Construction of mucA mutants

The alginate operon (algD) responsible for alginate biosynthesis is transcribed using sigma factor RpoE (σE). When the bacteria finds itself in a favorable environmental condition, it does not need to biosynthesize alginate. Then the anti-sigma factor, MucA, sequesters the RpoE sigma factor at the membrane level. The alginate operon cannot be transcribed. On the other hand, when the bacteria finds itself under stress conditions, such as water stress for example, it will tend to produce more alginate. Thus a regulatory cascade intervenes, allowing the release of RpoE by mucA. Thus the sigma factor is released and can allow transcription of the alginate operon.

In the article Pulcrano, Giovanna et al. The new microbiologica, 2012, mucoid strains of P. aeruginosa (bio synthesizing more alginate than normal) were isolated and sequenced. The researchers noticed that these mucoid strains had one thing in common: the anti-sigma factor was truncated at its C-Terminal domain.

This mutation therefore prevented the sequestration of RpoE while keeping the bacteria alive, because the complete deletion of the mucA gene is lethal. Thus, in this condition the bacteria is able to produce alginate constitutively.

Transformation in E. coli

Our idea was therefore to reproduce this mutation in our bacterium P. putida. This is an experiment that has never been done but which seemed to us to be the best solution for our bacteria to over-produce alginate. So we created 4 mutants in order to maximize our chances of obtaining one that will produce a high level of alginate. To do this, we attempted to reproduce the same mutants identified in the article by truncating the protein at the same locations identified in the article. We could not create exactly the same ones because the mucA gene in P. aeruginosa is 585 base pairs while that of P. putida is 591 base pairs.

We chose to create the following mutants:

mucA*1 → 240 base pairs
mucA*2 → 360 base pairs
mucA*3 → 399 base pairs
mucA*4 → 450 base pairs

Our method was to introduce a stop codon at the place where we wanted the gene to be mutated in order to obtain a truncated protein. For this we used PCR to construct the mutated gene. To allow the wild gene to be replaced by our construction we used homologous recombination. So we did 2 PCRs which introduced a stop codon into the mucA gene and which had floating tails homologous to the genome as well as homologous SLIC regions to allow cloning.

We paid close attention to the reading frame, ensuring that the introduction of the stop codon did not shift it. We also took care to keep the RBS of mucB so that it could still be translated.

pcr-diluted-undiluted-dna — Figure 1: PCR with diluted and undiluted DNA of the different fragments of our construct

stop inserting strategy — Figure 2: Diagram explaining the strategy for inserting the STOP codon into the mucA gene

Once the assembly of our 2 PCR fragments was carried out with the NEBbuilder 2X HiFi DNA assembly Mix Kit, we proceeded to clone the fragment into the suicide plasmid PKNG101.

cloning slic method — Figure 3: Diagram showing the cloning of our fragment with the SLIC method into the PKNG101 plasmid

Then we transformed competent E. coli cc118 λpir strains with this plasmid.

cloning results — Figure 4: Cloning results

Then we verified our clones by colony PCR. We tested 6 clones per build because the gel well was too small for the volume used. We repeated 3 clones of each construction on another gel with larger wells to confirm the correct size. We extracted the plasmids from the 2 clones of each construct that looked good and sent them for sequencing. Every clones were good except the mucA*1

pcr-results-colony — Figure 5: Results of PCR on colonies

Conjugation in P. putida

conjugation figure 1 — Figure 6: Diagram of the mutator plasmid and homologous recombination. Black arrows represent possible homologous recombination events. Designed with Biorender

Once we had cloned our fragments of interest (the different mutated mucAs) into the suicide plasmid pKNG101 carried by the E. coli CC118 strain, we introduced this mutator plasmid into our chassis bacterium P. putida by conjugation. Firstly, we brought into contact the donor strain CC118 carrying the mutator plasmid and the mobilizing strain prk2013 then we added the recipient strain P. putida. To ensure that we eliminated E. coli cells and only selected Pseudomonas strains, we cultured on PIA medium supplemented with Streptomycin. The culture results obtained the next day are presented in the figures below. as negative controls, we cultivated the donor, recipient and mobilizing strains in PIA SM 2000.

