Best New Basic Part Nomination

We modified a diguanylate cyclase (DGC) native to Pseudomonas species IsoF to stop c-di-GMP from binding to the negative allosteric binding site which effectively enhanced the enzyme’s activity.

DGCs which produce c-di-GMP contain a conserved GG(D)EF domain which contains a so-called I-site (Inhibitory site), the allosteric inhibitory site where c-di-GMP binds.1 By disrupting this binding site, the negative feedback of the enzyme can be inhibited, allowing continuous c-di-GMP production and therefore increasing its intracellular concentration. Nabanita De. et al. 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 enzyme 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.2

Based on their findings we wanted to apply the same mechanism to increase c-di-GMP even further. We did this by performing a sequence alignment of the WspR to our DGC of choice PisoF_00565 to locate the GGDEF domain where we planned on introducing our mutation. This way we could identify the appropriate arginine residues to target and modify them to alanine residues (R196A and R240A).

Sequence Alignment of WspR wt and WspR mutation R198A to our DGC IsoF wt and DGC IsoF R196A

Figure 1: Sequence Alignment of WspR wt and WspR mutation R198A to our DGC IsoF wt and DGC IsoF R196A.
Created with BioRender.com

Experimental Success

Our following experimental findings indicated that the R196A mutation showed a higher enzyme activity than the R240A. Therefore we chose the R196A as our nominee for best basic part. For more information please visit our Engineering and Results page.

Fluorescence Intensity from c-di-GMP Assay across different Strains.
Fluorescence Intensity from c-di-GMP Assay across different Strains.

Figure 2,3: Fluorescence Intensity from c-di-GMP Assay across different Strains.

To test whether our DGC mutant produces a higher level of c-di-GMP compared to the wildtype enzyme, as well as other DGCs, we performed c-di-GMP assays (see figure 2).

Our mutation, DGC PisoF_00565 R196A labelled 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 construct is functioning as expected and that the introduction of our DGC increases c-di-GMP levels. (R196A: t = 6.4794, df = 7.7563, p-value = 0.0002199).

Further we aimed to assess whether the mutation of the DGC PisoF we created indeed leads to increased activity compared to the wild-type DGC. The R196A mutation of the DGC PisoF showed a significantly higher c-di-GMP level than the wildtype DGC PisoF 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 or rather 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.

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 mutation and our control, confirming that this variation is not merely background noise:

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

This represents 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, this makes our approach of c-di-GMP regulation broadly applicable.

Outlook

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 has implications for leveraging the advantages biofilms have to offer by upregulation of c-di-GMP production, as we did in our project. However, in environments such as hospitals biofilms pose a major challenge, contributing to persistent infections.4 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] Eike H. Junkermeier and Regine Hengge. (2023). Local signaling enhances output specificity of bacterial c-di-GMP signaling networks. microLife, (4, 1-11)

[2] Nabanita De, Michelle Pirruccello, Petya Violinoca Krasteva, Narae Bae, Rahul Veera Raghavan, Holger Sondermann. (2008). Phosphorylation-Independent Regulation of the Diguanylate Cyclase WspR. PLoS Biology, (6(3):e67).

[3] Soyoung Park and Karin Sauer. (2022). Controlling biofilm development through cyclic di-GMP signaling. Adv Exp Med Biol., (1386, 69-94).

[4] Nunez, C., Kostoulias, X., Peleg, A. Y., Short, F. & Qu, Y. (2023). A comprehensive comparison of biofilm formation and capsule production for bacterial survival on hospital surfaces. Biofilm, 5, 100105.