Part:BBa_K5250004

DGC PisoF_00565 R196A

A mutation of a diguanylate cyclase (DGC) PisoF_00565 gene enzyme from the metabolism of Pseudomonas species IsoF.

Usage and Biology

The wild type diguanylate cyclase (DGC) 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 adheres to surfaces and produces a matrix of polysaccharides. 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. Various DGCs contain a conserved GG(D)EF domain and within this domain there is a so-called I-site (inhibitory site). The I-site is an allosteric binding site specific for c-di-GMP. When c-di-GMP binds there, it reduces the enzyme’s activity and thereby prevents further production of c-di-GMP. C-di-GMP thus acts as a non-competitive inhibitor for the DGC. This negative feedback serves as a self-regulatory mechanism of the intracellular c-di-GMP concentration. Targeting this inhibitory site and preventing c-di-GMP from binding to the I-site may result in an increase in the enzyme’s activity.

Altering the inhibitory site and thus preventing c-di-GMP from binding to it has been successfully done in the DGC from Pseudomonas aeruginosa, WspR [1]. Nabanita De. et al. [1] discovered that c-di-GMP binds to two arginine residues (R242 and R198) within the GGDEF domain of the WspR diguanylate cyclase. They created two site-directed mutants of the DGC enzyme PP_1494 by substituting one of the arginine residues by alanine. The purified enzymes showed higher activity and no detectable c-di-GMP was bound to the I-site, indicating that the binding of c-di-GMP and therefore inhibition of the enzyme was disrupted in both mutations [1]. This strategy has never been tried on DGCs native to P. sp. IsoF. Therefore we decided to explore this approach in PisoF_00565, a DGC native to P. sp. IsoF. We created two mutants of the PisoF_00565 DGC sequence. Alpha Fold was used to identify the GGDEF domain in the sequence of PP_1494 and therefore also in PisoF_00565. We then performed our own sequence alignment using Benchling to identify the I-site within the GGDEF domain of our target DGC PisoF_00565. After identifying the binding site, we also mutated the corresponding arginine into an alanine. In one of the mutants, the arginine residue at position 196 was altered to an alanine. This is the DGC PisoF_00565 R196A.

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). ) 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. Here we will focus on the results obtained from PisoF_00565 DGC R196A

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. 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.

First, we wanted to assess whether the c-di-GMP level increases when the strains are induced with rhamnose, as our constructs are only activated when induced with rhamnose. If a change in c-di-GMP levels is observed, it would suggest that this change results from the introduced DGC. Our mutation, DGC PisoF_00565 R196A, labeled as DGC PisoF R196A in the graph, showed a significant increase in activity when induced with rhamnose compared to the condition without rhamnose. This suggests that our constructs were functioning as expected and that the introduction of a DGC increases c-di-GMP levels. (R196A: t = 6.4794, df = 7.7563, p-value = 0.0002199)

When comparing the difference in average c-di-GMP levels between the rhamnose and no rhamnose conditions, we observed a larger difference between our DGC mutations and our control, confirming that this variation is not merely due to background noise:

• No Plasmid (Rhamnose) - No Plasmid (No Rhamnose): 96.00 RFU
• DGC R196A (Rhamnose) - DGC R196A (No Rhamnose): 240.33 RFU
• DGC R240A (Rhamnose) - DGC R240A (Rhamnose): 144.533 RFU

Further we aimed to assess whether the mutation of the DGC PisoF_00565 we created indeed leads to increased activity compared to the wild-type DGC. The mutation of the DGC PisoF_00565 R196A, in which we exchanged an arginine at position 196 with alanine, showed a significantly higher c-di-GMP level than the wildtype DGC PisoF_00565 sequence (t = 2.5872, df = 8.9978, p-value = 0.02936).

Our mutated enzyme therefore shows higher activity than the wildtype enzyme. This indicates that we successfully mutated DGC PisoF_00565, specifically the enzyme’s I-site, by removing its negative regulation through c-di-GMP. So by altering the sequence of the DGC we prevented c-di-GMP from binding to the negative allosteric site, which enhanced the enzyme’s activity.

This has never been done or shown before for any DGC native to P. sp. IsoF.

Biofilm staining

Description

Since a biofilm, among other things, consists of a matrix of different polysaccharides, we decided to measure the production of polysaccharides as our second method to quantify biofilm formation. We decided to stain the polysaccharides produced by the different P. sp. IsoF strains, using a Congo Red derived dye that stains different polysaccharides and was kindly provided by our host lab. The fluorescence of the stained strains was measured under a microscope.

Experiment

We evaluated the effect of our engineered DGCs, including the P. sp. IsoF R196A mutation, and the PDE knock-down strain. All of those are expressed after a rhamnose inducible promoter (BBa_K914003) from the iGEM Parts registry. We prepared two square plates with the Congo red derived dye. 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 one set of overnight cultures of these strains with rhamnose, while the second uninduced set of cultures served as a control to test whether the rhamnose itself influences 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 2: Left: Biofilm staining assay. Right: 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. 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 mutated DGC PisoF_00565 (DGC PisoF R196) did not show the same increase, but only displayed a modest increase in fluorescence compared to the controls.

Throughout our different experiments, we observed that the engineered strains 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.

To further analyze the results, we applied a same linear model with bacterial strain as the explanatory variable (10 categories) and mean fluorescence as the response variable. As noted, the strain containing DGC PisoF R196A did not lead to a significant increase in fluorescence or polysaccharide production, despite the increase observed in the c-di-GMP assays.

Outlook

Our findings represent a novel approach, as it has to our knowledge, not been attempted before for any DGC native to P. sp. IsoF. As various DGCs contain the conserved GG(D)EF domain, our approach of c-di-GMP regulation is broadly applicable. This strategy is not only new to P. sp. IsoF, but it’s also an efficient way to enhance the activity of DGCs. By altering a single amino acid, we are potentially able to regulate biofilm formation through the upregulation of c-di-GMP.[3] This approach leverages the advantages biofilms have to offer through upregulation of c-di-GMP production. However, in environments such as hospitals biofilms pose a major challenge, contributing to persistent infections. [1] To combat this it is crucial to have a fundamental understanding of the enzyme's structure, its I-site and ultimately the negative feedback caused by c-di-GMP. For example, one could synthesize a c-di-GMP agonist with a higher affinity for the I-site to downregulate the enzyme and therefore decrease biofilm production. This could lay the groundwork for new treatments aimed at biofilm-related infections.

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

[3]Beat Christen, Matthias Christen, Ralf Paul, Franziska Schmid, Marc Folcher, Paul Jenoes, Markus Meuwly and Urs Jenal. (2006). Allosteric Control of Cyclic di-GMP Signaling. THE JOURNAL OF BIOLOGICAL CHEMISTRY, (281(42), pp. 32015-32024).