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OmpR and RcsB abolish temporal and spatial changes in expression of flhD in Escherichia coli biofilm.

Samanta P, Clark ER, Knutson K, Horne SM, Prüß BM - BMC Microbiol. (2013)

Bottom Line: Intriguingly, rcsB expression did not correlate inversely with flhD expression, yet a mutation in rcsB abolished time dependence of flhD expression as well.This increase was paralleled by reductions in biofilm amounts at four tested time points.Our data lead to the conclusion that FlhD/FlhC and its regulation by OmpR and RcsB may be our first target mechanism for the development of novel biofilm prevention and treatment techniques.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Biofilms are communities of bacteria that are characterized by specific phenotypes, including an increased resistance towards anti-microbials and the host immune system. This calls for the development of novel biofilm prevention and treatment options to combat infectious disease. In Escherichia coli, numerous global regulators have been implicated in the control of biofilm associated cell surface organelles. These include the flagellar regulator FlhD/FlhC, the osmoregulator EnvZ/OmpR, and the colanic acid activator RcsCDB. Using flow cell technology and fluorescence microscopy, we determined the temporal expression from flhD::gfp, ompR::gfp, and rcsB::gfp in E. coli biofilm, as well as the impact of the negative regulation of flhD by OmpR and RcsB. Spatial gene expression was investigated from flhD::gfp.

Results: The temporal gene expression profile for flhD yielded an early peak at 12 h, a minimum of expression at 35 h, and a second increase in expression towards 51 h of biofilm development. In contrast, the ompR profile showed a peak at 35 h. A mutation in ompR abolished time dependence of flhD expression after the initial growth period of 12 h. Intriguingly, rcsB expression did not correlate inversely with flhD expression, yet a mutation in rcsB abolished time dependence of flhD expression as well. Spatially, expression of flhD was highest in the outermost layer of the biofilm in the parent strain. In ompR and rcsB mutants, flhD was expressed throughout the biofilm. Mutations in both, ompR and rcsB increased flhD expression throughout all temporal and spatial experiments. This increase was paralleled by reductions in biofilm amounts at four tested time points.

Conclusion: Our data lead to the conclusion that FlhD/FlhC and its regulation by OmpR and RcsB may be our first target mechanism for the development of novel biofilm prevention and treatment techniques.

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Temporal expression of flhD, ompR, rcsB in AJW678 and flhD in the ompR and rcsB mutant strains. A. Fluorescence was quantified as percent area of the images that were fluorescent, averages and standard deviations were determined. The x-axis indicates the time (hours) of biofilm formation. The y-axis indicates the total fluorescence intensity in percent area for the different strains at the different time points. The yellow, black, and blue lines are showing the gene expression profile of BP1470 (AJW678 flhD::gfp), BP1432 (AJW678 ompR::gfp), and BP1462 (AJW678 rcsB::gfp), respectively. The red line is the temporal expression profile of BP1531 (flhD::gfp ompR::Tn10), the orange line that of BP1532 (flhD::gfp rcsB::Tn5). The purple line is our housekeeping strain BP1437 which contains the aceK::gfp fusion plasmid. B. Confidence bands were calculated using the loess procedure. Upper and lower lines of each colors are indicating the highest and the lowest level of the total fluorescence intensity. The color code is identical to A.
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Figure 2: Temporal expression of flhD, ompR, rcsB in AJW678 and flhD in the ompR and rcsB mutant strains. A. Fluorescence was quantified as percent area of the images that were fluorescent, averages and standard deviations were determined. The x-axis indicates the time (hours) of biofilm formation. The y-axis indicates the total fluorescence intensity in percent area for the different strains at the different time points. The yellow, black, and blue lines are showing the gene expression profile of BP1470 (AJW678 flhD::gfp), BP1432 (AJW678 ompR::gfp), and BP1462 (AJW678 rcsB::gfp), respectively. The red line is the temporal expression profile of BP1531 (flhD::gfp ompR::Tn10), the orange line that of BP1532 (flhD::gfp rcsB::Tn5). The purple line is our housekeeping strain BP1437 which contains the aceK::gfp fusion plasmid. B. Confidence bands were calculated using the loess procedure. Upper and lower lines of each colors are indicating the highest and the lowest level of the total fluorescence intensity. The color code is identical to A.

Mentions: The images from Figure 1 were converted into quantitative data by calculating the percent area of the images that were fluorescent. The resulting expression profile for flhD showed a peak at 12 h (Figure 2A, yellow line, blue triangles). Fluorescence was lowest at 35 h and increased again towards 51 h. We also noticed a small single point peak at 3 h, which is in agreement with the occasional high fluorescence of small numbers of individual bacteria that was visualized on the images (Figure 1). Since fluorescence from the green fluorescence protein reporter is indicative of flhD expression, we conclude that flhD expression was highest at 12 h, lowest at 35 h, and increased again towards 51 h.


