The wild-type strain Pseudomonas fluorescens SBW25 does not grow on creatinine

Objectives

The pathway for creatinine degradation is well characterized in some Pseudomonas bacteria, e.g., P. putida ¹. However, creatinine metabolization by our strain of interest P. fluorescens has not been investigated so far. Our objective was to assess whether the wild-type strain P. fluorescens was able to metabolize creatinine and the downstream compounds of the pathway.

Method

Hypothesis

Results

To quantify the degradation of sarcosine by the WT P. fluorescens strain, we monitored sarcosine uptake using Nuclear Magnetic Resonance (NMR) spectroscopy (500 MHz NMR spectrometer Bruker). NMR analysis was conducted on the supernatants collected during the exponential growth phase of the bacteria. A sarcosine uptake rate of 5.2 mmol.h⁻¹.gDW⁻¹ was obtained (Figu. 2), while the measured growth rate under this condition was 0.19 h⁻¹ (n=1). The cultures used for this analysis were grown in Erlenmeyer flasks, providing more effective aeration and agitation compared to microplate experiments, which may explain the higher growth rate.

To validate the predictability of our metabolic model, we used the experimentally measured sarcosine uptake rate as an input parameter and the model predicted a growth rate of 0.20 h⁻¹, which is in perfect agreement with the experimentally observed rate of 0.19 h⁻¹. These results strongly suggest that the sarcosine degradation pathway is functioning optimally in the WT strain, and that our whole-cell model is able to give accurate predictions about metabolic fluxes.

The results from Figure 1 also demonstrate that P. fluorescens is unable to utilize glycine as a sole carbon and nitrogen source in vivo. However, P. putida is able to degrade creatinine following the creatinine metabolic pathway which ends with glycine conversion and provides a source of carbon and nitrogen^{2, 3} (Figure 3). The obtained results from P. fluorescens are different, except for the sarcosine oxidase (SoxA) which catalyzes the conversion of sarcosine into glycine with co-production of formaldehyde and H₂O₂:

It is important to note that, although P. fluorescens cannot grow on creatinine, creatine, or glycine as sole carbon and nitrogen sources, it may still be capable of utilizing creatinine or glycine as nitrogen sources. To explore this hypothesis, we supplemented the M9 minimal medium with sarcosine, creatine, or creatinine along with citrate, which served as primary carbon source. Citrate, a key intermediate in the TCA cycle, allows bacteria to produce energy and biosynthetic precursors. In addition, modeling simulations demonstrated that citrate can be co-consumed with creatinine.

This was experimentally demonstrated. The WT strain significantly grew on creatinine supplemented with citrate (Figure 4, left), exhibiting a clear exponential phase. In contrast, no growth was observed when citrate was provided along with creatine (Figure 4, center). Bacterial growth was also observed on glycine and citrate (0.28 ± 0.01 h⁻¹ (n=9), Figure 4, right), with a growth curve slightly delayed compared to that of creatinine and citrate (0.29 ± 0.01 h⁻¹ (n=9), Figure 4, left).

These findings suggest that P. fluorescens is unable to use creatinine, creatine, or glycine as sole carbon and nitrogen sources. The WT strain can utilize creatinine and glycine only as nitrogen sources when citrate is provided as the primary carbon source, but not creatine.

New questions arise

Construction of the plasmid pSEVA438-Ptet cloned with the operon creA-crnA and soxA genes

Objectives

The initial design aimed at cloning the genes creA, crnA, and soxA into pSEVA438-Ptet vector to obtain the pSEVA438-Ptet-creA-crnA-soxA plasmid for transformation of P. fluorescens.

Method

We experimentally tested these hypotheses and found that P. fluorescens SBW25 was only able to grow on sarcosine as a sole source of carbon and nitrogen (56 mM) with a maximal growth rate of 0.16 ± 0.01 h–1 (mean ± standard deviation, from 9 biological replicates), but not on creatinine (44 mM), creatine (38 mM), nor glycine (67 mM) (Figure 1).

