bars represent +/− the standard error. All differences are statistically significant (p<0.05, student’s t test), except among the strains at 1 h time point and between the strains Rev1Δfla and WT at 48 and 72 hours.
Figure 7.
Scanning electron microscopy (SEM) of biofilms formed at the air-liquid interface.
Different strains were grown on glass cover slips for 2 hours (left panels) and 72 hours (right panels) followed by processing for SEM as described in the Materials and Methods. The scale bar represents 10 µm.
Figure 8.
Confocal scanning laser micrographs (CSLM) of B. bronchiseptica biofilm development.
CSLM of biofilms formed at the air-liquid interface of different strains grown on glass coverslips after 6 hours (left panels), 48 hours (middle panels), and 72 hours (right panels). The cells were tagged with GFP and thus are green. For each micrograph, the middle panel represents the x–y plane, and the adjacent top and side panels represent the x–z and y–z planes, respectively.