Figure 7: Selection of the Pseudomonas putida transformants strains in PIA supplemented with Streptomycin.

We consider the clones obtained in the PIA SM 2000 medium as clones having undergone the first recombination event given that they are P. putida clones resistant to streptomycin (resistance gene carried by the plasmid pKNG101).

In order to force the 2nd recombination event, we cultivated a few random clones obtained from PIA SM2000 which we isolated on LB medium without NaCl supplemented with 6% sucrose. The colonies obtained were repatched on LB, LB supplemented with Streptomycin, LB without NaCl supplemented with 6% sucrose. The results obtained are presented in the figures below :

Figure 8: Selection of clones having undergone the 2nd recombination event. The SM sensitive ones are retained.

The clones likely to have undergone the 2nd recombination event are supposed to be sensitive to streptomycin because this would mean that they have excised the mutator plasmid (lethal in the presence of sucrose because of the Levane-sucrase gene carried in the pKNG101 plasmid). Therefore the clones which will have grown on LB and LB ssNaCl 6% sucrose are retained. However, we cannot ensure that the latter have the mucA mutated as we desire, it could be that the 2nd recombination event would have led to the WT phenotype. To screen the mutants, we carried out PCR on colonies using the pairs of primers from table () and the results obtained are presented below :

The mutant	Forward Primer	Reverse Primer
mucA1	F1	R_SLIC mucB
mucA2	F2	R_SLIC mucB
mucA3	F3	R_SLIC mucB
mucA4	F4	R_SLIC mucB

Figure 9: PCR on colonies of the different mucA mutants to screen the 2nd recombination event

The PCR results on colonies show the amplification of the fragments comprising the truncation point of mucA at different locations (corresponding to mucA1, mucA2, mucA3 and mucA4), but the primers used can just as well hybridize on a wild-type mucA therefore the results obtained do not confirm that our strains have a truncated mucA, for this, it will be necessary to carry out another PCR on colonies using new primers which would hybridize on the regions deleted during the truncation.

PCR verification

Given that we were unable to confirm the acquisition of mutant strains via PCR solely based on amplicon size, we have devised a novel approach to distinguish strains that have undergone homologous recombination events resulting in the integration of the mutated mucA gene from those reverting to a wild-type (wt) phenotype.

To address our issue, since the mutated mucA gene corresponds to a shortened wt mucA gene, we contemplated conducting colony PCR under two different conditions based on the choice of primer pairs (Figure 10).

Figure 10: **Schematic representation of wt and mutant *mucA* genes and primers at their hybridization regions.**The reverse complementary primer (in black) is the same for all matrix under both conditions. The green forward primers correspond to the positive control PCR based on the considered gene, while the red primer is used for the negative control PCR across all matrix. Diagrams were generated using Biorender, with gene schematics from Snapgene.

The so-called positive control PCR corresponds to a colony PCR using primers that anneal to the boundaries of the target mutant gene. The negative control PCR, on the other hand, utilizes primers that anneal to the wt mucA gene, where the red forward primer hybridizes to a region unique to the wild-type gene. Thus, confirmation of a mutant strain will be achieved if an amplification of the expected size for the mutant gene is observed in the positive control PCR, and no amplification is detected in the negative control PCR. Both PCR conditions will be conducted on a wild-type strain as a control, and amplification in both conditions is expected, further ruling out the hypothesis that non-amplification in the negative control PCR is due to it not functioning.

Figure 11: **Results of colony PCR analyzed by agarose gel electrophoresis (1%).** Colony PCR was performed under both control conditions for a colony of *P. putida* wild-type (WT), colony 4 of the mucA1 strain (mucA14), and colony 4 of the mucA3₄ strain obtained after homologous recombination. The marker size (bp) is indicated on the left.

Following the colony PCR controls, we only obtained a mutant mucA3 strain of P. putida corresponding to colony 4 obtained after homologous recombination. As depicted in Figure 11, amplification is observed in both PCR conditions for the wild-type strain, confirming that the PCR worked. For the mucA3₄ strain, amplification is only visible in the positive control PCR since those primers anneal to the gene boundaries. Furthermore, the amplicon size matches that of the mutant mucA4 gene, 450 bp. No amplification is detected in the negative control PCR because the forward primer's hybridization region is only present in the wild-type gene, indicating that the strain has indeed replaced the wild-type mucA gene with the mutated mucA following homologous recombination.

