Objectives

To investigate why GFP expression was high in the absence of signal in our engineered DSF system.

DSF-Induced Phosphorylation of RpfG Mediates GFP Expression

We constructed models similar to those used in the PQS system to investigate the signal-response behaviour of the DSF system. Our results verified what we had expected to happen. Phosphorylation of RpfG is induced by DSF binding to a cell receptor. RpfG[P] then degrades c-di-GMP which relieves the inhibition of Clp. Clp then activated the expression of GFP through the engineered manA promoter. In the absence of a DSF signal, RpfG remained in its unphosphorylated form and GFP expression was repressed.

DSF-Independent Activation of the manA Promoter

Our experimental results revealed that in the absence of DSF, the manA promoter was active in our engineered system and hence GFP expression upregulated.

Like the BDSF pathway, the DSF system contains a phosphorelay system. Our first hypothesis, therefore was that the expression of GFP was dependent on signal-independent phosphorylation of RpfG.

However, as shown in Fig 1 the model predicted that increasing the level of phosphorylated RpfG would have no significant effect on the production of GFP in our engineered cells.

Our second hypothesis was that enhanced inhibition of Clp by elevated levels of c-di-GMP could repress GFP expression. However, on setting c-di-GMP levels in the model to a high level, GFP was still expressed (albeit at a lower low level), Fig 2. This can be explained by Clp having a higher binding affinity for the promoter than c-di-GMP. Since both these reactions are reversible, there will still be sufficient Clp free in the cytoplasm to activate GFP expression.

The model predictions so far still did not explain the high expression levels reported by the wet team. Thus bringing us to our third hypothesis - our cells were over-expressing Clp.

An analysis of our model for the DSF system revealed that the steady state levels of free Clp and c-di-GMP are only dependent on their (constitutive) rate of production and degradation (and not dependent on the Clp - c-di-GMP binding affinities nor the promoter binding affinity). As shown in Fig 3 we see that the model predict that an over-expression of Clp results in a corresponding high level of GFP production.

Conclusion

Our models were used to test different plausible hypotheses for the DFS-signal independent expression of GFP in our engineered cells. We conclude that over-expression of Clp could be responsible for the experimental observations.