Part:BBa_K5246007
CB2/CB2A HfsG Glycosyltransferase
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 part is also a part of the holdfast tetrad assembly operon BBa_K5246041 and a part of full polymerization and export operon BBa_K5246043.
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] HfsG gene encodes a cytoplasmic protein of 309 aa homologous to family 2 glycosyltransferases. Transferrs sugar units from UDP-GlcNAc to oligosaccharide, catalyzes the polymerization of GlcNAc. C. crescentus HfsG mutants were completely devoid of holdfast material -->This part is also a part of the holdfast tetrad assembly operon BBa_K5246041 and a part of full polymerization and export operon BBa_K5246043.
This part also has a his-tagged variant BBa_K5246025.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 825
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 28
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 480
- 1000COMPATIBLE WITH RFC[1000]
Experimental characterization
Bioinformatic analysis
CDD and protein BLAST analysis suggest that HfsG is a glucosyltransferase family 2 protein. Proteins of this family are involved in cell wall biosynthesis. HfsG is similar to the WecA protein in E.Coli that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate. In the case of HfsG, it catalyzes the transfer of UDP-GlcNAc to the sugar acceptor made earlier in the holdfast synthesis pathway.
Protein topology analysis using DeepTMHMM suggests that HfsG is a globular protein located in the cytoplasm. AlphaFold 3 structures further confirm it. A pTM score above 0.5 suggests that the predicted overall structure may closely resemble the true protein fold, while ipTM indicates the accuracy of the subunit positioning within the complex. Values higher than 0.8 represent confident, high-quality predictions (Fig. 1).
HfsG is family 2 glycosyltransferase similar to WecA of E. coli. This globular protein transfers UDP-GlcNAc to the acceptor molecule. Our conclusions are in agreement with existing research. [7][8][9]
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, Harry D. and Brun, Y.V. (2008) ‘Characterization of the Caulobacter crescentus holdfast polysaccharide biosynthesis pathway reveals significant redundancy in the initiating glycosyltransferase and polymerase steps’, Journal of Bacteriology, 190(21), pp. 7219–7231. doi:10.1128/jb.01003-08.
8. Hardy, G.G. et al. (2018) ‘Mutations in sugar-nucleotide synthesis genes restore holdfast polysaccharide anchoring to Caulobacter crescentus holdfast anchor mutants’, Journal of Bacteriology, 200(3). doi:10.1128/jb.00597-17.
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