Part:BBa_K5246004

CB2/CB2A HfsD 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 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 HfsD from Caulobacter crescentus encodes an integral outer membrane protein of 247 amino acids which is essential for holdfast export and transfer to the anchoring proteins.

===Sequence and Features=== BBa_K5246004 SequenceAndFeatures ===Experimental characterization=== ====Bioinformatic analysis==== Conservative Domain Database analysis identified HfsD as part of the polysaccharide biosynthesis/export family, which is linked to PEP-CTERM system proteins involved in polysaccharide export. Additionally, HfsD shows significant similarity to E. coli Wza periplasmic proteins, which play a role in cell membrane and wall formation. Protein BLAST indicated similarities with several E.Coli export channel proteins. Using the DeepTMHMM tool to analyze its transmembrane structure, it was predicted that HfsD is probably globular and does not cross the inner membrane. Instead, it is positioned outside of it in the periplasm. This is consistent with the CDD findings of similarities between HfsD and periplasmic Wza proteins. AlphaFold 3 resulted in a high-quality structure of HfsD with some less well-characterized regions (orange and yellow). 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). https://static.igem.wiki/teams/5246/registry/hfsd-1.png Fig. 1. Alphafold 3 structure showing 8 subunits structure of hfsD. On the left-folded structure with confidence scores above, on the right-predicted aligned error plot of the structure Considering HfsD similarity with PEP-CTERM-associated proteins and the fact that it should be a part of the export complex, we tried to fold it together with HfsA and HfsB (Fig.2). https://static.igem.wiki/teams/5246/registry/hfsd-2.png Fig. 2. Alphafold 3 structure showing hfsD in complex with hfsA and hfsB. On the left-folded structure with confidence scores above, on the right-predicted aligned error plot of the structure Due to AlphaFold 3's computational limitations, we could not fully assemble the entire export apparatus. However, the confidence in the predicted structures indicates that HfsA, HfsB, and HfsD together form a tunnel-like complex in the membrane for polysaccharide export. Conservative Domain Database and protein BLAST analyses identified HfsD as part of the polysaccharide export family, similar to E. coli Wza proteins. DeepTMHMM predicted that HfsD is globular and located in the periplasm. The predicted Alphafold 3 structures suggest that HfsA, HfsB, and HfsD form a tunnel-like complex for polysaccharide export. Our findings correspond with earlier research [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. 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. 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.