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CB2/CB2A HfsK Acetyltransferase, 6xHis tag for purification

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 protein is part of the Tetrad assembly system BBa_K5246043 and operon responsible for addition of N-acetyl-D-glucosamine and deacetylation BBa_K5246042.

Part was used in Vilnius-Lithuania iGEM 2024 project "Synhesion" https://2024.igem.wiki/vilnius-lithuania/.

This part also has a non 6xhis-tagged variant BBa_K5246011.

Biology and usage

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].

HfsK in particular is responsible for deacetylation N-acetylglucosamine.

HfsK

The HfsK gene from Caulobacter crescentus encodes a 359 amino acid acetyltransferase protein. HfsK is a c-di-GMP effector involved in holdfast biogenesis. Cells lacking HfsK form highly malleable holdfast structures with reduced adhesive strength that cannot support surface colonization. HfsK is a soluble protein that associates with the cell membrane during most of the cell cycle but is transferred to the cytosol in the process of holdfast synthesis. HfsK deacetylates N-acetyl-glucosamine from the holdfast, which results in better adhesive properties.

Usage

Proteins of the holdfast synthesis system assemble a short chain of sugar monomers in a specific sequence on a lipid carrier - a glycolipid.

Glycolipids are predominantly located on the extracellular surface of eukaryotic cell membranes and are responsible for various functions such as receptors for viruses and other pathogens, allowing them to enter a specific host cell that has unique glycolipid markers. This feature can let us use said glycolipids as labels for a precise and targeted liposome distribution throughout the body, delivering anything from cancer drugs to gene editing systems directly to the target cells.

To create a liposome labeling system, we had to select specific proteins that could be utilized for this purpose. Following bioinformatics analysis using the Conserved Domain Database, Protein BLAST, DeepTMHMM, and AlphaFold 3, we identified five proteins of interest from each strain: HfsG, HfsH, HfsJ, HfsK, and HfsL.

To utilize these enzymes, it was essential to develop a suitable purification strategy. For efficient cloning, we chose Golden Gate assembly. For efficient purification, we selected immobilized ion affinity chromatography (IMAC) as our purification method, based on recommendations from one of the few available papers where C. crescentus proteins were expressed and purified from E. coli. We opted for conventional 6x histidine tags (his-tag) to facilitate straightforward purification. It was crucial to determine the appropriate terminus for 6xHis-tag insertion to avoid disrupting the protein conformation and lessening purification efficiency.

Sequence and Features

Experimental characterization

Bioinformatic analysis

Using CDD analysis, it was identified that HfsK is similar to the GNAT N-acetyltransferase family. Its domains suggest that HfsK is part of the Bcls superfamily. Acetyltransferases of this superfamily are usually involved in cellulose biosynthesis. Protein BLAST did not give conclusive results, which could result from a unique HfsK protein amino acid sequence and structure.

DeepTMHMM's protein topology predictions showed that HfsK is most likely a globular protein located on the cytoplasmic side of the membrane.

High confidence scores of AlphaFold 3 structures suggest that HfsK is likely a globular protein. 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).

To summarise, HfsK is most likely a globular N-acetyltransferase. Earlier evidence, combined with our findings, suggests that it plays a role in the deacetylation of N-acetylglucosamine within the holdfast synthesis pathway. [7][8][9]

Protein expression

CB2 strain

We chose the BL21(DE3) strain for adjustable and efficient expression of target proteins since the system's proteins were best expressed in this strain. Given the lack of time, we went with conditions optimized beforehand in earlier experiments for the whole pathway expression: temperature of 37°C, induction with 0.5 mM IPTG concentration, and expression for 3 hours.

After SDS-PAGE gel analysis, we concluded that we successfully expressed all HfsG, HfsH, HfsJ, HfsK, and HfsL proteins from C. crescentus CB2 and CB2A strains. We noticed that HfsJ and HfsL glycosyltransferases were visible in lower quantities compared to the other proteins. Both of these protein expression conditions need to be further investigated and optimized.

HfsK is visible on the right side of the gel (Fig. 2).

Table 1. C. crescentus protein sizes in kDa

Protein Name	Size (kDa)
HfsG	34
HfsH	27.9
HfsJ	34.7
HfsK	43.3
HfsL	33.3

CB2A strain

We chose the BL21(DE3) strain for adjustable and efficient expression of target proteins since the system's proteins were best expressed in this strain. Given the lack of time, we went with conditions optimized beforehand in earlier experiments for the whole pathway expression: temperature of 37°C, induction with 0.5 mM IPTG concentration, and expression for 3 hours.

After SDS-PAGE gel analysis, we concluded that we successfully expressed all HfsG, HfsH, HfsJ, HfsK, and HfsL proteins from C. crescentus CB2 and CB2A strains. We noticed that HfsJ and HfsL glycosyltransferases were visible in lower quantities compared to the other proteins. Both of these protein expression conditions need to be further investigated and optimized.

HfsK is visible on the right side of the gel (Fig. 2).

Table 2. C. crescentus protein sizes in kDa

Protein Name	Size (kDa)
HfsG	34
HfsH	27.9
HfsJ	34.7
HfsK	43.3
HfsL	33.3

Protein purification

CB2 strain

After successful expression, we proceeded to work on the purification of his-tagged proteins. We went with immobilized metal ion affinity chromatography (IMAC). Adapting protocols from the little research that was available, we used HisPur Ni-NTA Spin Columns (Thermo Scientific). Equilibration, wash, and elution buffers contained 10 mM Tris pH 7.4, 150 mM NaCl, and 10 mM, 75 mM, and 500 mM imidazole, respectively.

HfsK protein was successfully purified and is clearly seen in the elution fraction.

CB2A strain

After successful expression, we proceeded to work on the purification of his-tagged proteins. We went with immobilized metal ion affinity chromatography (IMAC). Adapting protocols from the little research that was available, we used HisPur Ni-NTA Spin Columns (Thermo Scientific). Equilibration, wash, and elution buffers contained 10 mM Tris pH 7.4, 150 mM NaCl, and 10 mM, 75 mM, and 500 mM imidazole, respectively.

HfsK protein was successfully purified and is clearly seen in the elution fraction.

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. Chepkwony, N.K. and Brun, Y.V. (2021) ‘A polysaccharide deacetylase enhances bacterial adhesion in high-ionic-strength environments’, iScience, 24(9), p. 103071. doi:10.1016/j.isci.2021.103071.
8. Sprecher, K.S. et al. (2017) ‘Cohesive properties of the Caulobacter crescentus holdfast adhesin are regulated by a novel C-di-GMP effector protein’, mBio, 8(2). doi:10.1128/mbio.00294-17.
9. Hershey, D.M., Fiebig, A. and Crosson, S. (2019) ‘A genome-wide analysis of adhesion in Caulobacter crescentus identifies new regulatory and biosynthetic components for holdfast assembly’, mBio, 10(1). doi:10.1128/mbio.02273-18.