Results

We succeeded in cloning the Ptet promoter into pSEVA438 and the creA-crnA operon (BBa_K5108009) into the pSEVA438-Ptet backbone, as shown in Figure 6. The construct was confirmed by Sanger sequencing (Genewyz, Germany). However, we failed to introduce the bidirectional terminator LUZ7 T50 (BBa_K4757058) and the soxA gene into the pSEVA438-Ptet-creA-crnA plasmid. As we demonstrated that P. fluorescens could grow on sarcosine, the soxA gene should be unnecessary for bacteria to grow on creatinine. This is why we decided to directly transform P. fluorescens with the pSEVA438-Ptet-creA-crnA plasmid.

The engineered strain Pseudomonas fluorescens SBW25 can express creatinine amidohydrolase (CrnA) and creatinase (CreA)

Objectives

The objective was to verify the overproduction of the creatinine amidohydrolase (CrnA) and creatinase (CreA) in P. fluorescens by activation of the Pm promoter with the inducer m-toluic acid.

Method

Hypothesis

Results

The obtained SDS-PAGE is presented in Figure 7.

Both soluble and insoluble fractions contained MBPeGFP, with the majority of protein being in the soluble fraction independently of the presence of the inducer. Although transcriptional leakage was clearly observed without the inducer, MBPeGFP was overproduced when the Pm promoter was activated with 0.5 mM of m-toluic acid, confirming the functionality of the Pm promoter in P. fluorescens. The presence of insoluble MBPeGFP can be caused by its overexpression leading to protein aggregation. SDS-PAGE analysis of the cell lysate derived from P. fluorescens transformed with pSEVA438-Ptet-creA-crnA revealed a clearly visible band at the expected size of CreA in both soluble and insoluble fractions when its expression is induced. As expected based on the leaky expression of MBPeGFP, CreA is also produced in lower quantities without the inducer in the soluble protein fraction. In contrast, there is no visible band at the expected size of CrnA, suggesting that the crnA gene is not or poorly expressed.

New questions arise

The engineered strain Pseudomonas fluorescens SBW25 expressing creatinine amidohydrolase (CrnA) and creatinase (CreA) grows on creatinine

Objectives

Our goal was to implement the metabolic pathway of creatinine degradation in P. fluorescens and to assay the growth of the engineered strain on creatinine, creatine, or sarcosine.

Method

Hypothesis

Results

Growth curves of three different strains of Pseudomonas fluorescens were monitored on M9 minimal medium supplemented with creatinine, creatine, or sarcosine as sole carbon and nitrogen sources:

• the wild-type strain (negative control),
• a strain transformed with the empty plasmid pSEVA438-pTET (negative control),
• a strain transformed with the plasmid pSEVA438-pTET creA-crnA (engineered strain).
• The results are reported in Figure 8.

No growth was observed in either the WT or the negative control strains when cultured with creatinine, confirming that our strain P. fluorescens SBW25 does not naturally metabolize this compound. In contrast, the engineered strain harboring the creA and crnA genes exhibited clear growth on creatinine, demonstrating that the introduced metabolic pathway is functional with a growth rate of 0.06 h⁻¹ (Figure 9).

In analogy with the results obtained with creatinine, neither the WT nor the negative control strains grew on creatine. However, the strain engineered with creA-crnA operon showed moderate growth, confirming that the transformed bacteria could also metabolize creatine. The same growth has been observed for growth on creatine as a sole source of carbon and nitrogen.

However, the growth rate on sarcosine (0.10 h⁻¹) was higher than on creatinine, indicating that the pathway for creatinine degradation may not be as efficient as for sarcosine. This suggests potential possibilities to optimize the metabolic pathway, as differences in growth rates point to bottlenecks or suboptimal expression of the enzymes involved in creatinine degradation.

