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Caulobacter crescentus CB2/CB2A HfsA-HfsB-HfsD-HfsF-HfsC-HfsI Polysaccharide export apparatus

Introduction

Vilnius-Lithuania iGEM 2024 project Synhesion aspires to create biodegradable and environmentally friendly adhesives. We were inspired by bacteria, which naturally produce adhesives made from polysaccharides. Two bacteria from aquatic environments - C. crescentus and H. baltica - harness 12 protein synthesis pathways to produce sugars, anchoring them to the surfaces. We aimed to transfer the polysaccharide synthesis pathway to industrially used E. coli bacteria to produce adhesives. Our team concomitantly focused on creating a novel E. coli strain for more efficient production of adhesives.

This is the complete holdfast polymerization and export apparatus. Parts of this composite can be found:BBa_K5246044 and BBa_K5246045.

This part was used in Vilnius-Lithuania iGEM 2024 project "Synhesion" https://2024.igem.wiki/vilnius-lithuania/.

Biology and Usage

Biology

Caulobacter crescentus is a common freshwater gram-negative oligotrophic bacterium of the clade Caulobacterales. Its distinguishing feature is its dual lifestyle. Initially, C. crescentus daughter cells are in a “swarmer” cell phase, which has a flagellum, enabling them to perform chemotaxis. After the motile phase, they differentiate into “stalked” cells. This phase features a tubular stalk with an adhesive structure called a holdfast, allowing them to adhere to surfaces and perform cell division. [1][2]

Caulobacterales synthesize a polysaccharide-based adhesin known as holdfast at one of their cell poles, enabling tight attachment to external surfaces. It is established that holdfast consists of repeating identical units composed of multiple monomers. Current literature agrees that in Caulobacter crescentus, these units form tetrads composed of glucose, an unidentified monosaccharide (either N-mannosamine uronic acid or xylose), N-acetylglucosamine, and N-glucosamine. These units are polymerized and exported to the outer membrane of the cell, where they function as anchors, securing the bacterium to a surface[3][4]. The C. crescentus holdfast is produced via a polysaccharide synthesis and export pathway similar to the group I capsular polysaccharide synthesis Wzy/Wzx-dependent pathway in Escherichia coli. The holdfast synthesis (hfs) genes include those encoding predicted glycosyltransferases, carbohydrate modification factors, and components of a wzy-type polysaccharide assembly pathway. [4][5][6] The synthesis of holdfast polysaccharides (Fig.1) occurs through a mechanism analogous to the Wzx/Wzy-dependent group I capsular polysaccharide biosynthesis pathway observed in Escherichia coli. The process is initiated in the cytoplasm by the glycosyltransferase (1) HfsE, which transfers an activated glucose-phosphate from UDP to an undecaprenyl-phosphate (Und-P) lipid carrier (1) [7]. Subsequent monosaccharide residues are added to the lipid carrier to form a repeating unit by the action of three glycosyltransferases: (2) HfsJ (adding N-mannosamine uronic acid or D-xylose), (3) HfsG (adds N-acetylglucosamine) and (4) HfsL (most likely adding another N-acetylglucosamine) [8]. Then some of the N-acetyl-D-glucosamine within these repeat units undergoes enzymatic modification through the activity of the deacetylases (5) HfsH and HfsK, which “incorporates” into the tetrad of another saccharide - D-glucosamine [9]. The completed repeat of four monomers is then flipped over the inner membrane to the periplasm by flippase HfsF (6) [8]. In the periplasm, the repeat unit is transferred to copolymerases HfsC and HfsI (7), which assemble holdfast into a mature polysaccharide [10]. Subsequently, following the polymerization, holdfast saccharides are exported through a multi-protein export channel made of HfsB, HfsA, and HfsD (8-10) [11]. After excretion, holdfast polymer is relocated to the anchoring Hfa group of proteins (11), where they function by holding the mature polysaccharide on the cell's surface of, e.g. C. crescentus or H. baltica , and securing it to the surface [8].

Usage

Genes from this composite part are responsible for holdfast polymerization and export from the cell. HfsF (BBa_K5246006 ) is a flippase that catalyzes the translocation of undecaprenol diphosphate-linked K-repeating units formed at the cytoplasmic side of the inner membrane across this membrane. In the case of holdfast synthesis pathway, it flipps tetrasaccharide (glucose--mannosaminuronic acid--N-acetyl-D-glucosamine--D-glucosamine) linked to a lipid carrier over the inner membrane to the periplasm and transfers it to the co-polymerases HfsC and HfsI (BBa_K5246003 ) (BBa_K5246009 ) after polymerization assembled polysaccharide is transffered to the export apparatus that consists of HfsB, HfsA, HfsD (BBa_K5246002 ) (BBa_K5246001 ) (BBa_K5246004 ) and transported out of the cell.

