Part:BBa_K1973010
miniTn7BB-Gm-nahR-Psal-nasF-yhjH
nahR (Pseudomonas putida, see BBa_K1031610) is expressed under the constitutive Pr promoter. It encodes a regulatory protein that activates the Psal promoter in the presence of salicylate. yhjH (see BBa_K777121) is expressed from the Psal promoter (see BBa_J61051) in the presence of this molecule. nasF (see BBa_K1973001) is an attenuator of transcription elongation (terminator) from Klebsiella pneumoniae that decreases the basal expression from the Psal promoter. The miniTn7 tool allows the stable integration of this module in the chromosome of a wide range of bacteria (see https://parts.igem.org/Genome_Integration).
Having as an objective the implementation of a system that allows us to control externally the synthesis and turn-down of c-diGMP, we had cloned both genes pleD* and yhjH separately under the control of inducible expression system nahR-Psal, which is regulated by salicylate (parts BBa_K1973011 and BBa_K1973004, respectively). This allows us to have high levels of expression under inducing conditions and low ones in non-inducing conditions. To obtain lower expression levels, we used a variant of our constructs that contained the nasF attenuator, cloned between the promoter and the gene of interest (parts BBa_K1973018 and BBa_K1973008, respectively). This sequence limits the transcription, diminishing the levels of both basal and inducible expression, limiting toxicity in the case the product of our gene of interest is deleterious (Cebolla et al., 2001). These constructs were cloned in miniTn7BB-Gm device, allowing its stable integration in attTn7 site in P. putida and other Gram-negatives genome (parts BBa_K1973023, BBa_K1973022, BBa_K1973012 and BBa_K1973010, respectively). For this, we made use the transposition functions of the miniTn7 device using the helping vector pTNS2. We therefore had four new strains that contained the new functions provided by these genetic pieces.
In order to determine whether the production of both enzymes has any impact on the intracellular levels of c-diGMP, the plasmid pCdrA::gfpC was introduced in all the strains containing DGC or PDE constructs. This plasmid counts with a GFP fusion to the PcdrA promoter from Pseudomonas aeruginosa, which is inducible by c-diGMP.
The addition of salicylate did not affect in a significant way the expression of PcdrA-gfpmut3 fusion in the wild type or any of the strains containing the attenuator, whereas the levels of fluorescence were significantly higher in the strain expressing pleD* without attenuator. Furthermore, the fluorescence levels were significantly lower in the strain expressing yhjH without attenuator. As a summary, our results point out that the induction with salicylate of the synthesis of PleD* increases the intracellular concentration of c-diGMP, whereas the synthesis of YhjH decreases the intracellular concentration of this second messenger.
Figure 1. Absorbance and fluorimetry data. Graphic representation of the bacterial growth (dots) and accumulated fluorescence (squares) produced by PcdrA promoter fusion with stable GFP protein in all the assayed strains in both induction (blue) and non-induction (orange) conditions. Fluorescence levels are similar to those of the wild type in all situations except for the strain producing PleD* without attenuator in induction conditions. There were growing differences between the wild type and strains producing both PleD* and YhjH without the attenuator under induction conditions.
c-diGMP is involved in the necessary signalling for both formation and dispersal of biofilm, promoting its formation whenever the levels are high enough and provoking its dispersal whenever the intracellular concentrations are low (Monds & O’Toole, 2009).
In order to monitor both planktonic and biofilm growth of the studied strains, dilution-based growth curves were performed. This method uses a series of dilutions in a multi-well plate to recapitulate the temporal evolution of both planktonic and biofilm populations (López-Sánchez et al., 2013).
In the presence of salicylate, the PleD* producing strain without the attenuator system showed a slower growth accompanied by 7 times higher levels of biofilm than those of the wild type strain. In the other hand, Yhjh producing strain without the attenuator system showed a normal planktonic growth, however showing a great delay on biofilm formation. These experiments suggest that the expression of a diguanylate cyclase, which produces high c-diGMP levels, provokes a biofilm super formation and its non-dispersal, as well as a slower growth, whereas low levels of c-diGMP provoked by the expression of a phosphodiesterase provoke a delay in biofilm formation.
Figure 2. Dilution-based growth and biofilm curves. Graphical representation of both growth and biofilm formation in the different studied strains, always compared with the wild type strain carrying the miniTn7 device with no genetic construction in it. Dashed lines correspond to growth whereas solid lines refer to the biofilm development. Orange dots refer to wild type strain, whereas blue dots refer to the different producing strains. Strains hosting the attenuator did not have differences towards the wild type. YhjH producing strain shows a delay in biofilm formation in inducing conditions. PleD* producing strain shows an increase in biofilm formation and its non-dispersal.
For further information, check out wiki page http://2016.igem.org/Team:UPO-Sevilla/Experiments .
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