In the present paper, we have described the development of two similar series of fluorescent reporters that can form the basis for genetic and chemical biology approaches to alteration of the level of c-di-GMP in P. aeruginosa
and potentially other bacteria as well. The reporters are based on transcriptional fusions between the c-di-GMP-responsive cdrA
promoter and gfp
. The cdrA
promoter was chosen because transcriptome analyses indicate that the level of cdrA
transcription is regulated by the level of c-di-GMP in the bacteria (9
). Without prior knowledge of the exact location of the regulatory elements within the cdrA
promoter region, the Copenhagen group of reporters was constructed by amplifying 200 bp upstream of the start codon while at the same time omitting the sequence likely containing the native RBS. With a fusion of the amplified promoter region to gfp
containing an optimized RBS, we were able to see a clear difference in the fluorescence level in the ΔwspF
compared to the wspF
wild-type background strain of P. aeruginosa
. This difference is attributed to their known difference in internal c-di-GMP levels (24
), and it was corroborated by LC-MS/MS analyses of the c-di-GMP content in the Δpel
background strains used in this study.
The Seattle group of reporters was created by amplifying a larger part of the cdrA
promoter, including the native RBS and a small part of the coding sequence, and fusing it to promoterless GFP vectors. These reporters responded positively to an increased level of c-di-GMP in the same manner as the Copenhagen reporters, yielding a clear indication that the binding site for the c-di-GMP-responsive regulatory element had been included in both groups of constructs and that they function as expected. The regulatory element was shown to be the transcriptional regulator FleQ, as a fleQ
mutant harboring a c-di-GMP reporter showed a high fluorescent output similar to that of a wspF
mutant harboring the c-di-GMP reporter. This result is consistent with previous observations linking c-di-GMP to increased cdrA
transcription via sequestration of FleQ (23
). We anticipate that the c-di-GMP level reporters may work in other bacterial species that produce FleQ homologs. It is also likely that the reporters will work in other bacterial species if they are modified so that the P. aeruginosa fleQ
gene is included in the reporter construct.
Traditionally, measurements of c-di-GMP levels have been made directly on the molecule using thin-layer chromatography or mass spectrometry on cellular extracts. These labor-intensive methods have the advantage of measuring the amount of c-di-GMP directly but are unsuitable for screening purposes. In addition, fluorescence-based measurements of c-di-GMP levels have been described recently. One study utilizes the discovery that thiazole orange fluoresces upon formation of a specific complex with c-di-GMP (38
). The method requires lysis of the bacteria but is less laborious than the standard methods. However, it is sensitive to the presence of nucleic acids which bind thiazole orange and gives rise to fluorescence. Another method employs a genetically engineered c-di-GMP binding protein fused to two fluorescent proteins at the N and C termini (11
). Binding of c-di-GMP induces a conformational change that decreases the amount of fluorescence observed due to fluorescence resonance energy transfer (FRET) between the fluorescent proteins. The method itself is ingenious, but the small amount of fluorescence usually obtained via FRET might be less than that required for sufficient sensitivity during screening. In contrast to these methods, we have created a cell-based c-di-GMP-sensitive reporter system similar to the ones employed in the discovery of quorum-sensing inhibitors in P. aeruginosa
. An analogous approach has also recently been described in E. coli
). The approach relies on increased binding of Congo red to cellulose produced by cells in response to elevated levels of c-di-GMP resulting from overexpression of the DGC AdrA. Compared to the present setup, the visual inspection of Congo red binding gives the assay a limited dynamic range less suitable for high-throughput screening where subtle differences in c-di-GMP levels are also of relevance.
The reporters in this study were constructed using both stable and unstable GFP and were made plasmid based or transposon integrated. From the epifluorescence microscope observations, it became clear that the plasmid-based reporters, as expected, showed the highest level of fluorescence and were best suited for screening purposes. In addition, testing of the plasmid-based reporter strains showed that the use of stable GFP gave adequate fluorescence levels, making the plasmid-based reporter with stable GFP the reporter of choice in the screening for compounds affecting c-di-GMP metabolism. The other reporter strains could still be employed in later steps of the process. The unstable GFP could be used to monitor the change in c-di-GMP levels during longer periods of growth in flow-cell setups investigating biofilm formation over time. Experiments using these reporters should provide information regarding spatiotemporal expression of cdrA in P. aeruginosa biofilms and would be valuable in describing the antipathogenic effects of specific compounds that decrease fluorescence from the reporter in the initial screen.
The deletion of wspF
used to increase the level of c-di-GMP causes increased polysaccharide production and therefore intense clumping of cells grown in liquid culture, making growth monitoring by means of optical density impossible. For correct measurements of growth, the background strains used here carried mutations abolishing polysaccharide production. However, the inability to produce Pel and Psl polysaccharides results in a fluorescent output from the reporters lower than what is seen with polysaccharide-producing strains harboring the reporters (data not shown). This is in accordance with previously published reverse transcription-PCR data on cdrA
expression showing the same trend (9
). It thus appears that the presence of polysaccharides somehow affects c-di-GMP metabolism in P. aeruginosa
Besides being able to distinguish the two background strains with different levels of c-di-GMP on the basis of their fluorescent output, it was crucial that the reporter showed sufficient sensitivity to gauge intermediate c-di-GMP levels stemming from changes in the metabolism caused by c-di-GMP level-altering compounds. We tested this by treatment of the c-di-GMP-overproducing reporter strain with the NO-releasing compound SNP. SNP has previously been shown to reduce c-di-GMP levels 2-fold in P. aeruginosa
at two different μM concentrations (6
). In our study, we could observe different degrees of changes in the fluorescence output over a range of concentrations, indicating that the reporter was sensitive enough to distinguish small changes in c-di-GMP levels. Compared to the untreated reporter strain, the total decrease in fluorescence was roughly 2- to 3-fold for the lower concentrations of SNP tested, which is comparable to previous observations for the cellular c-di-GMP levels (6
contains 33 GGDEF domain proteins (including domains with degenerate motifs) (33
), of which one-fourth have been found, at present, to be active DGCs (24
). This has raised the question whether the intracellular pool of c-di-GMP is compartmentalized or global and how the phenotypes related to c-di-GMP signaling are regulated. Recent research shows that the two DGCs RoeA and SadC regulate different aspects of biofilm formation (37
), indicating that c-di-GMP might be sequestered as local pools that are regulated by distinct DGCs and PDEs. This suggests that potent antipathogenic drugs targeting c-di-GMP production should be able to inhibit several DGCs. This could complicate our choice of working with a reporter background where WspR is the dominating DGC, due to the wspF
deletion, contributing to the increased level of c-di-GMP. The use of this reporter background to discover DGC inhibitors will likely result in hits that mainly affect WspR. We showed, however, that overexpression of another DGC, TpbB, caused a similar increase in the fluorescent readout from the reporters, indicating that such constructs could favorably be used to test initial drug candidates against several DGCs, determining if they display a broad activity increasing the desired antipathogenic effect.
A comparative analysis of the residues comprising the active site of the different P. aeruginosa DGCs shows a high degree of similarity indicating that they share the same conformation and thus should be able to bind the same inhibitor (unpublished analysis). We therefore believe that it will be possible to identify compounds that inhibit several DGCs in P. aeruginosa and other bacteria and thus will be suitable leads in the development of potent antipathogenic compounds. In our future work, we will use the constructed reporters in screens for antipathogenic drugs and to obtain knowledge on basic P. aeruginosa biofilm biology.