-->This part works as a system for holdfast synthesis together with part BBa_K5246043.

Sequence and Features

Experimental characterization

Part assembly

All of the proteins composing this system are responsible for polysaccharide polymerization and export. Since the system's proteins are found in the membrane, we concluded that using a low-copy plasmid would decrease the probability of inclusion body formation. Their formation would diminish the functionality of our system, as the proteins would not allow the polysaccharide to be exported outside the bacteria.

We had to assemble this part to further create the holdfast synthesis pathway in E. coli together with composite part: BBa_K52460463, we had to assemble this part first into a backbone of pACYC-Duet-1. We designed a strategy to maximize the success of plasmid assembly by first assembling plasmids with 3 genes (part:BBa_K5246044) and, after verifying the sequences, integrating 3 left genes (part:BBa_K5246045) into that backbone (Fig. 2). In this way, we prevented Golden Gate assembly errors by trying to construct plasmids from 8 or more fragments.

The BBa_K5246044 assembly was done using Golden Gate assembly with IIS AarI restriction enzyme sites introduced during PCR amplification. The backbone of pACYC-Duet-1 (Novagen) and fragments were amplified using Phusion Plus DNA polymerase, as the genome of C. crescentus has a high GC% content making the appearance of non-specific products during PCR amplification more common and primer design more challenging (Fig. 3). Since hfsA gene had an AarI RE site directly in the gene, this site was domesticated during side directed mutagenesis.

Due to the high amount of non-specific products, the fragments were gel-purified. Vectors and fragments composing this operon were mixed in equimolar amounts with GG reaction components and incubated as described in the protocol. The reaction was later transformed into E. coli Mach1 (Thermo Scientific) competent cells. The assembly was then confirmed with restriction digest analysis (Fig. 4), and positive colonies were sequenced.

After acquiring sequence-verified clones, we further cloned the 3 remaining genes: hfsF,hfsC, hfsI, this was done the same way as the first 3 genes. After transformation into E. coli , obtained colonies were once again screened with cPCR (Fig. 5) and restriction digestion analysis (Fig. 6). After choosing positive colonies full plasmids with hfsA-hfsB-hfsD-hfsF-hfsC-hfsI were once again sequenced.

Fig. 6. Restriction digest analysis of C. crescentus CB2 pACYC-hfsA-hfsB-hfsD-hfsF-hfsC-hfsI. On the left - expected in silico profile of restriction digest with EcoRI, HindIII and XhoI, on the right - digested plasmids - 5,8,10,12,13 positive cPCR colonies, M - molecular weight ladder, GeneRuler DNA Ladder Mix (Thermo Scientific).

Holdfast polymerization and export apparatus usage for holdfast synthesis

Holdfast synthesis system expression optimization

The intricate holdfast synthesis pathway involves numerous proteins that must be efficiently co-expressed in Escherichia coli. After obtaining plasmids used for full holdfast synthesis pathway assembly, we had to optimize the expression of the whole system in E. coli. Previously, only three studies have tried to recombinantly express C. crescentus proteins in E. coli for unassociated studies with our project's goal [12][13][14]. Since the E. coli strains and protein expression conditions were unrelated to each other, and before our project, no one in iGEM besides the 2009 iGEM ULB-Brussels team ever tried expressing more than two C. crescentus proteins in E. coli at the same time, we had no solid foundation for expression and chose to experiment with different E. coli strains and conditions. Therefore, it was essential to optimize the conditions for simultaneous protein expression by trying different media, temperatures, IPTG concentrations, and expression times on multiple E. coli strains. We used SDS-PAGE analysis of cell lysates and HPLC-MC proteomics to verify the expression results.

Results overview: Optimal C.crescentus protein expression was achieved in the BL21(DE3) strain cultivated in the LB medium. The most favorable conditions included an incubation temperature of 37°C, induction with 0.5 mM IPTG, and an expression duration of 3 hours at 37°C.