Alginate quantification assays

Since we aim for a higher quantity of alginate production by bacteria than the wild-type strains under conditions of membrane stress where alginate is naturally produced (1) and under normal conditions, we have designed mucA mutants that affect alginate production with the hope of achieving a substantial quantity.

To address this challenge, we conducted alginate quantification assays and compared the values of the wild-type strains under LB conditions and LB + NaCl hydraulic stress conditions with those of the mutants.

Figure 12: The concentration of Alginate as a function of the strain

Based on the results presented, it was observed that under LB conditions, there was no detectable alginate production. However, under stress conditions (LB + NaCl), significant alginate production was observed, with values of 17.59 µg/mL, 15.13 µg/mL, and 68.78 µg/mL of alginate for LB + 0.1 M, LB + 0.3 M, and LB + 0.5 M NaCl conditions, respectively. These results suggest a correlation between NaCl concentration and alginate production.

This difference can be explained by the fact that water stress induces a stress response in bacteria, including an increase in alginate production. Bacteria produce alginate as a component of biofilm which allows protection against adverse environmental conditions, aiding in their resistance to stress[1]. The mutant mucA34 consistently showed alginate production. The strain produces 37 µg/mL. It appears that this strain likely produces more alginate than the wild-type strain under LB conditions (approximately 30 times more), although its production is lower than that of the wild-type strain under stress conditions.

Indeed, we thought it is normal to observe higher alginate production under stress conditions compared to mutants, as alginate production is a multifactorial process, and the mutants have primarily affected only the regulatory part of this process. The next step will be to integrate alg8 to influence the metabolic part and optimize polymerization towards alginate production. The goal is to ensure a consistently stable and high alginate production with the mutants. With these modifications, it is possible to achieve relatively stable and high alginate production.

Growth of P. putida on glycerol medium

In order to make our product as competitive as possible, we had the idea of developing a culture medium for our P. putida bacteria that was as inexpensive as possible. Thus, we were able to obtain several liters of 80% raw vegetable glycerin, which are a byproduct from the Saipol company. The composition of this product is:

82% glycerol
3% ash
13% water
1.5% sodium
0.4% fat

We chose to test this composition as a medium which we have diluted with different amounts of water, in comparison with LB medium and laboratory glycerol.

Medium	0h	1h	2h	3h	4h	5h	6h
5% Saipol glycerol	0	0,04	0,09	0,18	0,19	0,17	0,17
10% Saipol glycerol	0,01	0,02	0,05	0,08	0,07	0,08	0,07
20% Saipol glycerol	0,02	0,04	0,05	0,06	0,05	0,07	0,08
40% Saipol glycerol	0	0,06	0,07	0,06	0,12	0,06	0,06
5% Lab glycerol	0,02	0,02	0,03	0,03	0,05	0,05	0,05
10% Lab glycerol	0,03	0	0	0	0,01	0,01	0,01
20% Lab glycerol	0,02	0,01	0	0	0	0	0
40% Labo glycerol	0,01	0,02	0,01	0,01	0,01	0	0
LB	0,02	0,1	0,42	1,3	3,4	4,1	4,5

Figure 13: Growth of *P. putida* on glycerol medium

We can notice that the medium with 5% glycerol from Saipol stands out from the others. In fact, this allowed better growth of the bacteria.

Figure 14: Growth of *P. putida* on glycerol medium compared to growth on LB

On the other hand, in comparison with the growth of P. putida in LB medium, the growth in glycerol medium seems insignificant.

But the data obtained must be taken into consideration because there is still growth, although slight, with around 5% Saipol glycerol.

Subsequently, we considered improving this culture medium by providing different carbon sources, amino acids or sugars, always coming from industrial waste, allowing our medium to remain as cheap as possible.

