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C.Crescentus CB2/CB2A hfsE-hfsJ-hfsG - part of the polysaccharide tetrad assembly system

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 a part of the complete holdfast tetrad assembly system BBa_K5246043 used in Vilnius-Lithuania iGEM 2024 project "Synhesion" https://2024.igem.wiki/vilnius-lithuania/. This part can also be used separately for glycolipids consisting of glucose, mannosaminuronic acid, and N-acetyl-D-glucosamine synthesis, but this feature needs more detailed characterization.

Usage and 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] -->Full functional project operon assembly with this part and full operon characterization can be found in composite part BBa_K5246043.

Sequence and Features

Experimental characterization

Part cloning

All of the proteins composing this system are responsible for tetrad assembly. Since the system's proteins are found in the cytoplasm, we concluded that using a high-copy plasmid would ensure sufficient protein concentration for tetrasaccharide repeat synthesis and activation of the whole pathway. This would provide adequate substrate supply for polysaccharide polymerization and export.

To assemble specifically this part into BBa_K5246043 to then further assemble the holdfast synthesis pathway in E. coli , we had to assemble this part first into a backbone of pRSF-Duet-1. We designed a strategy to maximize the success of plasmid assembly by first assembling plasmids with 3 genes and, after verifying the sequences, integrating 3 left genes into that backbone (Fig. 1). In this way, we prevented Golden Gate assembly errors by trying to construct plasmids from 8 or more fragments.

The assembly was done using Golden Gate assembly with IIS AarI restriction enzyme sites introduced during PCR amplification. The backbone of pRSF-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. 2). 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 protocol. The reaction was later transformed into E. coli Mach1 (Thermo Scientific) competent cells. The assembly was then confirmed with colony PCR (Fig.3) and restriction digest analysis (Fig. 4) and positive colonies were sequenced.

-->Full functional project operon assembly with this part and full operon charazterization can be found in composite part BBa_K5246043.

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