Using sarcosine as a substrate, all three strains could grow, as expected. Growth rates (Figure 9) of the strains transformed with the empty plasmid (0.11 h⁻¹) or with the pSEVA438-Ptet-creA-crnA plasmid (0.10 h⁻¹) were lower than that of the WT strain (0.15 h⁻¹), indicating that plasmid introduction had an adverse effect on the growth rate. This could be explained by a metabolic burden on the bacteria caused by the production of CreA and CrnA , along with the cost of maintaining antibiotic resistance.

Overall, these results clearly demonstrate that transformation of P. fluorescens with the plasmid pSEVA438-pTET-creA-crnA enabled the bacteria to grow on creatinine and creatine, whereas the WT and negative control strains could not utilize these nutrients. This validates our engineering approach and the constructed DNA creatinine parts.

New questions arise

NMR Analysis of Creatinine, Creatine, and Sarcosine Uptake and Degradation in engineered strain Pseudomonas fluorescens SBW25

Objectives

The objective of these experiments was to monitor and quantify the degradation of sarcosine, creatine, and creatinine by Pseudomonas fluorescens transformed with the creA-crnA plasmid, and to compare the experimental growth and degradation rates to those predicted by our in silico metabolic model.

Method

Hypothesis

Results

Sarcosine degradation:

As shown in Figure 10, the experimental data reveals a clear decrease in sarcosine concentration over time, which can be attributed to the uptake and subsequent degradation by the cells. A sarcosine uptake rate of 3.6 mmol.h⁻¹.gDW⁻¹ was obtained and the measured growth rate under this condition was 0.10 h⁻¹ (n=1). The lower growth rate observed in the transformed Pseudomonas fluorescens strain on sarcosine (0.10 h⁻¹) compared to the wild-type (0.19 h⁻¹) in the same condition (growth in Erlenmeyer flasks) can likely be attributed to the metabolic burden imposed by the plasmid carrying the creA-crnA genes.

To validate the predictability of our metabolic model, we used the experimentally measured sarcosine uptake rate as an input parameter and the model predicted a growth rate of 0.11 h⁻¹, which is in perfect agreement with the experimentally observed rate.

Creatine degradation:

The degradation of creatine as the only substrate was also monitored by NMR (Figure 11). The experimental data fit well with the simulated kinetics, further validating our metabolic model for creatine degradation. A creatine uptake rate of 2.05 mmol.h⁻¹.gDW⁻¹ was obtained and the measured growth rate under this condition was 0.05 h⁻¹ (n=1). To validate the predictability of our metabolic model, we also used the experimentally measured creatine uptake rate as an input parameter and the model predicted a growth rate of 0.08 h⁻¹, which remains in agreement with the experimentally observed rate.

Creatinine Degradation:

Lastly, the degradation of creatinine was measured (Figure 12). Although the uptake rate of creatinine was higher than that of sarcosine and creatine, the observed growth rate was, in fact, lower. This indicates a lack of optimisation of the creatinine degradation, which may be due to a low expression of the gene crnA, as discussed above. A creatinine uptake rate of 3.58 mmol.h⁻¹.gDW⁻¹ was obtained and the measured growth rate under this condition was 0.03 h⁻¹ (n=1). To validate the predictability of our metabolic model, we also used the experimentally measured creatinine uptake rate as an input parameter and the model predicted a growth rate of 0.14 h⁻¹, which is higher in comparison to the experimentally observed rate.

Conclusion:

These results demonstrate that Pseudomonas fluorescens carrying the creA-crnA plasmid can degrade sarcosine, creatine, and creatinine, and utilize each of them as a sole source of carbon and nitrogen. As summarized in Table 1, the experimental data from NMR were in good agreement with the simulations for sarcosine and creatine uptake, validating our model and overall approach.

New questions arise

To go further in optimizing the project, we wanted to reduce oxidative stress-related damages by overexpressing the catalase (KatB) that degrades H2O2 which is produced during glycine synthesis from sarcosine. H₂O₂ generates superoxide radicals, which can damage cellular components, including DNA, proteins, and lipids. In addition, we knew that regolith is a hostile environment for both plants and bacteria. To help P. fluorescens overcome the stress it could be exposed to, we intended to stimulate the global stress response by overexpressing the native stress factors hfq and rpoS.

