Difference between revisions of "Part:BBa K5246012"
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===Introduction===
 
===Introduction===
 +
Vilnius-Lithuania iGEM 2024 project <HTML><b><a href="https://2024.igem.wiki/vilnius-lithuania" target="_blank">Synhesion</a></b></html> 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 - <I> C. crescentus </I> and <I> H. baltica </I> - harness 12 protein synthesis pathways to produce sugars, anchoring them to the surfaces. We aimed to transfer the polysaccharide synthesis pathway to industrially used <I>E. coli</I> bacteria to produce adhesives. Our team concomitantly focused on creating a novel <I>E. coli</I> strain for more efficient production of adhesives.
  
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This part is also a part of the holdfast tetrad assembly operon <html><a href="https://parts.igem.org/Part:BBa_K5246042" target="_blank">BBa_K5246042</a></html> and a part of full holdfast tetrad assembly operon <html><a href="https://parts.igem.org/Part:BBa_K5246043" target="_blank">BBa_K5246043</a></html>.
  
 
===Usage and Biology===
 
===Usage and Biology===
−
TBA
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<i>Caulobacter crescentus</i> is a common freshwater gram-negative oligotrophic bacterium of the clade <i>Caulobacterales</i>. Its distinguishing feature is its dual lifestyle. Initially, <i>C. crescentus</I> 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]
 +
 
 +
<p><i>Caulobacterales</i> 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 <i>Caulobacter crescentus</i>, 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 <i>C. crescentus</i> holdfast is produced via a polysaccharide synthesis and export pathway similar to the group I capsular polysaccharide synthesis Wzy/Wzx-dependent pathway in <i>Escherichia coli</i>.
 +
The holdfast synthesis (<i>Hfs</i>) genes include those encoding predicted glycosyltransferases, carbohydrate modification factors, and components of a wzy-type polysaccharide assembly pathway. [4][5][6]
 +
 
 +
<b>HfsL</b> in particular is responsible for N-acetyl-D-glucosamine transfer to the acceptor molecule.
 +
 
 +
Gene HfsL from Caulobacter crescentus encodes a protein of 305aa. HfsL is one of the glucotransferases involved in the holdfast synthesis pathway and is responsible for addition of N-acetyl-D-glucosamine that is fourth monomer on the sugar tetrad in the beginning of holdfast synthesis
 +
 
 +
<html>
 +
<body>
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<p>
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This part also has a his-tagged variant <a href="https://parts.igem.org/Part:BBa_K5246029">BBa_K5246029</a>.
 +
</p>
 +
</html>
  
 
===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K5246012 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5246012 SequenceAndFeatures</partinfo>
  
−
 
−
===Functional Parameters===
 
−
<partinfo>BBa_K5246012 parameters</partinfo>
 
  
 
===Experimental characterization===
 
===Experimental characterization===
  
 +
====Bioinformatic analysis====
 +
 +
CDD analysis showed specific hits in glycosyl transferase family 2. This diverse family transfers sugar from UDP-glucose, UDP-N-acetyl-galactosamine, GDP-mannose, or CDP-abequose to a range of substrates. Protein BLAST further supports these findings and suggests that HfsL is most likely a family 2 glycosyltransferase, which has a domain very similar to the poly-beta-1,6-N-acetyl-D-glucosamine synthase domain of biofilm PGA synthase.
 +
 +
DeepTMHMM analysis suggests that the protein is likely globular and positioned on the inner side of the cell membrane. The AlphaFold3 structure provides additional evidence supporting its globular shape. 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 sum up, HfsL is most probably a globular family 2 glycosyltransferase, responsible for N-acetyl-D-glucosamine transfer to the acceptor molecule, as is further verified by existing research. [7][8][9]
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<center> https://static.igem.wiki/teams/5246/registry/hfsl.png </center>
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<center> <b> Fig. 1. </b> AlphaFold 3 structure showing  </center>
  
 
===References===
 
===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
 +
<br>
 +
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
 +
<br>
 +
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
 +
<br>
 +
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
 +
<br>
 +
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
 +
<br>
 +
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
 +
<br>
 +
 +
7. 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.
 +
<br>
 +
8. Chepkwony, N.K., Hardy, G.G. and Brun, Y.V. (2022) ‘HFAE is a component of the holdfast anchor complex that tethers the holdfast adhesin to the cell envelope’, Journal of Bacteriology, 204(11). doi:10.1128/jb.00273-22.
 +
<br>
 +
9. Chepkwony, N.K., Berne, C. and Brun, Y.V. (2019) ‘Comparative analysis of ionic strength tolerance between freshwater and marine Caulobacterales adhesins’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00061-19.

Latest revision as of 16:35, 1 October 2024


CB2/CB2A HfsL Glycosyltransferase

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_K5246042 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] HfsL in particular is responsible for N-acetyl-D-glucosamine transfer to the acceptor molecule. Gene HfsL from Caulobacter crescentus encodes a protein of 305aa. HfsL is one of the glucotransferases involved in the holdfast synthesis pathway and is responsible for addition of N-acetyl-D-glucosamine that is fourth monomer on the sugar tetrad in the beginning of holdfast synthesis

This part also has a his-tagged variant BBa_K5246029.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 235
    Illegal NgoMIV site found at 378
    Illegal NgoMIV site found at 609
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental characterization

Bioinformatic analysis

CDD analysis showed specific hits in glycosyl transferase family 2. This diverse family transfers sugar from UDP-glucose, UDP-N-acetyl-galactosamine, GDP-mannose, or CDP-abequose to a range of substrates. Protein BLAST further supports these findings and suggests that HfsL is most likely a family 2 glycosyltransferase, which has a domain very similar to the poly-beta-1,6-N-acetyl-D-glucosamine synthase domain of biofilm PGA synthase.

DeepTMHMM analysis suggests that the protein is likely globular and positioned on the inner side of the cell membrane. The AlphaFold3 structure provides additional evidence supporting its globular shape. 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 sum up, HfsL is most probably a globular family 2 glycosyltransferase, responsible for N-acetyl-D-glucosamine transfer to the acceptor molecule, as is further verified by existing research. [7][8][9]

hfsl.png
Fig. 1. AlphaFold 3 structure showing

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. 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.
8. Chepkwony, N.K., Hardy, G.G. and Brun, Y.V. (2022) ‘HFAE is a component of the holdfast anchor complex that tethers the holdfast adhesin to the cell envelope’, Journal of Bacteriology, 204(11). doi:10.1128/jb.00273-22.

9. Chepkwony, N.K., Berne, C. and Brun, Y.V. (2019) ‘Comparative analysis of ionic strength tolerance between freshwater and marine Caulobacterales adhesins’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00061-19.