Difference between revisions of "Part:BBa K5246013"
 
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===Introduction===
 
===Introduction===
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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 - <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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Part was used in Vilnius-Lithuania iGEM 2024 project "Synhesion" <HTML><b><a href="https://2024.igem.wiki/vilnius-lithuania" target="_blank">https://2024.igem.wiki/vilnius-lithuania/</a></b></html>.
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<html>
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<p style="font-size: 1em;">
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This part also has a non 6xhis-tagged variant <a href="https://parts.igem.org/Part:BBa_K5246012">BBa_K5246012</a>.
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</p>
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</html>
  
 
===Usage and Biology===
 
===Usage and Biology===
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<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 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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The HfsA gene encodes a 496 amino acid protein in Hirschia baltica, which as a complex togethery with HfsB controls the polymerisation of holdfast pollysaccharide. HfsA, HfsB, HfaD and HfsE proteins in combination are resposible for holdfast association with the cell envelope.
 
The HfsA gene encodes a 496 amino acid protein in Hirschia baltica, which as a complex togethery with HfsB controls the polymerisation of holdfast pollysaccharide. HfsA, HfsB, HfaD and HfsE proteins in combination are resposible for holdfast association with the cell envelope.
  
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===Experimental characterization===
 
===Experimental characterization===
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This part needs extensive characterization.
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====Bioinformatic analysis====
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Conservative Domain Database analysis revealed that the HfsA protein contains domains characteristic of the GumC superfamily, which typically function in exopolysaccharide export within the cell wall or membrane. Additionally, it shows significant similarity to the PEP-CTERM superfamily, proteins that are generally involved in determining polysaccharide chain length.
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NCBI protein BLAST analysis revealed significant similarities between HfsA and a capsular polysaccharide biosynthesis protein from the ABC transporter family of <i>Caldimonas thermodepolymerans</i>.
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Using the DeepTMHMM tool to analyze its transmembrane structure, it was predicted that HfsA spans the membrane twice, embedding itself firmly in the cell envelope.
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With AlphaFold 3, we assessed different configurations of HfsA subunits, focusing on ipTM and pTM scores. 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 We hypothesise that HfsA transmembrane protein consists of 8 subunits with confidence scores of ipTM = 0.72 and pTM = 0.73.
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Based on this data, we hypothesize that HfsA is a transmembrane protein responsible for exporting polysaccharides from the cell. Similar findings were proposed by earlier research. [1][2][3][4]
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===References===
 
===References===
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1. Kurtz, H.D. and Smith, J. (1994) ‘The Caulobacter crescentushold fast: Identification of holdfast attachment complex genes’, FEMS Microbiology Letters, 116(2), pp. 175–182. doi:10.1111/j.1574-6968.1994.tb06697.x.  
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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
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<br>
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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
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<br>
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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
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<br>
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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
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<br>
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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
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<br>
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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
 
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2. 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.  
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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.  
 
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3. 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.  
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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.  
 
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4. Toh, E., Kurtz, H.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. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2580695/
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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 17:15, 1 October 2024


HB HfsA 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.


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_K5246012.

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]. The HfsA gene encodes a 496 amino acid protein in Hirschia baltica, which as a complex togethery with HfsB controls the polymerisation of holdfast pollysaccharide. HfsA, HfsB, HfaD and HfsE proteins in combination are resposible for holdfast association with the cell envelope.

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
    COMPATIBLE WITH RFC[25]
  • 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.