Real Time, Spatial, and Temporal Mapping of the Distribution of c-di-GMP during Biofilm Development

Abstract

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a dynamic intracellular signaling molecule that plays a central role in the biofilm life cycle. Current methodologies for the quantification of c-di-GMP are typically based on chemical extraction, representing end point measurements. Chemical methodologies also fail to take into consideration the physiological heterogeneity of the biofilm and thus represent an average c-di-GMP concentration across the entire biofilm. To address these problems, a ratiometric, image-based quantification method has been developed based on expression of the green fluorescence protein (GFP) under the control of the c-di-GMP-responsive cdrA promoter (Rybtke, M. T., Borlee, B. R., Murakami, K., Irie, Y., Hentzer, M., Nielsen, T. E., Givskov, M., Parsek, M. R., and Tolker-Nielsen, T. (2012) Appl. Environ. Microbiol. 78, 5060-5069). The methodology uses the cyan fluorescent protein (CFP) as a biomass indicator and the GFP as a c-di-GMP reporter. Thus, the CFP/GFP ratio gives the effective c-di-GMP per biomass. A binary mask was applied to alleviate background fluorescence, and fluorescence was calibrated against known c-di-GMP concentrations. Using flow cells for biofilm formation, c-di-GMP showed a non-uniform distribution across the biofilm, with concentrated hot spots of c-di-GMP. Additionally, c-di-GMP was found to be localized at the outer boundary of mature colonies in contrast to a uniform distribution in early stage, small colonies. These data demonstrate the application of a method for the in situ, real time quantification of c-di-GMP and show that the amount of this biofilm-regulating second messenger was dynamic with time and colony size, reflecting the extent of biofilm heterogeneity in real time.

Keywords: Pseudomonas aeruginosa (P. aeruginosa); bacteria; biofilm development; bioreporter; cyclic di-GMP (c-di-GMP); development; image based quantification; nitric oxide.

FIGURE 1.

Comparison of different cdrA reporter constructs in P. aeruginosa. Overnight grown, planktonic cultures of P. aeruginosa PAO1 with different versions of the cdrA::gfp reporter construct were compared for fluorescence intensity. Reporter activity was determined based on analysis of confocal images, where the mean intensity was calculated as the total image intensity per cell. Error bars, S.D. (n = 15). a.u., arbitrary units.

FIGURE 2.

Chromatic aberration analysis. A 6-μm bead expressing cyan and green colors was imaged using the same imaging parameters as ratiometric imaging. A, the fluorescent signals in the xy plane showed complete overlap between cyan and blue. B, the chromatic aberration in the yz dimension was significant, showing non-overlapping cyan and green images. C, corrected chromatic aberration in the yz plane, showing overlapping cyan and green images. D, ratio image created with the non-corrected bead, showing intensity changes as a 16-color LUT. E, ratio image of the corrected bead, showing identical ratios across the entire bead. The white color represents a ratio of 1 in the 16-color LUT.

FIGURE 3.

Comparison of c-di-GMP reporter-based fluorescence of high c-di-GMP-producing mutants versus wild type P. aeruginosa. The fluorescence intensity per cell is shown for the wild type P. aeruginosa, P. aeruginosa ΔwspF and P. aeruginosa ΔwspFΔpslBCDΔpelA. Error bars represent the standard deviation (n = 3). Magnification: 20×. Scale bar: 10 μm.

FIGURE 4.

Effect of c-di-GMP-modulating compounds on the expression of the PcdrA::gfp (ASV) in P. aeruginosa. Bacteria were grown in 24-well plates in a Tecan plate reader with continuous shaking at 37 °C. Optical density (OD₆₀₀) (A) and fluorescence intensity (B) were measured every 10 min, and fluorescence intensity was normalized with the optical density and is presented as RFU. ●, P. aeruginosa ΔwspFΔpslBCDΔpelA P_cdrA::gfp (ASV), 125 μm SNP; ■, P. aeruginosa ΔwspFΔpslBCDΔpelA P_cdrA::gfp (ASV), 250 μm SNP; ▴, P. aeruginosa P_cdrA::gfp (ASV); ▾, P. aeruginosa P_cdrA::gfp (ASV), 20 μg/ml tellurite; ♦, P. aeruginosa P_cdrA::gfp (ASV), 50 μg/ml tellurite. Shown are representative data from three biological replicates. Error bars, S.D. (n = 3).

FIGURE 5.

Measurement of CFP and GFP signal separately on a P. aeruginosa wild type biofilm undergoing starvation. A–C, before the start of carbon starvation; D–F, after 30 min of starvation; G, CFP gray values before and after treatment; H, GFP gray values before and after treatment; I, ratio of GFP/CFP before and after the induction of starvation.

FIGURE 6.

c-di-GMP reporter PcdrA::gfp (ASV) calibration. Shown are ratiometric images of planktonic P. aeruginosa P_cdrA::gfp (ASV) (A), a biofilm of P. aeruginosa P_cdrA::gfp (ASV) (B), a planktonic culture of P. aeruginosa ΔwspF P_cdrA::gfp (ASV) (C), and a biofilm of P. aeruginosa ΔwspF P_cdrA::gfp (ASV) (D). E, a comparison of image quantification versus chemical quantification. F, c-di-GMP concentration plotted versus the average weighted pixel frequency. R² = 0.90827, x intercept = 0.07099, y intercept = 0.00527, and slope = −0.70655. Magnification, ×20. The color scale represents the c-di-GMP concentration normalized to protein concentration in a 16-color LUT, where white represents ∼75 pm/μg protein and black represents 0.38 pm/μg protein. Planktonic cultures were grown at room temperature in M9-glucose medium in a shake flask overnight, and 2 ml of culture were centrifuged in Corning® Costar® cell culture plates (24-well, flat bottom) at 811 × g for 10 min. Confocal images were captured after adjusting the correction ring in the ×20 lens for the thickness of the plate.

FIGURE 7.

c-di-GMP distribution across a developing P. aeruginosa biofilm in a flow cell. A, day 2; B, day 3; C, day 4; D, day 5; E, histogram of biofilm development on days 2, 3, 4, and 5. F, average weighted pixel intensity as determined by the ratiometric method. The color scale represents the c-di-GMP concentration normalized to protein concentration, where white represents ∼75 pm/μg protein, and black represents 0.38 pm/μg protein. Shown are representative data from three biological replicates. Error bars, S.D. (n = 3).