Difference between revisions of "Part:BBa K5246002"

Latest revision as of 12:57, 29 September 2024

CB2/CB2A HfsB 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 HfsB from Caulobacter crescentus that encodes a protein from complex that in combination with hfsA is responsible for controlled polymerization of holdfast polysaccharide. HfsB is a polysaccharide secretion autokinase.

Sequence and Features

Bioinformatical characterization

Bioinformatic analysis

Conservative Domain Database analysis revealed that the HfsB protein contains domains characteristic of the CpaE-like family. Members of this group contain proteins similar to the cpaE protein of the Caulobacter pilus assembly. Additionally, it resembles the eps_fam family, which typically describes the capsular exopolysaccharide proteins in bacteria. A number of these proteins regulate the exopolysaccharide biosynthesis (EPS).

NCBI protein BLAST analysis identified similarities between HfsB and several membrane-associated tyrosine-protein kinases. Using the DeepTMHMM tool to analyze its transmembrane structure, it was predicted that HfsB does not wholly cross the membrane. Instead, it is positioned on the inner side, only associated with the membrane, congruous with earlier hypotheses proposed in the literature.

When analyzed independently, protein structure prediction using AlphaFold 3 produced unclear results for HfsB. Since HfsB is part of the polysaccharide export complex, we attempted to fold it with HfsA, significantly improving the outcome. HfsB only folds correctly in the presence of HfsA, supporting the hypothesis that these two proteins function together as a single export unit. 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).

In summary, HfsB is probably a component of the membrane polysaccharide export apparatus that closely interacts with HfsA and is required for holdfast synthesis. [7][8][9]

-->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 <be> 7. 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.
8. Brown, P.J.B. et al. (2008) ‘Complex regulatory pathways coordinate cell-cycle progression and development in Caulobacter Crescentus’, Advances in Microbial Physiology, pp. 1–101. doi:10.1016/s0065-2911(08)00001-5.