KRX(DE3)

To determine the best conditions for the whole system expression, we first used E. coli KRX(DE3) strain. We tried expressing separate plasmids pRSF-(1)HfsE-hsfJ-hfsG-(2)hfsH-hfsK-hfsL (BBa_K5246043) and pACYC-(3)hfsA-hfsB-hfsD-(4)hfsF-hfsC-hfsI (BBa_K5246046) with different IPTG concentrations - 0.1, 0.25, 0.5, 0.75 and 1 mM - and 0.1% rhamnose with protein expression for 3h at 37°C after induction. As we saw, some bands, corresponding to our protein sizes, were appearing in pACYC-(3)hfsA-hfsB-hfsD-(4)hfsF-hfsC-hfsI (BBa_K5246046) expression (Fig. 7.1), but we were not sure if they were our system proteins, therefore for pRSF-(1)HfsE-hsfJ-hfsG-(2)hfsH-hfsK-hfsL (BBa_K5246043) we used negative control with empty pRSF vector and expressed the proteins in similar conditions. We saw that pRSF-(1)HfsE-hsfJ-hfsG-(2)hfsH-hfsK-hfsL (BBa_K5246043) operon proteins were also expressed (Fig. 7.2) with minimal IPTG concentration impact on protein amount.

Fig. 7.1. SDS-PAGE analysis of pACYC-hfsA-hfsB-hfsD-hfsF-hfsC-hfsI (BBa_K5246046) expression in KRX(DE3) at different IPTG concentrations at 37°C for 3h. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26610 (Thermo Scientific).

Fig. 7.2. SDS-PAGE analysis of pRSF-(1)HfsE-hsfJ-hfsG-(2)hfsH-hfsK-hfsL (BBa_K5246043) expression in KRX(DE3) at different IPTG concentrations at 37°C for 3h. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

We decided to use the lower IPTG concentrations - 0.25, 0.5, and 0.75 mM - for gene expression induction of the full system, as it is more cost-effective for upscale in the future. But, unfortunately, full system expression at different temperatures and expression times did not provide clear bands of proteins in SDS-PAGE gel analysis (Fig. 8. 1-5).

Fig. 8.1. SDS-PAGE analysis of CB2 system expression in KRX(DE3) at different IPTG concentrations for 3h at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

Fig. 8.2. SDS-PAGE analysis of CB2 system expression in KRX(DE3) at different IPTG concentrations for 3h at 30°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

Fig. 8.3. SDS-PAGE analysis of CB2 system expression in KRX(DE3) at different IPTG concentrations overnight at 30°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

Fig. 8.4. SDS-PAGE analysis of CB2 system expression in KRX(DE3) at different IPTG concentrations for 3h at 22°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

Only some proteins, in the size range of 30-50 kDa, appeared, but in general, the results of expression of the whole system were inconclusive, leading to the need to test another E. coli strain.

BL21(DE3)

The next E. coli strain we tested was BL21(DE3). Since the IPTG concentration appeared not to make that big of an impact on the expression, we settled on IPTG concentrations of - 0.25, 0.5, and 0.75 mM - in this way covering a wide range of them and accelerating the optimization effort, if the system would be expressed. We also decided to yet again test different expression temperatures - 37°C, 30°C, 16°C - before and after gene expression induction.

Initially, we tested pACYC-(3)hfsA-hfsB-hfsD-(4)hfsF-hfsC-hfsI (BBa_K5246046) operon expression at 37°C for 3h, which did not give promising results (Fig. 9.1), as we could not see distinguishable differences before and after induction. Nevertheless, we proceeded with pRSF-(1)HfsE-hsfJ-hfsG-(2)hfsH-hfsK-hfsL (BBa_K5246043) operon expression at the same conditions, which appeared to be working (Fig. 9.2), as we could see stark differences between empty E. coli and our operon lysates. As with the expression in the KRX strain, we could not see many differences between the IPTG concentrations.

Fig. 9.1. SDS-PAGE analysis of pRSF-(1)HfsE-hsfJ-hfsG-(2)hfsH-hfsK-hfsL expression in BL21(DE3) at different IPTG concentrations at 37°C for 3h. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

Fig. 9.2. SDS-PAGE analysis of pACYC-(3)hfsA-hfsB-hfsD-(4)hfsF-hfsC-hfsI expression in BL21(DE3) at different IPTG concentrations at 37°C for 3h. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26610 (Thermo Scientific). Note: negative control cultures for 0.25 mM and 0.75 mM were contaminated and subsequently not used for SDS-PAGE analysis.

We advanced with the whole system expression. After analyzing expression at 37°C for 3h conditions, we saw that there were pronounced differences between the uninduced system and the system after 3 hours (Fig. 10.1). As with KRX(DE3) expression, we saw that IPTG concentration used for gene expression induction did not make a big impact for overall expression.

