Fungal Sensing

The pathogenic fungus Fusarium oxysporum cubense (foc) will secrete fusaric acid, a toxin needed to invade the banana plant via the roots. Pseudomonas putida, which serves us as a platform for the development of our biological control agent, naturally is a root colonizing bacteria and will therefore be found in the rhizosphere. Here the engineered Pseudomonas putida will be able to sense fusaric acid using a fusaric acid induced promoter located near an endogenous fusaric acid pump. Resistance to fusaric acid is crucial in this process and Pseudomonas putida has been shown to already possess a good base level of resistance. To be able to transfer our system to different platforms, we aim to introduce fusaric acid resistance in Escherichia coli.

Resistance

Different gene cluster known to be related to fusaric acid resistance will be isolated from multiple different organisms. FusABCDE from Burkholderia cepacia [1], FDT-123 from Klebsiella oxytoca [2], FuaABC from Stenotrophomonas maltophilia [3] and PP1263-5 from Pseudomonas putida itself. These clusters will then be expressed under an IPTG inducible promoter (BBa_J04500) or a ramose inducible promoter (BBa_K914003) in order to test the resistance levels. Since all of the clusters contain multiple illegal PstI sites, it was decided to insert the cluster directly in the SpeI site of the biobrick suffix.
PP1263-5 from Pseudomonas putida could be isolated from the wild type strain used at our own facilities. The original strains referred to in the research papers for the other three organisms were not available. Therefore homologues genes, with high levels of sequence identity (>98%) were identified and obtained from microbial collections.

Fusaric Acid Sensing

A fusaric acid efflux pump within Pseudomonas putida is encoded by an operon consisting of four genes. This operon is controlled by a LysR-type gene (pp1262) which is located upstream of the operon [4, 5]. This gene inhibits the binding of RNA polymerase to the promoter in the intergenic region between pp1262 and the operon. Fusaric acid will block this inhibition allowing activity of the operon (Figure 1). Hence, pp1262 and the intergenic region are isolated and put into BioBrick form, effectively together acting as a Fusaric Acid inducible Promoter (FAiP).

In all available sources it is mentioned that fusaric acid resistance by this cluster is provisional. So we decided to test and characterize the fusaric acid sensitivity and expression by different levels of fusaric acid. The promoter will is tested using GFP and RFP reporter genes (BBa_I13507, BBa_I13504, BBa_J23100) as output. Each BioBrick contains an RBS, a reporter gene and two terminators. The new constructs will be expressed in P. putida to test for expression in the presence of fusaric acid. Since trace amounts of fusaric acid are lethal to E. coli, this is not used as a host to test the expression.

Resistance

All clusters but FuaABC were successfully isolated by PCR but cloning proved to be more difficult. Initial attempts to ligate the cluster behind BBa_J04500 in psb1C3 and transform it into E. Coli yielded no results. Although colony PCR showed a positive colony in some cases of the FDT cluster, plasmids isolated from these colonies were not of the right size to contain the inserts. Sequencing one of these plasmids with the VR and VF2 primers showed insertion of a small fragment containing the end and start of the primer used to PCR the cluster and a small palindromic sequence of 8bp. (ATCGATGCTA)
Membrane proteins can potentially be toxic when expressed at a too high rate. Therefore the construct was inserted in to a low copy number backbone (pSB3K3 / pSB4K5). Additionally the three genes of the smallest cluster (FDT-123) were separately expressed to see if one of them was the cause of the possible toxicity.
Successful transformants were obtained in pSB4K5 for the whole FDT cluster, FDT-2 and FDT-3 under a pLac promoter. Once again the whole FDT cluster showed up successful on colony PCR, but failed to show the expected results when a restriction digest was done on isolated plasmids. Sequencing showed again inclusion of both the primers and a small palindromic sequence (TAGCATCGAT/ATCGATGCTA).
Given that both FDT-2 and FDT-3 were successfully cloned but the whole cluster or FDT-1 separately did not yield any results, FDT-1 , which has homologues proteins in all 3 clusters is the most likely cause for toxicity.

Fusaric Acid Sensing

To isolate the pp1262 gene and the downstream intergenic region, two promoters containing the RFC10 parts were used in a PCR reaction on the genomic DNA of P. putida KT2440. The product was ligated into pSB1C3 and transformed into chemically competent E. coli cells. The part was digested again and ligated upstream of several different reporter genes. BBa_I13507, which contains an RBS, mRFP and two terminators was successfully transformed. However, after sequencing it became clear that this BioBrick did not contain mRFP, making it unsuitable for this experiment. BBa_I13504, which contains an RBS, GFP and two terminators was used for further experiments. This construct was ligated and transformed into E. coli. Sequensing confirmed the successful assembly. This construct should only show green fluorescence in the presence of fusaric acid, and no fluorescence when fusaric acid is absent.
The E. coli cells, containing the construct were grown on agar plates containing different concentrations of fusaric acid. At 25 µg/ml, there was no significant difference between the transformed cells and the control cells. At higher concentrations the cells died, due to their inability to pump the acid out of the cell.
The construct was transformed into a different backbone (pSB1K3) and inserted into electrocompetent P. putida cells. The transformed cells were again grown on plates containing different concentrations of fusaric acid. A slight difference in colour could be observed between the transformed cells and the wild-type cells, indicating promoter is only active when fusaric acid is present. (See Figure 2.)

A growth experiment was performed in a 96-wells plate, containing 0, 10, 25, 50, 100 and 250µg/ml fusaric acid and both transformed P. putida cells and wild-type cells. After 18h, at a concentration of 50 µg/ml fusaric acid, the transformed cells showed more fluorescence than the wild-type cells, while both strains showed no fluorescence when no fusaric acid was added, indicating that the promoter is not active when no fusaric acid is present. After washing with PBS since GFP did not include an excretion tag, the wild-type showed no significant fluorescence, whereas the transformed cells clearly did, (see Figure 3.) indicating that the promoter is functional.

The transformed P. putida cells did not survive a fusaric acid concentration of 100µg/ml or higher, possibly because of overexpression of the inhibitor, which would not only inhibit the activity of the GFP, but also of the original fusaric acid efflux pump in the original genome of P. putida.
A new growth experiment was set up, using M9 medium instead of LB medium. In this experiment both the WT P. putida and P. putida containing BBa_K1493000 are grown in a 96-wells plate at 0, 15, 30, 45, 60 and 75µg/ml fusaric acid. Furthermore, DH5α E. coli cells (WT and BBa_K741002) were also grown in the same plate, but without fusaric acid. The transformed E. coli has a well characterized promoter with the same GFP gene downstream. (See Figure 4.) By comparing the fluorescence of our fusaric acid induced promoter at different fusaric acid concentrations to this constitutive promoter, a characterization can be performed. pLac is used as a constitutive promoter, which has shown activity without induction of IPTG.

Up to now, the regulation of this fusaric acid efflux pump was only theoretical. However, these experiments do not only show that the functioning of this promoter is indeed regulated by the induction of fusaric acid, but they also show that this regulator can actually easily be used for other purposes, such as detecting Fusarium oxysporum and producing anti-fungal agents in its presence. A fusaric acid detection system has not been developed before now, and the implications of this part could very likely exceed this iGEM project.