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CB2/CB2A HfsE Integral membrane glycosyltransferases

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 holdfast tetrad assembly 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] Encodes a protein of 512 amino acids, integral membrane glycosyltransferases from the polyisoprenylphosphate hexose-1-phosphate transferase (PHPT) family , transferrs hexose-1-phosphate residues from UDP-hexoses to the lipid carrier molecule undecaprenol phosphate in the inner membrane. In C.Crescentus catalyzes the first step in polysaccharide biosynthesis by transferring glucose-1-phosphate residues from UDP-glucoses to the lipid carrier molecule undecaprenol phosphate in the inner membrane Studies using hfsE mutants showed minimal changes in holdfast synthesis and bacterial adhesion. Through further genome analysis two paralogs, pssY and pssZ, were identified as potential substitutes for the hfsE protein in holdfast synthesis. However, double combination mutants did not have a substantial decrease of neither surface adherence nor inability to synthesize holdfast, but had a slight misplacement at the tip of the stalk. While hfsE, pssY and pssZ triple deletion mutant, was unable to produce holdfast or adhere to surfaces. [8]" -->This part is also a part of the holdfast tetrad assembly operon BBa_K5246041 and a part of full holdfast tetrad assembly operon BBa_K5246043.

Sequence and Features

Experimental characterization

Bioinformatic analysis

According to CDD, protein is similar to undecaprenyl-phosphate glucose phosphotransferases found in E. coli. Most of the genes for these proteins are found within large operons dedicated to the production of complex exopolysaccharides such as the enterobacterial O-antigen. It also has some overlap with other bacterial sugar-transferases.

Protein BLAST further supports the prediction of HfsE being a UDP-glucose-carrier transferase because of its similarities with multiple glucose transferases.

HfsE is likely a transmembrane protein that traverses the membrane six times, with a portion of it exposed on the cytoplasmic side.

AlphaFold 3 structure confidence scores suggest that it is a protein made mostly of alpha helices. 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 prediction (Fig.1).

Altogether, HfsE is a membrane protein that is responsible for the first glucose addition to an undecaprenyl-phosphate lipid carrier in the holdfast synthesis pathway, similar to bacterial glucose transferases. Analogous HfsE function is proposed in the literature by earlier 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. Patel, K.B. et al. (2012) ‘Functional characterization of UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferases of Escherichia coli and Caulobacter crescentus’, Journal of Bacteriology, 194(10), pp. 2646–2657. doi:10.1128/jb.06052-11.
8. Chepkwony, N.K., Berne, C. and Brun, Y.V. (2019b) ‘Comparative analysis of ionic strength tolerance between freshwater and marine Caulobacterales adhesins’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00061-19.