Since the CB2 system was expressing, we tried different temperatures to optimize protein expression further. Remarkably, decreasing the expression temperature to 30°C and expression overnight did not make a significant difference as the proteins were still expressed in similar amounts to that of 37°C (Fig. 10.2). Expression of 16°C overnight produced some of the expected bands but not in the same capacity as expression at higher temperatures (Fig.10.3).

Fig. 10.1. SDS-PAGE analysis of CB2 system expression in BL21(DE3) at different IPTG concentrations for 3h at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

Fig. 10.2. SDS-PAGE analysis of CB2 system expression in BL21(DE3) at different IPTG concentrations overnight at 30°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

Proteomic analysis of samples induced by 0.5 mM IPTG from separate parts and the whole system, revealed that proteins responsible for holdfast polymerization - hfsC and hfsI - were not expressed (Fig. 11 (a),(b)). In addition, protein levels during full system expression dropped notably compared to separate part expression. However, proteins were still expressed in slightly higher quantities than in control (Fig. 11. (c)).

Nevertheless, as later experiments showed, these proteins were probably substituted by paralogous proteins found in E. coli as the system without 2 parts was still producing a polysaccharide (see BBa_K5246003 and BBa_K5246009 ). We reason that in the future, we should first test whether the separate proteins - hfsC and hfsI - are expressed and at what conditions before assembling new plasmids with different operon orders or additional promoters. T7/lac could serve as a good starting point, other considerations could involve separately inducible or constitutive promoters available in iGEM Parts Registry.

C41(DE3)

Once the system was successfully expressed in BL21(DE3) strain, we proceeded to optimize the expression further by testing another E. coli strain - C41(DE3). We decided to test separate system parts and the whole CB2 system with different IPTG concentrations - 0.25, 0.5, and 0.75 mM. Since we saw that the proteins were best expressed at 37°C in KRX(DE3) and BL21(DE3) strains, we settled on testing only this temperature. SDS-PAGE analysis of cell lysates before and after gene expression induction revealed that proteins were expressed in separate parts of the system and the whole system (Fig. 12. 1-3). Regrettably, the quantity was visibly less than that seen in BL21(DE3) strain indicating that this strain is not suitable for efficient system expression.

Fig. 12.1. SDS-PAGE analysis of pACYC-(3)hfsA-hfsB-hfsD-(4)hfsF-hfsC-hfsI expression in C41(DE3) at different IPTG concentrations at 37°C for 3h. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26610 (Thermo Scientific)

Fig. 12.2. SDS-PAGE analysis of pRSF-(1)HfsE-hsfJ-hfsG-(2)hfsH-hfsK-hfsL expression in C41(DE3) at different IPTG concentrations at 37°C for 3h. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

Rosetta(DE3) pLysS

Even though to this point we found E. coli strain with, in our perspective, sufficient system expression, we were determined to improve the expression even more. A study was conducted, where C. crescentus hfsJ gene was expressed in E. coli to determine its interaction with hfiA (inhibitor of holdfast development), we investigated the strain they used for expression - Rosetta (DE3) pLysS - for our own purpose [4]. Since we decided to use this strain, we had to carefully reconsidering our CB2 systems’ design, as the pRARE plasmid, native to Rosetta, contains the same antibiotic resistance - chloramphenicol - and the same origin of replication - p15A - as one of our system’s plasmids - pACYC-(3)hfsA-hfsB-hfsD-(4)hfsF-hfsC-hfsI (BBa_K5246046). For this we decided to change the backbone’s antibiotic resistance and origin of replication (ori) (Fig.13).

As a donor of new antibiotic resistance and origin of replication we choose pBAD-PhoCl2f plasmid, which was kindly gifted to us by VU LSC Institute of Biotechnology. This particular plasmid contains the ampicillin resistance gene and ColE1 origin of replication compatible with our pRSF operon and pRARE plasmid.

To save time we decided to utilize Golden Gate assembly for resistance/ori switching. We successfully acquired plasmids with new resistance/ori, which, after testing them with colony PCR, restriction analysis (Fig. 14), were sequenced by whole plasmid sequencing by SeqVision. Since after transformation into electrocompetent Rosetta cells they grew without any problems on LB agar plates with 3 different antibiotics, we came to a conclusion that the resistance/ori switch worked.

To test the expression we used different IPTG concentrations - 0.25, 0.5 and 0.75 mM - and our standard expression temperature of 37°C with checking the total expressed protein amount after 3 hours by SDS-PAGE analysis. Unfortunately, we saw very low or almost no protein expression in separate plasmids, leading to the same happening when we tried expressing the whole system (Fig. 15. 1-3). We reason that this might be due to increased metabolic strain on E. coli due to a whole additional plasmid introduced into the system during expression. This E. coli strain might be suitable for specific C. crescentus protein expression but, sadly, is not fitting for our needs.

