Difference between revisions of "Part:BBa K5246003"

 
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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 export and polymerization operon <html><a href="https://parts.igem.org/Part:BBa_K5246045" target="_blank">BBa_K5246045</a></html> and a part of full polymerization and export operon <html><a href="https://parts.igem.org/Part:BBa_K5246046" target="_blank">BBa_K5246046</a></html>.
  
 
===Usage and Biology===
 
===Usage and Biology===
Gene from Caulobacter crescentus HfsC, encodes a protein of 422 amino acids that polymerases repeats of monomers into a mature holdfast polymer, deletion of polysaccharide polymerase gene hfsC in C.Crescentus didn't cause holdfast synthesis defects, because of its paralogue - HfsI. Double mutants of HfsC and HfsI cause severe holdfast synthesis defects
+
<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 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]
 +
 
 +
<HTML><P>Gene from <I>Caulobacter crescentus</I> <I>HfsC</I> encodes a protein of 422 amino acids that polymerases repeat monomers into a mature holdfast polymer. Deletion of polysaccharide polymerase gene HfsC in <I>C. crescentus</I> did not cause holdfast synthesis defects because of its paralogue - <HTML><b><a href="https://parts.igem.org/Part:BBa_K5246009" target="_blank">HfsI</a></b></html>. Double mutants of HfsC and HfsI cause severe holdfast synthesis defects. [7]</P>
  
 
===Sequence and Features===
 
===Sequence and Features===
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CDD analysis showed that only part of the protein resembles established conservative domains. The predicted domain is part of the O-antigen ligase family, which is a group of proteins responsible for outer membrane lipopolysaccharide synthesis in <i>E. coli</i>.  
 
CDD analysis showed that only part of the protein resembles established conservative domains. The predicted domain is part of the O-antigen ligase family, which is a group of proteins responsible for outer membrane lipopolysaccharide synthesis in <i>E. coli</i>.  
  
Protein BLAST also showed partial similarities with E.Coli O-antigen ligases suggested by the CDD analysis.
+
Protein BLAST also showed partial similarities with <I>E. coli</i> O-antigen ligases suggested by the CDD analysis.
  
 
DeepTMHMM predicted that the protein is embedded in the membrane, crossing it approximately 12 times. This prediction is supported by structural evidence from AlphaFold 3, which shows 12 helices. 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).
 
DeepTMHMM predicted that the protein is embedded in the membrane, crossing it approximately 12 times. This prediction is supported by structural evidence from AlphaFold 3, which shows 12 helices. 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).
  
Considering the data, HfsC is most probably a membrane protein that functions as a polysaccharide ligase due to its similarity to O-antigen ligase; this hypothesis is further supported by earlier research. [1][2]
+
Considering the data, HfsC is most probably a membrane protein that functions as a polysaccharide ligase due to its similarity to O-antigen ligase; earlier research further supports this hypothesis [8][9].
  
 
<center> https://static.igem.wiki/teams/5246/registry/hfsc.png </center>
 
<center> https://static.igem.wiki/teams/5246/registry/hfsc.png </center>
  
<center> <b> Fig. 1. </b> Alphafold 3 structure </center>
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<center> <b> Fig. 1. </b> Alphafold 3 structure of HfsC. On the left-folded structure with confidence scores above, on the right-predicted aligned error plot of the structure </center>
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 +
 
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<b>--> More about this part's functionality as a part of a system you can see in this composite part <html><a href="https://parts.igem.org/Part:BBa_K5246046" target="_blank">BBa_K5246046</a></html>.</b>
  
 
===References===
 
===References===
1. Smith, C.S. et al. (2003a) ‘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.  
+
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. Toh, E., Kurtz, H. D., & 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), 7219–7231. https://doi.org/10.1128/jb.01003-08
 +
<br>
 +
8. Smith, C.S. et al. (2003a) ‘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.
 +
<br>
 +
9. 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.
 
<br>
 
<br>
2. 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.
 

Latest revision as of 13:06, 29 September 2024


CB2/CB2A HfsC Polysaccharide polymerase

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 and polymerization operon BBa_K5246045 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 from Caulobacter crescentus HfsC encodes a protein of 422 amino acids that polymerases repeat monomers into a mature holdfast polymer. Deletion of polysaccharide polymerase gene HfsC in C. crescentus did not cause holdfast synthesis defects because of its paralogue - HfsI. Double mutants of HfsC and HfsI cause severe holdfast synthesis defects. [7]

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1072
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1072
    Illegal NotI site found at 745
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 491
    Illegal BglII site found at 863
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1072
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1072
    Illegal NgoMIV site found at 12
    Illegal NgoMIV site found at 109
    Illegal NgoMIV site found at 732
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental characterization

Bioinformatic analysis

CDD analysis showed that only part of the protein resembles established conservative domains. The predicted domain is part of the O-antigen ligase family, which is a group of proteins responsible for outer membrane lipopolysaccharide synthesis in E. coli.

Protein BLAST also showed partial similarities with E. coli O-antigen ligases suggested by the CDD analysis.

DeepTMHMM predicted that the protein is embedded in the membrane, crossing it approximately 12 times. This prediction is supported by structural evidence from AlphaFold 3, which shows 12 helices. 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).

Considering the data, HfsC is most probably a membrane protein that functions as a polysaccharide ligase due to its similarity to O-antigen ligase; earlier research further supports this hypothesis [8][9].

hfsc.png
Fig. 1. Alphafold 3 structure of HfsC. On the left-folded structure with confidence scores above, on the right-predicted aligned error plot of the structure


--> 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. Toh, E., Kurtz, H. D., & 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), 7219–7231. https://doi.org/10.1128/jb.01003-08
8. Smith, C.S. et al. (2003a) ‘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.
9. 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.