Part:BBa_K5250006

DGC PisoF_00565

The wild type diguanylate cyclase (DGC) PisoF_00565 enzyme from the metabolism of Pseudomonas species IsoF produces the second messenger cyclic di-GMP which is involved in biofilm formation. A biofilm is a community of bacteria that adhere to surfaces and produce a matrix of polysaccharides. [1] These are influenced by c-di-GMP. We decided to elevate the intracellular c-di-GMP levels of Pseudomonas species IsoF through the introduction of a DGC [2].

This has been done before from Hailing Nie et. al. in Pseudomonas putida KT2440. The DGC PP_1494 was overexpressed and showed the highest increase in biofilm formation among other DGC enzymes in the experiment [2]. Pseudomonas putida KT2440 is a model organism with a high degree of similarity to P. sp. IsoF, and commonly used by our host lab. Therefore, we assume that also a DGC native to P. sp. isoF (PisoF_00565) that is homologous to PP_1494 will affect the biofilm production in a similar way.

Characterization

In our project we compared the activity of five different DGCs, two wild type DGCs (PisoF_00565 and WspR) with three mutations (PisoF_00565 R196A, PisoF_00565 R240A, DGC WspR R242A). To test our DGC overexpression module, we applied a c-di-GMP assay and a biofilm staining experiment in which we evaluated the five different DGCs and one PDE knock-down strain.

Cyclic di-GMP assay

Experiment

For the c-di-GMP assay we decided to use the Kit from Lucerna Technologies which is based on a fluorescence signal generated by c-di-GMP. The c-di-GMP sensor used in this kit consists of a c-di-GMP riboswitch and a Spinach aptamer. When c-di-GMP binds to the riboswitch, it stabilizes the Spinach aptamer. This allows the fluorophore DFHBI-1T from the kit to bind and emit a fluorescent signal. The fluorescence is then measured using a fluorescence plate reader.

Results

Figure 1: Fluorescence intensity of the strains tested in the c-di-GMP assay.

Figure 1. above shows the results from the first two c-di-GMP assays put together in one graph. The first six strains are our PDE and DGC constructs that we created with the intention of enhancing biofilm production. We included three negative controls - wild type P. sp. IsoF, P. sp. IsoF with the pBBR1MCS5 backbone, and a strain that overexpresses a PDE plasmid. We also included a positive control, P. sp. IsoF DGC YedQ - a very potent DGC that causes a drastic increase in the biofilm production. All of these controls were also provided by our host lab. Despite these strains being known to work, and therefore used as controls, they exhibited unexpected c-di-GMP values. After analyzing our controls, we decided to handle our results with caution.

P. sp. IsoF containing a DGC PisoF_00565 showed a visible increase in c-di-GMP levels, however the difference was not significant. This indicates that different DGC enzymes can lead to different increases in intracellular c-di-GMP levels, but that in general the intracellular c-di-GMP concentration is elevated by introducing a DGC.

Biofilm staining

Experiment

Since our goal is to enhance biofilm formation, we found a method to quantify the biofilm components produced by Pseudomonas species isoF strains. Although biofilms consist of various components, in our assay we focused specifically on staining the polysaccharides in the biofilm as a way to quantify biofilm production. We used Congo red-derived dye, kindly provided by our host lab, and measured the fluorescence of our stained strains under a microscope.

We evaluated the effect of all of our engineered DGCs and the PDE knock-down strain. All of those are expressed after the rhamnose inducible promoter (BBa_K914003) from the iGEM Parts registry. We prepared two square plates, stained them with the dye and pipetted 10uL of each strain we wanted to test on them. 40% rhamnose was added in only one of the plates. As negative controls we took a wild type P. sp. IsoF and a P. sp. IsoF strain with an empty pBBR1MCS5 plasmid, and PisoF DGC YedQ as a positive control.

We induced with 40% rhamnose one set of overnight cultures of these strains, while the second uninduced set of cultures served as a control to test whether the rhamnose itself influenced intracellular c-di-GMP concentration. We adjusted the cultures to an optical density of 1 and plated each adjusted strain onto the respective induced or uninduced plate. After a 3-day incubation period, the fluorescence of the stained polysaccharides of each strain was measured using a fluorescence microscope. The images were processed and analyzed using imageJ. Two repeats were made for each strain and condition (rhamnose or no rhamnose).

Results

Figure 1: Fluorescence data of the biofilm staining assay. The figure illustrates the mean fluorescence for each strain.

As expected, the negative control strains showed the lowest fluorescence and the YedQ positive control showed the highest fluorescence. Among the other tested strains, three showed a significant increase in fluorescence: P. sp. isoF DGC PisoF_00565 wild type, P. sp. isoF DGC WspR and P. sp. DGC WspR R242A.

The strain expressing the mutated WspR DGC (P. sp. WspR R242A) showed the highest fluorescence level, even exceeding the positive control (P. sp. isoF YedQ). The wild type DGC PisoF_00565 also shows very high fluorescence, contradicting the above mentioned results from the c-di-GMP assay. The detected fluorescence in the wild type DGC PisoF_00565 biofilm is statistically significant. This means that this strain leads to a significant increase in the production of polysaccharides and therefore biofilm.

Throughout our different experiments we observed that the strains carrying different DGCs had reduced growth when induced with rhamnose. We have two hypotheses of why this could be the case. Firstly, it could be that rhamnose is toxic to bacteria in high concentrations. Alternatively, the overexpression of a DGC leading to the overproduction of c-di-GMP might be costly for the bacteria and thus inhibits their growth. Additionally, during the biofilm staining assay, we were unable to measure the OD of each strain, meaning that we don’t know how well each strain grew on the plate. As a result, we could not normalize the polysaccharide production for the strains’ OD, which might have affected the results.

References

[1] De, N., Pirruccello, M., Krasteva, P. V., Bae, N., Raghavan, R. V., & Sondermann, H. (2008). Phosphorylation-Independent Regulation of the Diguanylate Cyclase WSPR. PLoS Biology, 6(3), e67. https://doi.org/10.1371/journal.pbio.0060067

[2] Nie, H., Xiao, Y., He, J., Liu, H., Nie, L., Chen, W., & Huang, Q. (2019). Phenotypic–genotypic analysis of GGDEF/EAL/HD‐GYP domain‐encoding genes in Pseudomonas putida. Environmental Microbiology Reports, 12(1), 38–48. https://doi.org/10.1111/1758-2229.12808