Fig. 15.1. SDS-PAGE analysis of pRSF-(1)HfsE-hsfJ-hfsG-(2)hfsH-hfsK-hfsL expression in Rosetta(DE3) pLysS at different IPTG concentrations at 37°C for 3h. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

Fig. 15.2. SDS-PAGE analysis of pACYC-(3)hfsA-hfsB-hfsD-(4)hfsF-hfsC-hfsI expression in Rosetta(DE3) pLysS at different IPTG concentrations at 37°C for 3h. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26610 (Thermo Scientific).

HMS174(DE3)

Since one of the strains used for more efficient polysaccharide production (see down below) was E. coli HMS174(DE3), we also expressed CB2 and an empty system in previously optimized conditions. We also analyzed protein production after overnight incubation with 1% glucose. After performing SDS-PAGE gel analysis, we saw that this strain also expresses some of the proteins of the CB2 system (Fig. 16). Remarkably, after overnight incubation with 1% glucose, more protein bands appear, corresponding to, e.g., hfsH or hfsD, proteins, indicating that it is likely that prolonged incubation does not negatively impact protein production. This might explain why some of the strains, previously expressing few proteins after 3 hours, were still forming rings after overnight incubation, as this strain also formed rings (see explanation in holdfast synthesis part down below).

Holdfast biosynthesis

Following the optimization of protein expression, we examined the conditions required for successful holdfast polysaccharide production. Previous experiments demonstrated that the presence of the proteins does not guarantee holdfast synthesis, prompting further investigation into the underlying factors.

Results overview: The addition of 1% glucose and incubation overnight at 30°C are required for polysaccharide synthesis. E. coli cultures produce ring-like structures after incubation. Holdfast is primarily produced in the ring cells, detectable by Wheat Germ agglutinin lectin, and contains considerable amounts of N-acetyl-D-glucosamine. Cells producing the holdfast are morphologically different, forming biofilm-like structures. The best substrate for polysaccharide production is glucose, with mannose as a second choice, altough more expensive.

Holdfast synthesis requires glucose

Multiple experiments revealed that having the proteins alone is insufficient for holdfast synthesis. We attempted to purify the holdfast from the media (see Experiments for detailed purification protocol), but the results were not as expected (Fig. 18). No holdfast was produced under the given conditions, despite the presence of all the proteins. No differences were observed between the control and target samples in the dot blot assay of the purification samples.

So we considered adding precursors for the biosynthesis pathway to facilitate its’ activation. We hypothesized that we should be able to adapt methods, suitable for polymer production in E. coli [15][16]. Holdfast synthesis begins from UDP-D-glucose transfer to an UndP lipid carrier, so having an excess of available glucose could solve the problem. We designed a protocol based on previous studies [17][18][19]. Adding 1% (w/v) glucose after target protein expression, as the addition of glucose lowers expression levels by targeting lac operator, and incubating overnight at 30°C with a shaking speed lowered to 170 rpm resulted in distinct rings forming around the edges of the shaking media (Fig.19) [20].

In addition, for further experiments, e.g. holdfast purification, we expressed and synthesized ring-like formations, later deemed to be the holdfast, in larger flasks. This increase in size - from 500 mL to 1 L flask did not diminish the ability to form the rings (Fig. 20). Not only that but after multiple day incubation and consistently lowering the shaking speed, we were able to get multiple rings to form around the 1 L flask walls. This information was later used to produce rings for other experimental applications where larger amounts of starting material were required.

Polysaccharides are produced only in the part of the population

Wheat Germ Agglutinin (WGA) specifically binds to N-acetyl-D-glucosamine - a characteristic component of the holdfast, found in abundance in the polysaccharide [21]. This allowed us to determine the localization of the holdfast. We tested various samples, including cells from the media, the supernatant, material from the ring, sonicated cells, and the supernatant of the sonicated cells. After performing the dot blot assay (see see Experiments for detailed protocol), we saw that the holdfast is produced (indicated by considerable presence of N-acetyl-D-glucosamine) but is localized exclusively within the ring material (Fig. 21).