Oxidative stress has an impact on bacterial growth

Objectives

The first objective was to observe how P. fluorescens reacted to oxidative stress.

Method

Hypothesis

Results

As shown in Figure 13.A, growth of Pseudomonas fluorescens was affected by paraquat in a concentration dependent manner, with a strong inhibition from 1 mM.

Calculated growth rate dropped from 0.16 h⁻¹ at 0 mM of paraquat (control) down to 0.03 h⁻¹ at 10 mM (Figure 13.B), following the reduction of biomass measured at OD600. This result indicates that the WT strain is significantly affected by oxidative stress. Interestingly, bacteria still exhibited measurable growth at 0.1 mM paraquat, suggesting some natural ability to cope with moderate levels of oxidative stress, likely through the activation of detoxification mechanisms mediated by catalases.

New questions arise

Construction of the plasmid pSEVA244 cloned with the gene katB and transformation of Pseudomonas fluorescens SBW25

Objectives

The aim was to clone the gene katB into the plasmid pSEVA244 for transformation of P. fluorescens.

Method

Hypothesis

Results

After transformation in E. coli Stellar Competent Cells and isolation, the plasmid was digested using the enzymes EcoRI and HindIII, which validated the cloning (Figure 15). The correct sequence was validated by sequencing.

Attempts to overexpress a catalase in Pseudomonas fluorescens SBW25

Objectives

The objective was to verify the overproduction of the catalase KatB in P. fluorescens by activation of the Ptrc promoter with the inducer IPTG.

Method

Hypothesis

Results

The obtained SDS-PAGE is presented in Figure 16. Both soluble and insoluble fractions contained inducible MBPeGFP, with the majority of protein being in the soluble fraction independently of the presence of the inducer. SDS-PAGE analysis of the cell lysate derived from P. fluorescens transformed with pSEVA244-katB revealed a faint but visible band at the expected size of KatB in both soluble and insoluble fractions when its expression was induced (Figure 16). .

New questions arise

In this section, we describe our experiments with plants.

We addressed three questions:
• How do plants grow on regolith?
• Does P. fluorescens have a positive effect on plant growth on regolith?
• Do our modifications enabling creatinine consumption by P. fluorescens impact the growth of plants?

Plants grow poorly on regolith

Objectives

This first experiment aimed to observe the negative effect of lunar-simulant regolith on plant growth. It was an important step to ensure that our setup to grow plants was effective.

Method

Hypothesis

Results

On regular potting soil, we obtained green and healthy plants which grew vigorously (Figure 17). The plant on our lunar regolith simulant showed a slower development and a browner color. The smaller size and area of the rosette is due to the lack of nutrients present in the regolith. Under starving conditions, the leaves tend to be smaller¹¹ and the color browner. The change in color is due to the production of anthocyanins, a pigment indicative of abiotic stresses. Because the lightning, humidity, and temperature were designed to be as optimal as possible for the plant, the production of anthocyanins could be explained by the lack of nutrients on regolith¹².

This experiment confirmed that regolith is an unfavorable environment for plant growth. The use of a biostimulant to improve the development and limit stress is relevant.

Pseudomonas fluorescens WT increases the fraction of healthy leaves and plant hue on regolith

Objectives

The objective of this second experiment was to observe if P. fluorescens WT could relieve the negative effect of lunar-simulant regolith on plant growth.

Method

We monitored the growth of plants under three different conditions by varying the soil (fertile ground or regolith), and the presence or not of the WT strain on the regolith.

