Difference between revisions of "Part:BBa K5246023"
 
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<partinfo>BBa_K5246023 short</partinfo>
 
<partinfo>BBa_K5246023 short</partinfo>
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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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<p>
 
This HfsK gene from Hirschia baltica codes a 371 amino acid protein. This protein is a GNAT family N-acetyltransferase that catalyzes the transfer of an acetyl group from acetyl-CoA to a substrate. Research investigating the H. baltica genome found that hfsK and its paralogs are found outside the hfs locus.
 
This HfsK gene from Hirschia baltica codes a 371 amino acid protein. This protein is a GNAT family N-acetyltransferase that catalyzes the transfer of an acetyl group from acetyl-CoA to a substrate. Research investigating the H. baltica genome found that hfsK and its paralogs are found outside the hfs locus.
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Color coding corresponds to homologs and paralogs. Hash marks indicate genes found in a different location in the genome. [1] It is predicted that this is an intracellular protein.
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Color coding corresponds to homologs and paralogs. Hash marks indicate genes found in a different location in the genome. [1] It is predicted that this is an intracellular protein.</P>
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===Experimental characterization===
 
===Experimental characterization===
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<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_K5246033">BBa_K5246033</a>.
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</p>
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</html>
  
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====Bioinformatic analysis====
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This part needs more characterization.
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Using CDD analysis, it was identified that HfsK is similar to the GNAT N-acetyltransferase family. Its domains suggest 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.
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DeepTMHMM's protein topology predictions showed that HfsK is most likely a globular protein located on the cytoplasmic side of the membrane.
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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.
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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. [1][2][3]
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===References===
 
===References===
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1. 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.  
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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
 +
<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
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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
 +
<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>
 
<br>
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2. 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.  
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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.  
 
<br>
 
<br>
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3. 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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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 17:47, 1 October 2024


HB HfsK Acetyltransferase

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].

This HfsK gene from Hirschia baltica codes a 371 amino acid protein. This protein is a GNAT family N-acetyltransferase that catalyzes the transfer of an acetyl group from acetyl-CoA to a substrate. Research investigating the H. baltica genome found that hfsK and its paralogs are found outside the hfs locus. Color coding corresponds to homologs and paralogs. Hash marks indicate genes found in a different location in the genome. [1] It is predicted that this is an intracellular protein.

This part also has a his-tagged variant BBa_K5246033.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 988
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1038
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental characterization

This part also has a his-tagged variant BBa_K5246033.

This part needs more 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.