We made the conclusion that holdfast was located in the ring, but we still did not know the nature of the material, so we investigated if there were any living bacteria in it. Following the expression, after the rings formed, we resuspended the samples in LB media and streaked them on LB agar plates under four different conditions: (1) with appropriate antibiotics, (2) antibiotics + 0.5mM IPTG, (3) antibiotics + 1% glucose, and (4) antibiotics + 0.5mM IPTG + 1% glucose. The plates were then incubated overnight at 30°C. Variety of plates was supposed to show if there are any visible morphological differences between living bacterial cells from the ring, bacterial cells that express proteins of interest, and bacterial cells that not only express proteins but also, in theory, produce holdfast. It seems like ring formation consists of living bacteria that can grow on agar plates. Although they do not have any morphological differences (Fig. 22), they do appear to be more of a liquid-like appearance.

Following the discovery, we hypothesized that only part of the bacterial population is able to effectively express all 12 proteins, so we used plated bacteria from rings for standard protein expression and ran a SDS-PAGE analysis (Fig. 23). It is clearly noticeable that bacteria from the ring expresses proteins much better than the fresh ones.

Surprisingly, after incubating overnight, we did not notice any significant difference in the quantity of holdfast produced, based on ring appearance.

After understanding that the ring contains our target polysaccharides with bacteria, we further investigated the exact location of the holdfast. We expanded the dot blot assay method, including more samples from the ring: lysed ring cells, lysed ring cells’ supernatant, and lysed ring cells’ sediment. This lets us pinpoint the location of the polysaccharides and confirm where the holdfast is produced. Dot blot assays showed that holdfast is produced only in the ring cells mostly located in the soluble fraction of the lysate. These results were replicable for multiple biological repetitions (Fig.24.1-3).

The addition of glucose is required to activate the holdfast synthesis pathway. Even after the activation, only part of the bacterial population is capable of synthesizing polysaccharides, forming ring-like structures on surfaces. After the synthesis, most of the holdfast is attached to the cells.

Polysaccharides can not be purified using chemical purification

After successful holdfast biosynthesis, we aimed to purify the polysaccharides for further research and analysis. We adapted the only available C. crescentus holdfast chemical purification method and verified the results using dot blot assays with Heat Germ Aglutinnin (WGA) [22]. During the purification process, it was noticed that holdfast-producing cells stick together and can not be homogenized via pipetting or vortexing. Unfortunately, despite multiple attempts, we could not obtain pure holdfast material (Fig. 25.1-3.). None of the 3 attempts yielded significant differences between the CB2 system sample and the empty (control) sample. As the other experiments have showed, the E. coli with the CB2 system produces polysaccharides with distinct chemical features of holdfast attached to the cells, purification protocols specific for exopolysaccharide purification from the cell envelope could be tried upon the ring-formed cells but, sadly, not the protocol we used [23][24][25].

E. coli with holdfast synthesis pathway produce biofilm-like structures

Understanding that holdfast stays attached to the producing cells led us to the hypothesis that they ought to have some noticeable morphological differences. SEM analysis revealed that the polysaccharide-producing bacteria form tight conglomerates and differ morphologically compared to the control group. SEM pictures clearly show that holdfast-producing bacteria have altered cell wall morphology appearing bigger, and more “puffy” with uneven envelope topology. Moreover, the CB2 system containing bacteria seems to form large aggregates, sticking together into difficult-to-disrupt lumps. This characteristic was noticed beforehand in holdfast purification experiments, but only in SEM, it became clear that the underlying cause of the phenomenon is biofilm-like structures (Fig. 26. 1-4., Fig. 27. 1-3.) [18][19].

Fig.26.1. SEM pictures of negative control. Visible biofilm-like structures. Different locations of the same CB2 sample are shown.

Fig. 26.2. SEM pictures of holdfast-producing bacteria samples. Visible biofilm-like structures. Different locations of the same CB2 sample are shown.

Fig. 26.3. SEM pictures of holdfast-producing bacteria samples. Visible biofilm-like structures. Different locations of the same CB2 sample are shown.

Fig.26.4. SEM pictures of holdfast-producing bacteria samples. Visible biofilm-like structures. Different locations of the same CB2 sample are shown.

Fig.27.1. Close-up of Fig.26.4. showing the “crack” in the ring material revealing tightly associated bacteria forming biofilm-like structures. Picture analyzed using Fiji software.

Fig. 27.2. Close-up of Fig.26.4 showing the “crack” in the ring material revealing tightly associated bacteria forming biofilm-like structures. Picture analyzed using Fiji software.

Fig. 27.3. Close-up of Fig.26.4. showing the “crack” in the ring material revealing tightly associated bacteria forming biofilm-like structures. Picture analyzed using Fiji software.

Similar results were seen in flow cell bright-field microscopy, where it is noticeable that the CB2 sample has a considerable amount of big bacterial aggregates (Fig.28). Biofilm-like clumps noticeable in the video can not be homogenized by pipetting or vortexing, suggesting strong associations between cells. This could explain why CB2 sample bacteria were not evenly coating the glass slide, as the aggregates prevented enough bacteria from covering the bottom of the flow cell.

