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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, Pr├╝├č 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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Spatial gene expression of flhD in the parent strain. (A) and (B) are the 3D images constructed from the z-stacked images (bright field and fluorescence) at 12 hours (A) and 51 hours (B), using BP1470 (AJW678 pPS71). (C) is the quantitative representation of the spatial gene expression of flhD at 12 hours (dashed yellow line) and 51 hours (solid yellow line) of biofilm formation. The purple line is the spatial expression profile from the aceK::gfp fusion at 34 h.
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Figure 3: Spatial gene expression of flhD in the parent strain. (A) and (B) are the 3D images constructed from the z-stacked images (bright field and fluorescence) at 12 hours (A) and 51 hours (B), using BP1470 (AJW678 pPS71). (C) is the quantitative representation of the spatial gene expression of flhD at 12 hours (dashed yellow line) and 51 hours (solid yellow line) of biofilm formation. The purple line is the spatial expression profile from the aceK::gfp fusion at 34 h.

Mentions: From the temporal gene expression experiment, we knew that the highest expression of flhD was at 12 h and 51 h of biofilm formation. As a consequence, we performed the spatial gene expression experiment for flhD at those two time points. In both the 12 h (Figure 3A) and 51 h (Figure 3B) biofilms, the expression of flhD was highest at the outer layer of the biofilm. Fluorescence calculated from the individual images of the z-stacks showed that at 12 h, there was little or no expression of flhD within the first 2 μm from the surface that the biofilm had formed on (dotted yellow lines). Expression increased rapidly at 2 μm to approximately 50% coverage. In 51 h biofilms, there were three distinct intensity levels (solid yellow lines). Until 3 μm, the expression of flhD was very low; at 3.5 μm, the expression jumped to 50% and maintained this level until 6 μm; across the upper 2 μm of our biofilm, flhD expression increased to approximately 75% of the total area of the images. Our housekeeping gene in comparison was highly expressed all throughout the biofilm (purple lines).


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

Samanta P, Clark ER, Knutson K, Horne SM, Pr├╝├č BM - BMC Microbiol. (2013)

Spatial gene expression of flhD in the parent strain. (A) and (B) are the 3D images constructed from the z-stacked images (bright field and fluorescence) at 12 hours (A) and 51 hours (B), using BP1470 (AJW678 pPS71). (C) is the quantitative representation of the spatial gene expression of flhD at 12 hours (dashed yellow line) and 51 hours (solid yellow line) of biofilm formation. The purple line is the spatial expression profile from the aceK::gfp fusion at 34 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Spatial gene expression of flhD in the parent strain. (A) and (B) are the 3D images constructed from the z-stacked images (bright field and fluorescence) at 12 hours (A) and 51 hours (B), using BP1470 (AJW678 pPS71). (C) is the quantitative representation of the spatial gene expression of flhD at 12 hours (dashed yellow line) and 51 hours (solid yellow line) of biofilm formation. The purple line is the spatial expression profile from the aceK::gfp fusion at 34 h.
Mentions: From the temporal gene expression experiment, we knew that the highest expression of flhD was at 12 h and 51 h of biofilm formation. As a consequence, we performed the spatial gene expression experiment for flhD at those two time points. In both the 12 h (Figure 3A) and 51 h (Figure 3B) biofilms, the expression of flhD was highest at the outer layer of the biofilm. Fluorescence calculated from the individual images of the z-stacks showed that at 12 h, there was little or no expression of flhD within the first 2 μm from the surface that the biofilm had formed on (dotted yellow lines). Expression increased rapidly at 2 μm to approximately 50% coverage. In 51 h biofilms, there were three distinct intensity levels (solid yellow lines). Until 3 μm, the expression of flhD was very low; at 3.5 μm, the expression jumped to 50% and maintained this level until 6 μm; across the upper 2 μm of our biofilm, flhD expression increased to approximately 75% of the total area of the images. Our housekeeping gene in comparison was highly expressed all throughout the biofilm (purple lines).

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