This experiment also allowed us to define which metrics to use to quantify the growth of plants:
• Proportion of healthy leaves
  The proportion of healthy leaves were counted every day and used to analyze the plant’s survival.
• Plant area
  We determined the plant area for each plant. Pictures were taken every day and analyzed using ImageJ 2.7.0 version software. To calculate the plant area, the contour of each plant was cut out precisely on the image and the area was measured with the Measure tool in the Analyze tab.
• Color hue
  We determined the color for each plant. To measure the color, we used the Color_Histogram-2.7.0.jar plugin from ImageJ to extract the mean value of Red, Green and Blue pixels present in each pixel of the cropped plant. Using the rgb2hsv command from the grDevices package on R Studio, we converted RGB space (Red/Green/Blue) to HSV space (Hue/Saturation/Value). Only the Hue value was used in our analysis, which correlates with chlorophyll content and is suitable to estimate the stress¹³.

Hypothesis

Results

Images of plants at different stages are shown for the tested conditions in Figure 19. Addition of P. fluorescens on regolith moderately increased the number of leaves per plant after 14 days compared to the control without bacteria.

To collect more quantitative information about the impact of stress on the phenotype of plants, we also analyzed the proportion of healthy leaves per plant, the rosette area, and the plant hue. While the percentage of healthy leaves per plant decreased to 40% after 8 days in the absence of bacteria on regolith, it remained higher than 80% for plants growing on soil or on a regolith containing bacteria (Figure 20).

Therefore, the addition of Pseudomonas fluorescens WT on regolith results in plants that wilts significantly less than on regolith only. Furthermore, after 14 days, plants grown on regolith without bacteria exhibited the browner color and the smallest size (Figure 21), two characteristics of stress.

From these results, we conclude that P. fluorescens improves the growth and health of the plants on regolith.

Transformed P. fluorescens may have a protecting role on plant when grown on regolith

Objectives

This last experiment aimed to examine whether (i) our modification of P. fluorescens affects plant growth and (ii) the use of chemical agents to maintain the plasmid in the strain and to trigger the creatinine pathway expression could have some impact too.

Method

Table reporting the tested conditions:

Condition	Comment
Regolith + No bacteria + Water	Negative control
Regolith + No bacteria + Mix	Negative control for the “Mix” condition. This experiment showed us the impact of the mix alone on plant growth.
Regolith + Empty plasmid + Mix	Negative control for the “creA-crnA” condition. This experiment showed us the impact of bacteria in the presence of creatinine on plant growth.
Regolith + creA-crnA plasmid + Mix	This experiment showed us the impact of bacteria in the presence of creatinine that was metabolized to sustain bacterial growth on plant growth.

Hypothesis

Results

After 8 days on regolith, it appeared that the four conditions led to different phenotypes (Figure 22). This required investigating each measured parameter separately (healthy leaves, plant surface, and hue). .

The proportion of healthy leaves per plant was clearly lower without P. fluorescens and in the presence of creatinine and streptomycin mix (Figure 23), pointing to a negative effect of one or both compounds. Notably, a clear positive effect of bacteria can be seen, leading to >60% of healthy leaves after 9 days. The strain with the functional creatinine pathway did not protect more than the strain with the empty plasmid. This suggests a possible detoxification of the soil through the degradation of streptomycin by the cells.

To strengthen these observations, we carried out a survival analysis by using the Kaplan-Meier estimator (Figure 24). This allowed us to estimate the probability to reach an event in function of time. Here, the event of interest we considered is the time an individual reached less than 45% of healthy leaves. The survival curves indicated that after 8 days, the probability to have less than 45% of healthy leaves was between 0.8 and 0.9 for all conditions, except the No bacteria+Mix condition that led to a probability of 0.25 (Figure 24A). This indicates that the probability to have a healthy plant is a lot lower without bacteria and with the mix.

We coupled this analysis with a Cox proportional Hazard model (Figure 24C), which allowed us to evaluate simultaneously the effect of several factors on survival. The results are given by the exp(coef), which corresponds to the Hazard Ratio (HR). HR gives an estimation of how often a condition reaches more than a half (55%) of wilted leaves over time. Here, the conditions No bacteria+Mix, the Empty plasmid+Mix and the creA-crnA plasmid+Mix were compared to the control No bacteria+Water.