SEM and bright field microscopy analysis revealed that the ring material of E. coli cells producing the holdfast are morphologically different and produce biofilm-like structures.

Different E. coli strains form holdfast polysaccharide

We decided to test E. coli strain testing, we used C41(DE3), Rosetta(DE3) pLysS, and HMS174(DE3), and repeated the expression conditions with different IPTG concentrations (0.25, 0.5, and 0.75 mM) with protein expression for 3h at 37°C. After the expression, we added 1% glucose and left the flasks to incubate overnight at 30°C, 170 rpm. All of the flasks, after incubation, had prominent rings in CB2 system flasks compared to the control (Fig. 29.1-3.). However, the formed rings were not as prominent as the ones formed in BL21(DE3).

Fig.29.1. Photos showing formed rings after overnight incubation with 1% glucose in E. coli C41(DE3) strain with different IPTG concentrations.

Fig. 29.2. Photos showing formed rings after overnight incubation with 1% glucose in E. coli Rosetta(DE3) pLysS strain with different IPTG concentrations.

This indicated that even though these strains were not producing visible amounts of CB2 system proteins, the low amount of the expressed system was still sufficient enough to promote ring formation. As we ran out of time, we did not perform dot blot assays of the formed rings, but it is likely that they would also contain holdfast with N-acetyl-D-glucosamine as they appear during the same conditions used for BL21(DE3) rings production.

Suitable substrate search for holdfast production

We chose sugars that could be relevant to the polysaccharide composition: glucose, mannose, fructose, saccharose, N-acetyl-D-mannosamine, D-glucuronic acid, D-glucosamine, and xylose. After performing holdfast synthesis with standard conditions (in BL21(DE3)) optimized in earlier iterations, we observed the formation of rings with some substrates (Fig. 30.1-8). Notably, D-glucosamine substrate - a direct component of holdfast polysaccharide does not produce prominent rings, which indicates that it is incorporated into the holdfast as a N-acetyl-D-glucosamine that, after deacetylation with hfsH, only then becomes D-glucosamine (Fig. 30.7.) [28].

Fig. 30.1. A photo of culture flasks containing D-glucose substrate (number 1) with formed rings after overnight incubation at 30°C. CB2 system - bacteria containing holdfast synthesis pathway, empty system - bacteria without holdfast production system.

Fig. 30.2. A photo of culture flasks containing D-mannose substrate (number 2) with formed rings after overnight incubation at 30°C. CB2 system - bacteria containing holdfast synthesis pathway, empty system - bacteria without holdfast production system.

Fig. 30.3. A photo of culture flasks containing D-fructose substrate (number 3) with formed rings after overnight incubation at 30°C. CB2 system - bacteria containing holdfast synthesis pathway, empty system - bacteria without holdfast production system.

Fig. 30.4. A photo of culture flasks containing D-saccharose substrate (number 4) with formed rings after overnight incubation at 30°C. CB2 system - bacteria containing holdfast synthesis pathway, empty system - bacteria without holdfast production system.

Fig. 30.5. A photo of culture flasks containing N-acetyl-D-mannosamine substrate (number 5) with formed rings after overnight incubation at 30°C. CB2 system - bacteria containing holdfast synthesis pathway, empty system - bacteria without holdfast production system.

Fig. 30.6. A photo of culture flasks containing N-glucoronic acid substrate (number 6) with formed rings after overnight incubation at 30°C. CB2 system - bacteria containing holdfast synthesis pathway, empty system - bacteria without holdfast production system.

Fig. 30.7. A photo of culture flasks containing D-glucosamine substrate (number 7) with formed rings after overnight incubation at 30°C. CB2 system - bacteria containing holdfast synthesis pathway, empty system - bacteria without holdfast production system.

Fig. 30.8. . A photo of culture flasks containing N-xylsoe substrate (number 8) with formed rings after overnight incubation at 30°C. CB2 system - bacteria containing holdfast synthesis pathway, empty system - bacteria without holdfast production system.

Then, we examined the amount of holdfast produced with each monosaccharide substrate by dot blot assay (Fig. 31). As with visual representation of glucose formed rings (Fig. 30.1.), it appears that glucose is still the best choice as a substrate for holdfast production, with mannose coming second and D-glucosamine - third.

Additionally, crystal violet staining (see Experiments) experiments yielded similar results (repeated in 3 biological replicates), with glucose and mannose samples producing the largest amount of “biofilm”. This data was used in the model construction to estimate optimal substrate and its concentration (see Model).

