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. pleD* (see BBa_K1973016) 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 http://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.
One of the most affected processes by the changes in the intracellular concentration of c-diGMP is the production of cellulose and other exopolysaccharides during biofilm formation (Monds & O’Toole, 2009). The increasing of c-diGMP levels provokes an increase in the production of these EPS, which contribute to the maturation of the biofilm’s macrocolonies (Pérez-Mendoza et al., 2014; Romero-Jiménez et al., 2015). In order to study the changes performed in the production of EPS that were induced by the levels of c-diGMP, we made use of a Congo Red adsorption assay (a dying that binds cellulose and other types of EPS).
Both the wild type and the empty miniTn7 device strains produced flat colonies with no remarkable dying adsorption, what suggests that they do not produce high quantities of Congo-Red-adsorbing polysaccharides. This phenotype did replicate in all strains in non-inducing conditions. In presence of salicylate, all colonies had again the same phenotype, except for the colony corresponding to the nahR-Psal-pleD* construction, which had a rugose-colony phenotype with high crests and an intense red dying. These data suggest that the PleD* producing strain without the nasF attenuator produces high levels of EPS capable of adsorbing Congo Red, therefore the synthesis of them is regulated by c-diGMP in P. putida.
Figure 2. Congo Red assay. Congo Red assay in which the build Pseudomonas putida strains were tested, as well as the wild type strain and a wild type strain containing the empty miniTn7 device, kept as negative controls both in inducing (left) and non-inducing (right) conditions. The negative control strains kept a flat-colony, low-Congo-Red adsorption phenotype that was reproduced in all strains both in inducing and non-inducing conditions, except for the PleD* producing strain, which kept a rugose-colony, high-Congo-Red adsorption phenotype, with remarkable crests and high red coloration.
Flagellar motility is another of the aspects in bacterial physiology that becomes affected by the alteration in c-diGMP levels, high c-diGMP levels meaning an inhibition of the motility by the repression of the genes in charge of both the synthesis and functioning of flagella (Pérez-Mendoza et al., 2015; Romero-Jiménez et al., 2015). In order to check whether an alteration of c-diGMP levels had an effect in motility in the studied strains, a motility assay in soft-agar plates, or swimming, was performed. For this, the strains were inoculated in semi-solid plates both in presence and absence of salicylate, afterwards evaluating their capacity to move from the inoculation point to form a motility halo.
All the strains assayed did produce similar halos to that produced by the wild type, both in inducing and non-inducing conditions. The only exception was the PleD* producing strain without the nasF attenuator, which produced a halo similar to that of the wild type in the non-induced plate but did not show any halo at all in the induced plate. This strongly suggests the levels of c-diGMP produced by PleD* synthesis from the construction inhibit the synthesis or the functioning of flagella.
Figure 3. Swimming assay. Assay in which we can observe the swimming halos produced by the bacteria when swimming, both in inducing (right) and non-inducing (left) conditions. The wild type strain produces a halo that is similar to the ones produced by the rest of strains both in non-inducing and inducing conditions, except for the PleD* producing strain without the nasF attenuator, which shows a non-motile phenotype in inducing 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 4. 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 .
Sequence and Features
- 10Illegal prefix found in sequence at 4367
Illegal suffix found in sequence at 1
- 12Illegal EcoRI site found at 4367
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 4373
- 21Illegal EcoRI site found at 4367
Illegal BglII site found at 3051
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Illegal BglII site found at 4972
Illegal BamHI site found at 5584
Illegal BamHI site found at 6741
- 23Illegal prefix found in sequence at 4367
Illegal suffix found in sequence at 2
- 25Illegal prefix found in sequence at 4367
Illegal XbaI site found at 4382
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NgoMIV site found at 4804
Illegal NgoMIV site found at 5999
Illegal NgoMIV site found at 6146
Illegal AgeI site found at 6482
- 1000Illegal BsaI site found at 1681
Illegal BsaI site found at 5657
Illegal SapI.rc site found at 2763