OmpR and RcsB abolish temporal and spatial changes in expression of flhD in Escherichia coli biofilm.

Samanta P, Clark ER, Knutson K, Horne SM, Prüß BM - BMC Microbiol. (2013)

Temporal expression of flhD, ompR, rcsB in AJW678 and flhD in the ompR and rcsB mutant strains. A. Fluorescence was quantified as percent area of the images that were fluorescent, averages and standard deviations were determined. The x-axis indicates the time (hours) of biofilm formation. The y-axis indicates the total fluorescence intensity in percent area for the different strains at the different time points. The yellow, black, and blue lines are showing the gene expression profile of BP1470 (AJW678 flhD::gfp), BP1432 (AJW678 ompR::gfp), and BP1462 (AJW678 rcsB::gfp), respectively. The red line is the temporal expression profile of BP1531 (flhD::gfp ompR::Tn10), the orange line that of BP1532 (flhD::gfp rcsB::Tn5). The purple line is our housekeeping strain BP1437 which contains the aceK::gfp fusion plasmid. B. Confidence bands were calculated using the loess procedure. Upper and lower lines of each colors are indicating the highest and the lowest level of the total fluorescence intensity. The color code is identical to A.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750693&req=5

Figure 2: Temporal expression of flhD, ompR, rcsB in AJW678 and flhD in the ompR and rcsB mutant strains. A. Fluorescence was quantified as percent area of the images that were fluorescent, averages and standard deviations were determined. The x-axis indicates the time (hours) of biofilm formation. The y-axis indicates the total fluorescence intensity in percent area for the different strains at the different time points. The yellow, black, and blue lines are showing the gene expression profile of BP1470 (AJW678 flhD::gfp), BP1432 (AJW678 ompR::gfp), and BP1462 (AJW678 rcsB::gfp), respectively. The red line is the temporal expression profile of BP1531 (flhD::gfp ompR::Tn10), the orange line that of BP1532 (flhD::gfp rcsB::Tn5). The purple line is our housekeeping strain BP1437 which contains the aceK::gfp fusion plasmid. B. Confidence bands were calculated using the loess procedure. Upper and lower lines of each colors are indicating the highest and the lowest level of the total fluorescence intensity. The color code is identical to A.
Mentions: The images from Figure 1 were converted into quantitative data by calculating the percent area of the images that were fluorescent. The resulting expression profile for flhD showed a peak at 12 h (Figure 2A, yellow line, blue triangles). Fluorescence was lowest at 35 h and increased again towards 51 h. We also noticed a small single point peak at 3 h, which is in agreement with the occasional high fluorescence of small numbers of individual bacteria that was visualized on the images (Figure 1). Since fluorescence from the green fluorescence protein reporter is indicative of flhD expression, we conclude that flhD expression was highest at 12 h, lowest at 35 h, and increased again towards 51 h.

Bottom Line: Intriguingly, rcsB expression did not correlate inversely with flhD expression, yet a mutation in rcsB abolished time dependence of flhD expression as well.This increase was paralleled by reductions in biofilm amounts at four tested time points.Our data lead to the conclusion that FlhD/FlhC and its regulation by OmpR and RcsB may be our first target mechanism for the development of novel biofilm prevention and treatment techniques.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Biofilms are communities of bacteria that are characterized by specific phenotypes, including an increased resistance towards anti-microbials and the host immune system. This calls for the development of novel biofilm prevention and treatment options to combat infectious disease. In Escherichia coli, numerous global regulators have been implicated in the control of biofilm associated cell surface organelles. These include the flagellar regulator FlhD/FlhC, the osmoregulator EnvZ/OmpR, and the colanic acid activator RcsCDB. Using flow cell technology and fluorescence microscopy, we determined the temporal expression from flhD::gfp, ompR::gfp, and rcsB::gfp in E. coli biofilm, as well as the impact of the negative regulation of flhD by OmpR and RcsB. Spatial gene expression was investigated from flhD::gfp.

Results: The temporal gene expression profile for flhD yielded an early peak at 12 h, a minimum of expression at 35 h, and a second increase in expression towards 51 h of biofilm development. In contrast, the ompR profile showed a peak at 35 h. A mutation in ompR abolished time dependence of flhD expression after the initial growth period of 12 h. Intriguingly, rcsB expression did not correlate inversely with flhD expression, yet a mutation in rcsB abolished time dependence of flhD expression as well. Spatially, expression of flhD was highest in the outermost layer of the biofilm in the parent strain. In ompR and rcsB mutants, flhD was expressed throughout the biofilm. Mutations in both, ompR and rcsB increased flhD expression throughout all temporal and spatial experiments. This increase was paralleled by reductions in biofilm amounts at four tested time points.

Conclusion: Our data lead to the conclusion that FlhD/FlhC and its regulation by OmpR and RcsB may be our first target mechanism for the development of novel biofilm prevention and treatment techniques.

Show MeSH
Related in: MedlinePlus