Increasing holdfast production efficiency

We aimed to reduce the metabolic burden on the cell membrane for polysaccharide production. We decided to deactivate one of the metabolic pathways in E. coli that produces polysaccharides, allowing substrates to be redirected towards our desired polysaccharide synthesis.

Goal: Inactivate the Enterobacterial Common Antigen (ECA) pathway in E. coli, which produces polysaccharides, and evaluate its impact on polysaccharide production.

Results overview: One of the essential genes, wecA was knocked out from E.coli, and the ECA pathway was eliminated from these expression strains: BL21(DE3), Rosetta(DE3), and HMS174(DE3). The HMS174(DE3)ΔWecA strain, expressing the CB2 polysaccharide production system, exhibited slower growth than the wild-type strain, but it still produced a sufficient quantity of polysaccharides.

Holdfast Protein Expression and Production of Polysaccharides in HMS174(DE3)ΔwecA

CB2 holdfast system proteins are expressed, and polysaccharides were produced in HMS174(DE3)ΔWecA. We discovered that wecA gene deletion did not interfere with CB2 system protein production. Additionally, we compared it to the not edited HMS174(DE3) strain (Fig 32.).

We analyzed polysaccharide and ring production after overnight incubation with 1% glucose in an ECA-deficient HMS174(DE3)ΔWecA strain (Fig. 33). Although cells with the CB2 system grew slower than an empty control, they still produced rings with the polysaccharides, with thicker bands of the ring visible in figure 33.

Investigating the composition of the holdfast polysaccharide

We aimed to learn about the composition of holdfast polysaccharides by retracting one of the substrates synthesized by E.coli. We wanted to inactivate WecB - the enzyme that catalyzes the initial reaction in the production of the substrate UDP-N-acetyl mannosaminuronic acid (UDP-ManNAc), which is thought to be in the composition of our polysaccharide.

Results overview: The wecB gene was knocked out from E. coli expression strains BL21(DE3), Rosetta(DE3), and HMS174(DE3).

UDP-N-acetyl mannosaminuronic acid (UDP-ManNAc) is a sugar used to make polysaccharides in the CB2 system

CB2 holdfast system proteins are expressed but polysaccharides are not produced in HMS174(DE3)ΔwecB. We discovered that wecB gene deletion did not interfere with CB2 system protein production (Fig. 34).

We analyzed polysaccharide and ring production after overnight incubation with 1% glucose in the HMS174(DE3)ΔwecB strain. Cells with the CB2 system grew faster than unedited HMS174(DE3) and did not produce rings with polysaccharides. The flask with the E. coli containing the CB2 system looked the same as the E. coli without (Fig 35).

Additionally, we compared HMS174(DE3)ΔWecA with HMS174(DE3)ΔwecB containing CB2 system to see the difference of polysaccharide and ring production (Fig. 36).

FTIR analysis of the holdfast material

These experiments were done by our colleagues at the Vilnius University Center of Physical Sciences And Technology. Dr. Martynas Talaikis and Dr. Ilja Ignatjev. FTIR analysis reveals chemical properties of our holdfast. A shows the ATR-FTIR spectra of intact cell samples between the control and CB2 system samples (Fig.37.1.A). Since they are very similar, a difference spectrum was constructed by subtracting the control spectrum from the sample spectrum (Fig. 37.1.B) to highlight the differences. In the difference spectrum, the vibrational modes directed upwards belong to the CB2 sample. Vibrational modes of the amide functional group appear at 1618 cm-1 (Amide-I, C=O stretching), 1517 cm-1 (Amide-II, N-H in-plane bending), and 1235 cm-1 (Amide-III, N-H in-plane bending coupled with C-N stretching, and C-C stretching) were related to the amide fragment of N-Acetyl glucosamine. Another important vibration mode observed at 1169 cm-1 belongs to the C-O stretching vibration mode in glycosidic linkage, COH stretching, and C-H in-plane bending. Also, glycosidic linkage was observed at 978 cm-1 (symmetric C-O stretching), 931 cm-1 (C‑O stretching), and 833 cm-1 (C-C stretching of α-glycosidic linkage). All assignments were done according to literature [29][30]. All vibrational modes individually and collectively indicate the presence of polysaccharides. Figure 37.2 represent the control and sample spectra of cell debris with their difference spectra. The spectral assignment is similar to that presented in Figure 37.1. In both cases, the positive intensities in the difference spectra are related to sample-contained N-Acetyl glucosamine fragment and polysaccharides.

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