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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

BAIGT

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).

Part expression optimization

BL21(DE3)

KRX(DE3)

C41(DE3)

Rosetta(DE3) pLysS

Holdfast polymerization and export apparatus usage for holdfast synthesis

=Holdfast synthesis system expression optimization

BL21(DE3)

KRX(DE3)

C41(DE3)

Rosetta(DE3) pLysS

HMS174(DE3)

Holdfast biosynthesis

Holdfast synthesis requires glucose

Polysaccharides are produced only in the part of the population

Polysaccharides can not be purified using chemical purification

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

Different E. coli strains form holdfast polysaccharide

Suitable substrate search for holdfast production

Holdfast composition investigation

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

Increasing holdfast production efficiency

Investigating the composition of the holdfast polysaccharide

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

FTIR analysis of the holdfast material

References

1. Hendrickson, H., & Lawrence, J. G. (2000). Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites. FEMS Microbiology Reviews, 24(2), 177–183. https://doi.org/10.1111/j.1574-6976.2000.tb00539.x
2. Andrews, S. C., Robinson, A. K., & Rodríguez-Quiñones, F. (2004). Bacterial iron homeostasis. Journal of Bacteriology, 186(5), 1438–1447. https://doi.org/10.1128/jb.186.5.1438-1447.2004
3.Rabah, A., & Hanchi, S. (2023). Experimental and modeling study of the rheological and thermophysical properties of molybdenum disulfide-based nanofluids. Journal of Molecular Liquids, 384, 123335. https://doi.org/10.1016/j.molliq.2023.123335
4. Boutte, C. C., & Crosson, S. (2009). Bacterial lifestyle shapes stringent response activation. Journal of Bacteriology, 191(9), 2904-2912. https://doi.org/10.1128/jb.01003-08
5. Mackie, J., Liu, Y. C., & DiBartolo, G. (2019). The C-terminal region of the Caulobacter crescentus CtrA protein inhibits stalk synthesis during the G1-to-S transition. mBio, 10(2), e02273-18. https://doi.org/10.1128/mbio.02273-18
6.Thanbichler, M., & Shapiro, L. (2003). MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter. Journal of Bacteriology, 185(4), 1432-1442. https://doi.org/10.1128/jb.185.4.1432-1442.2003
7. Toh, E., Kurtz, H. D., & Brun, Y. V. (2008b). Characterization of the Caulobacter crescentus Holdfast Polysaccharide Biosynthesis Pathway Reveals Significant Redundancy in the Initiating Glycosyltransferase and Polymerase Steps. Journal of Bacteriology, 190(21), 7219–7231. https://doi.org/10.1128/jb.01003-08
8.Chepkwony, N. K., Hardy, G. G., & Brun, Y. V. (2022). HfaE Is a Component of the Holdfast Anchor Complex That Tethers the Holdfast Adhesin to the Cell Envelope. Journal of Bacteriology. https://doi.org/10.1128/jb.00273-22
9. Hershey, D. M., Fiebig, A., & Crosson, S. (2019). A Genome-Wide Analysis of Adhesion inCaulobacter crescentusIdentifies New Regulatory and Biosynthetic Components for Holdfast Assembly. mBio, 10(1). https://doi.org/10.1128/mbio.02273-18
10. Toh, E., Kurtz, H. D., & Brun, Y. V. (2008c). Characterization of the Caulobacter crescentus Holdfast Polysaccharide Biosynthesis Pathway Reveals Significant Redundancy in the Initiating Glycosyltransferase and Polymerase Steps. Journal of Bacteriology, 190(21), 7219–7231. https://doi.org/10.1128/jb.01003-08
11. Javens, J., Wan, Z., Hardy, G. G., & Brun, Y. V. (2013). Bypassing the need for subcellular localization of a polysaccharide export-anchor complex by overexpressing its protein subunits. Molecular Microbiology, 89(2), 350–371. https://doi.org/10.1111/mmi.12281