PROOF OF CONCEPT
Experiments of BBa_K1973023 (miniTn7BB-Gm-nahR-Psal-pleD*)
Experiments of BBa_K1973023 (miniTn7BB-Gm-nahR-Psal-pleD)
It has been demonstrated that in vivo synthesis of high levels of diguanylate cyclases (DGC) has as a result an increment of the intracellular concentrations for c-diGMP (Christensen et al., 2015; Romero-Jiménez et al., 2015). In order to determine whether the production of the enzyme has any impact on the intracellular levels of c-diGMP, the plasmid pCdrA::gfpC was introduced in all the strains containing DGC construct. This plasmid counts with a GFP fusion to the PcdrA promoter from Pseudomonas aeruginosa, which is inducible by c-diGMP. The GFP used for this fusion is gfpmut3, containing a stable derivative of this protein (Rybtke et al., 2012). The strains transformed with pCdrA::gfpC plasmid were grown in 10% diluted LB plates at 30ºC and shaking, both in presence and absence of salicylate, in an absorbance/fluorescence lector, allowing to measure 600 nm absorbance and fluorescence alongside the curve (Figure 1). From the experimental data, a GFP differential accumulation rate was calculated during growth exponential phase as it is described in the protocols. This allowed to compare the data objectively between the different strains assayed (Figure 2).
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 PleD* without the attenuator under induction conditions.
Figure 2. Differential accumulation rate. Graph in which are shown the data relating the differential accumulation rate with their error bars. The producing strains that had the nasF attenuator did present similar fluorescence levels as the wild type. The PleD* producing strain has an increase of up to 15 times in fluorescence over the wild type in induction conditions.
The growth and fluorescence levels of all strains were similar in the absence of salicylate. These results point out that the nahR-Psal expression system does not allow the production of physiologically significant levels of PleD* in non-inducing conditions, with independence of the presence of nasF attenuator. The addition of salicylate did not affect in a significant way to the growth of any strain, with the exception of the PleD* producing strain without the attenuating system. The growth rate of this strain was severely diminished. 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. As a summary, our results point out that the induction with salicylate of the synthesis of PleD* increases the intracellular concentration of c-diGMP.
Characterization of EPS production effect when arising c-diGMP levels
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). For this, the strains to study were inoculated in solid-medium plates supplemented with this dying (Fig. 3), adding sodium salicylate whenever necessary in order to induce the expression of the constructs within the strains.
Figure 3. 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.
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.
Characterization of flagellar motility when arising c-diGMP levels
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 (Fig. 4).
Figure 4. 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.
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 inhibits the synthesis or the functioning of flagella.
Characterization of the formation and dispersal of the biofilm when arising c-diGMP levels
c-diGMP is involved in the necessary signalling for both formation and dispersal of biofilm, promoting its formation whenever the levels are high enough (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) (Fig. 5).
Figure 5. 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. PleD* producing strain shows an increase in biofilm formation and its non-dispersal.
The pattern both in planktonic growth and biofilm formation from the wild type was essentially as described in (López-Sánchez et al., 2013). This strain showed an increase in biofilm mass coincident with the exponential phase of planktonic growth, and after getting a maximal in late exponential phase, biofilm dispersal was observed during stationary phase. Strains hosting attenuator nasF showed the same pattern as the wild type, both in inducing and non-inducing conditions, either in growth or biofilm development curves. The strain producing PleD* without the attenuator showed this same pattern in non-inducing conditions. However, in 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. 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.
Biofilm-producing bacteria are not only capable of preforming it over solid surfaces, but also can they produce it in the liquid-gas interphase of liquid cultures. This type of biofilm is named as pellicle. In order to characterize the effect on this phenotype of PleD* super production, a pellicle assay was performed to all the studied strains both in presence and absence of salicylate (Fig. 6).
As previously described (Jimenez-Fernandez et al., 2015), wild type strain showed a low-pellicle formation phenotype independently from the inductor presence. Similarly, there was no pellicle formation observed in the strain carrying the nasF attenuator. In contrast, the PleD* producing strain without the attenuator culture that had salicylate showed a high biomass quantity forming a pellicle. In addition, the culture was much less turbid than the others, and sediment of cell aggregate was observed at the bottom of the tube. These results suggest that the c-diGMP levels obtained with the construction in the PleD* producing strain without nasF induces pellicle formation and cell-cell interaction for aggregate formation.
Figure 6. Pellicle formation assay. Pellicle assay, both with no inductor (above) and with inductor (down), of the labelled strains. All strains show a low-pellicle formation phenotype except for PleD* producing strain without nasF in inducing conditions, which shows both a wide pellicle formation and cell aggregates at the bottom of the tube. Black arrows point the formation of pellicle over the walls of the tube and decanting of cell aggregates at the bottom of it.
P. putida is able to adhere to a variety of surfaces permanently on a short-time scale – a few minutes – and proliferate on them to produce microcolonies. In order to determine the effect on this capacity by the production of PleD*, adhesion assays were performed to the producing strains by means of phase contrast microscopy in a multi-well plate, both in presence and absence of salicylate. Results are shown in figure 7.
Fig. 7. Adhesion assay. Microscopy images of PleD* strain without transcription attenuator nasF with and without inductor. Wild type shows a microcolonies phenotype that fills the whole field. PleD* producer strain shows bacterial aggregates that are not uniformly distributed in induction conditions with salicylate.
In absence of salicylate all strains showed an adhesion and microcolonies formation phenotype similar to that of wild type. Producer strains carrying the attenuator showed an identical phenotype to that of wild type in presence of inductor. However, in induction conditions we observe important effects in PleD* producer strain phenotype without the attenuator nasF. PleD* producer strain cells were mainly forming aggregates, and planktonic cells were not seen. These results indicate that, in absence of nasF, PleD* synthesis induction causes the formation of aggregates that adhere steadily to the surface.