Difference between revisions of "Part:BBa K5246015"
 
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===Introduction===
 
===Introduction===
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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.
  
 
===Usage and Biology===
 
===Usage and Biology===
 +
<i>Hirschia baltica</i> is a common marine of the clade <i>Caulobacterales</i>. Its distinguishing feature is its dual lifestyle. Initially, <i>H. baltica</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]
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</p>
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<p style="font-size: 1em;">
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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].
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</p>
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<p style="font-size: 1em;">
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The <i>H. baltica</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>.
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</p>
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<p style="font-size: 1em;">
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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][9].
  
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Gene HfsC from Hirschia baltica, encoding a protein of 454 amino acids 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
+
 
 +
Gene HfsC from Hirschia baltica, encoding a protein of 454 amino acids polymeraze 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
  
 
===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K5246015 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5246015 SequenceAndFeatures</partinfo>
  
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===Functional Parameters===
 
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<partinfo>BBa_K5246015 parameters</partinfo>
 
  
 
===Experimental characterization===
 
===Experimental characterization===
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This part needs extensive characterization.
  
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====Bioinformatic analysis====
 
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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>.
 
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Protein BLAST also showed partial similarities with E.Coli O-antigen ligases suggested by the CDD analysis.
 
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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.
 
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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]
 
  
 
===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. 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>
 
<br>
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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.
+
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 17:25, 1 October 2024


HB 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.

Usage and Biology

Hirschia baltica is a common marine of the clade Caulobacterales. Its distinguishing feature is its dual lifestyle. Initially, H. baltica 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>

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 H. baltica 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][9]. Gene HfsC from Hirschia baltica, encoding a protein of 454 amino acids polymeraze 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

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 AgeI site found at 889
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental characterization

This part needs extensive characterization.


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.