Difference between revisions of "Part:BBa K5246001"

Latest revision as of 12:59, 29 September 2024

CB2/CB2A HfsA Part of export protein complex

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 export operon BBa_K5246044 and a part of full polymerization and export operon BBa_K5246046.

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]

Gene HfsA from Caulobacter crescentus encodes a protein of 502 amino acids from the complex that, in combination with HfsB, is responsible for the controlled polymerization of the holdfast polysaccharide.

Sequence and Features

Bioinformatical characterization

Bioinformatic analysis

Conservative Domain Database analysis revealed that the HfsA protein contains domains characteristic of the GumC superfamily, which typically function in exopolysaccharide export within the cell wall or membrane. Additionally, it shows significant similarity to the PEP-CTERM superfamily, proteins that are generally involved in determining polysaccharide chain length.

NCBI protein BLAST analysis revealed significant similarities between HfsA and a capsular polysaccharide biosynthesis protein from the ABC transporter family of Caldimonas thermodepolymerans. Using the DeepTMHMM tool to analyze its transmembrane structure, it was predicted that HfsA spans the membrane twice, embedding itself firmly in the cell envelope.

With AlphaFold3, we assessed different configurations of HfsA subunits, focusing on ipTM and pTM scores. 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. We hypothesize that the HfsA transmembrane protein consists of 8 subunits with confidence scores of ipTM = 0.72 and pTM = 0.73 (Fig.1).

Based on this data, we hypothesize that HfsA is a transmembrane protein that exports polysaccharides from the cell. Similar findings were proposed by earlier research. [7][8][9][10]

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--> More about this part's functionality as a part of a system you can see in this composite part BBa_K5246046.

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. Kurtz, H.D. and Smith, J. (1994) ‘The Caulobacter crescentus holdfast: Identification of holdfast attachment complex genes’, FEMS Microbiology Letters, 116(2), pp. 175–182. doi:10.1111/j.1574-6968.1994.tb06697.x.
8. Javens, J. et al. (2013) ‘Bypassing the need for subcellular localization of a polysaccharide export‐anchor complex by overexpressing its protein subunits’, Molecular Microbiology, 89(2), pp. 350–371. doi:10.1111/mmi.12281.
9. Smith, C.S. et al. (2003) ‘Identification of genes required for synthesis of the adhesive holdfast in Caulobacter crescentus’, Journal of Bacteriology, 185(4), pp. 1432–1442. doi:10.1128/jb.185.4.1432-1442.2003.
10. Toh, E., Kurtz, H.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. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2580695/