Sources

Chang, W. S., Van De Mortel, M., Nielsen, L., De Guzman, G. N., Li, X., & Halverson, L. J. (2007). Alginate production by Pseudomonas putida creates a hydrated microenvironment and contributes to biofilm architecture and stress tolerance under Water-Limiting conditions. Journal of Bacteriology, 189(22), 8290‑8299.

Alginate and CaCl₂ quantity optimization

We have tried to check how much alginate and CaCl₂ would be better to retain the water. This question is significant because eventually we want the cheapest solution achievable so that we can turn alginate into a commodity. Calcium and alginate won't be free, so we'll want to use the less of them possible for the best result possible.

raw data	T=0	T=1h	T=2h	T=3h
1% alginate 0.1M CaCl2	2	1,86	1,75	1,64
1% alginate 0.1M CaCl2	2	1,95	1,82	1,67
1% alginate 0.1M CaCl2	2	1,97	1,85	1,71
1% alginate 0.2M CaCl2	2	2	1,89	1,78
1% alginate 0.2M CaCl2	2	1,81	1,71	1,57
1% alginate 0.2M CaCl2	2	1,92	1,79	1,64
1% alginate 0.4M CaCl2	2	1,86	1,77	1,64
1% alginate 0.4M CaCl2	2	1,9	1,8	1,68
1% alginate 0.4M CaCl2	2	1,9	1,75	1,61
1% alginate 0.8M CaCl2	2	1,97	1,89	1,77
1% alginate 0.8M CaCl2	2	1,89	1,77	1,64
1% alginate 0.8M CaCl2	2	1,97	1,85	1,72
0.5% alginate 0.1M CaCl2	2	1,86	1,7	1,56
0.5% alginate 0.1M CaCl2	2	1,88	1,72	1,57
0.5% alginate 0.1M CaCl2	2	1,92	1,75	1,6
0.5% alginate 0.2M CaCl2	2	1,89	1,71	1,56
0.5% alginate 0.2M CaCl2	2	1,89	1,72	1,57
0.5% alginate 0.2M CaCl2	2	1,92	1,75	1,62
0.5% alginate 0.4M CaCl2	2	1,82	1,65	1,59
0.5% alginate 0.4M CaCl2	2	1,86	1,74	1,6
0.5% alginate 0.4M CaCl2	2	1,88	1,77	1,68
0.5% alginate 0.8M CaCl2	2	1,87	1,74	1,58
0.5% alginate 0.8M CaCl2	2	1,89	1,78	1,67
0.5% alginate 0.8M CaCl2	2	1,88	1,81	1,7

mean	0	1h	2h	3h
1% alginate 0.1M CaCl2	2	1,93	1,81	1,67
1% alginate 0.2M CaCl2	2	1,91	1,8	1,66
1% alginate 0.4M CaCl2	2	1,89	1,77	1,64
1% alginate 0.8M CaCl2	2	1,94	1,84	1,71
0.5% alginate 0.1M CaCl2	2	1,89	1,72	1,58
0.5% alginate 0.2M CaCl2	2	1,9	1,73	1,58
0.5% alginate 0.4M CaCl2	2	1,85	1,72	1,62
0.5% alginate 0.8M CaCl2	2	1,88	1,78	1,65

standard deviation	1h	2h	3h
1% alginate 0.1M CaCl2	0,06	0,05	0,04
1% alginate 0.2M CaCl2	0,10	0,09	0,11
1% alginate 0.4M CaCl2	0,02	0,03	0,04
1% alginate 0.8M CaCl2	0,05	0,06	0,07
0.5% alginate 0.1M CaCl2	0,03	0,03	0,02
0.5% alginate 0.2M CaCl2	0,02	0,04	0,06
0.5% alginate 0.4M CaCl2	0,03	0,06	0,05
0.5% alginate 0.8M CaCl2	0,01	0,04	0,06

Figure 15: Alginate and CaCl₂ quantity optimization

As we can see, neither the alginate or the CaCl₂ seems to have a significant effect. It's most likely because the protocol is wrong: we should not test how much the soil retain water for a few hours, but rather for a few weeks.

If we can restart these experiences, we may use in the future dedicated hardware such as Hydrop 2