For interpretation:
• HR = 1: No difference in the wilting compared to the control group over time.
• HR < 1: Reduction of the risk it gets 55% of wilted leaves compared to the control group over time.
• HR > 1: Increased risk of getting 55% of wilted leaves compared to the group control over time.

For the No bacteria+Mix, we obtained an HR=8, which means that this condition gives a high risk of getting 55% of wilted leaves compared to the No bacteria+Water condition. For the bacteria conditions, the HR < 1 indicates that transformation with either the empty plasmid or creA-crnA plasmid reduces the risk of getting 55% of wilted leaves compared to the No bacteria condition. This result suggests a negative effect of streptomycin.

Because the Cox proportional hazards model makes several assumptions, we needed to validate them for each of the covariable sets (Figure 24B). We proved the validity of our model by reaching p-values < 0.05 for each covariable.

The second metric used was the plant hue (Figure 25). The hue works like a binary metric: plants are either stressed (hue < 0.15) or not (hue > 0.15). For this study, dead plants have been arbitrarily set to a value of -0.025.
The negative control No bacteria+Water may have led to a high production of anthocyanins, hence the purple color (Figure 25, day 8). The three other conditions presented a more pronounced yellowing. With this color analysis, it is impossible to say whether yellowing or purpuling is a stronger stress mark. After 8 days, every condition showed signs of stress (mean hued < 0.15) on regolith. Notably, the conditions with bacteria transformed with either the empty-plasmid or creA-crnA displayed less stress than the condition No bacteria+Mix, with an averaged hue value at 0.14, corroborating the results described above.

To get a complete response of the plan phenotype to stress exposure, we combined the hue metric with plant surface (i.e., the third metric used in this analysis) (Figure 26). This provided a better discrimination of each plant state. At day 1, the hue indicates an absence of stress with some variability in the plant area. At day 8, the profiles are a lot more varied, indicating specific responses. The condition No bacteria+Mix clearly led to death. Condition No bacteria+Water resulted in a strong stress and lesser plant development. Conditions creA-crnA-plasmid+Mix and especially empy-plasmid+Mix resulted in greener hue and larger plant area.

These results demonstrate that our engineered strain of P. fluorescens is endowed with protective properties for the plant development on regolith. Further improvements will be necessary to take full advantage of the implemented creatinine pathway as it seems to create some more stress to plants than the strain transformed with the empty plasmid.

New questions arise

New potential research

To mitigate the oxidative stress caused by the release of H₂O₂ during creatinine degradation, an effective strategy would be to enhance the bacterial strain’s resistance to Reactive Oxygen Species. Overexpressing the catalase gene katB in P. fluorescens would be a way to degrade H₂O₂ and avoid its adverse effect on plant growth.

In order to avoid having to add Streptomycin to preserve the plasmid in the bacteria, chromosomal integration of creA-crnA should be considered.

In this BioMoon project, we choose to optimize the properties of Pseudomonas fluorescens as a biostimulant for growth in poor soil conditions like the lunar regolith.

Our main goal was to engineer the strain so that it could utilize non-recycled resources in a space base. We identified creatinine as a high-value compound unconsidered so far in spatial programs and that could serve as both carbon and nitrogen source. We demonstrated that Pseudomonas fluorescens is not able to naturally grow from creatinine. A synthetic creatinine pathway was successfully implemented in the bacterium and the properties of the resulting Pseudomonas fluorescens were thoroughly characterized both in silico and in wet lab experiments.

We set up a home-made system to ensure efficient and reproducible plant assays. We qualitatively and quantitatively analyzed the deleterious effect of regolith on plant growth, as well as the positive impact of Pseudomonas fluorescens. We managed to observe that our strain engineered with the creatinine pathway has kept most of its protective properties. We discussed how it could be optimized further to provide stronger stress resistance. Several strategies to reduce Pseudomonas fluorescens oxidative stress have been proposed and are currently in progress.

This work paves the way to future developments in using synthetic biology for the production of efficient biostimulants, both on Earth and in space. Please visit our entrepreneurship page to learn more about it.