Difference between revisions of "Part:BBa K5246008"
 
(8 intermediate revisions by one other user not shown)
Line 4: Line 4:
  
 
===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.
  
 +
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===
−
HfsH encodes a cytoplasmic deacetylase of 257 amino acids long. Deacetylase belongs to carbohydrate esterase family 4, catalyze the hydrolysis of N-linked acetyl groups from GlcNAc residues. C.Crescentus HfsH mutants were completely devoid of holdfast material
+
<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]
−
Another C. crescentus CB15 mutant, &#916;hfsH (YB2198), was used to study the role of deacethylation in adhesion efficiency.
+
 
−
Indeed, this mutant is lacking the gene hfsH, encoding a deacetylase that affects both cohesive and adhesive properties of the holdfast.30 C. crescentus &#916;hfsH produces smaller holdfasts compared to the wild-type and the &#916;hfaB strains. These fully acetylated holdfasts are not anchored properly to the cell envelope and are shed in the medium. [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 main strain used in this study was Caulobacter crescentus CB15 &#916;hfaB (YB4251),29 a mutant strain from C. crescentus CB15 wild-type
+
 
−
(YB135). This mutant has a clean deletion of the hfaB gene and therefore does not synthesize HfaB, one of the holdfast anchor proteins. This strain still produces a holdfast, but is unable to anchor it to the cell envelope. As a consequence, the newly synthesized holdfast is shed in the culture medium and on surfaces. [3]
+
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]
 +
 
 +
 
 +
<i>HfsH</i> encodes a cytoplasmic deacetylase of 257 amino acids long. Deacetylase belongs to carbohydrate esterase family 4 and catalyzes the hydrolysis of N-linked acetyl groups from GlcNAc residues. <i>C. crescentus HfsH</i> mutants were completely devoid of holdfast material
 +
Another <i>C. crescentus CB15</i> mutant, <i>&#916;hfsH</i> (YB2198), was used to study the role of deacetylation in adhesion efficiency.
 +
Indeed, this mutant lacks the gene HfsH, encoding a deacetylase that affects both the cohesive and adhesive properties of the holdfast. <i>C. crescentus &#916;HfsH</i> produces smaller holdfast than the wild-type and the </i>&#916;HfsB</i> strains. These fully acetylated holdfasts are not anchored properly to the cell envelope and are shed in the medium. [8]
 +
The main strain used in this study was <i>Caulobacter crescentus CB15 &#916;hfaB</i> (YB4251),29 a mutant strain from <i>C. crescentus</i> CB15 wild-type (YB135). This mutant has a clean deletion of the HfaB gene and, therefore, does not synthesize HfaB, one of the holdfast anchor proteins. This strain still produces a holdfast but cannot anchor it to the cell envelope. Consequently, the newly synthesized holdfast is shed in the culture medium and on surfaces. [9]
 +
 
 +
<html>
 +
<body>
 +
<p>
 +
This part also has a his-tagged variant <a href="https://parts.igem.org/Part:BBa_K5246026">BBa_K5246026</a>.
 +
</p>
 +
</html>
  
 
===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K5246008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5246008 SequenceAndFeatures</partinfo>
  
−
 
−
===Functional Parameters===
 
−
<partinfo>BBa_K5246008 parameters</partinfo>
 
  
 
===Experimental characterization===
 
===Experimental characterization===
  
 +
====Bioinformatic analysis====
 +
 +
Conserved domain database analysis suggests that HfsH is part of the carbohydrate esterase 4 superfamily and polysaccharide deacetylase family. Proteins of this family may catalyze the N- or O- deacetylation of a substrate. Protein BLAST results show high similarity to peptidoglycan N-acetylglucosamine deacetylase and other polysaccharide deacetylases.
 +
 +
Topology analysis with DeepTMHMM and AlphaFold3 structure showed that HfsH is most probably a globular protein located in the cytoplasm. 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).
 +
 +
HfsH is a globular polysaccharide deacetylase that catalyzes the deacetylation of N-acetylglucosamine in the holdfast synthesis pathway; previous research supports our conclusions. [7][8][9]
 +
 +
<center> https://static.igem.wiki/teams/5246/registry/hfsh.png </center>
 +
 +
<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. Wan, Z. et al. (2013) ‘The adhesive and cohesive properties of a bacterial polysaccharide adhesin are modulated by a deacetylase’, Molecular Microbiology, 88(3), pp. 486–500. doi:10.1111/mmi.12199.
 +
<br>
 +
8. 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>
 +
9. Liu, Q. et al. (2022) ‘The screening and expression of polysaccharide deacetylase from caulobacter crescentus and its function analysis’, Biotechnology and Applied Biochemistry, 70(2), pp. 688–696. doi:10.1002/bab.2390.

Latest revision as of 09:56, 30 September 2024


CB2/CB2A HfsH Deacetylase

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] HfsH encodes a cytoplasmic deacetylase of 257 amino acids long. Deacetylase belongs to carbohydrate esterase family 4 and catalyzes the hydrolysis of N-linked acetyl groups from GlcNAc residues. C. crescentus HfsH mutants were completely devoid of holdfast material Another C. crescentus CB15 mutant, ΔhfsH (YB2198), was used to study the role of deacetylation in adhesion efficiency. Indeed, this mutant lacks the gene HfsH, encoding a deacetylase that affects both the cohesive and adhesive properties of the holdfast. C. crescentus ΔHfsH produces smaller holdfast than the wild-type and the </i>ΔHfsB</i> strains. These fully acetylated holdfasts are not anchored properly to the cell envelope and are shed in the medium. [8] The main strain used in this study was Caulobacter crescentus CB15 ΔhfaB (YB4251),29 a mutant strain from C. crescentus CB15 wild-type (YB135). This mutant has a clean deletion of the HfaB gene and, therefore, does not synthesize HfaB, one of the holdfast anchor proteins. This strain still produces a holdfast but cannot anchor it to the cell envelope. Consequently, the newly synthesized holdfast is shed in the culture medium and on surfaces. [9]

This part also has a his-tagged variant BBa_K5246026.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 10
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 10
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 10
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 10
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 10
    Illegal NgoMIV site found at 68
    Illegal NgoMIV site found at 92
    Illegal NgoMIV site found at 96
    Illegal NgoMIV site found at 169
    Illegal NgoMIV site found at 295
    Illegal NgoMIV site found at 456
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental characterization

Bioinformatic analysis

Conserved domain database analysis suggests that HfsH is part of the carbohydrate esterase 4 superfamily and polysaccharide deacetylase family. Proteins of this family may catalyze the N- or O- deacetylation of a substrate. Protein BLAST results show high similarity to peptidoglycan N-acetylglucosamine deacetylase and other polysaccharide deacetylases.

Topology analysis with DeepTMHMM and AlphaFold3 structure showed that HfsH is most probably a globular protein located in the cytoplasm. 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).

HfsH is a globular polysaccharide deacetylase that catalyzes the deacetylation of N-acetylglucosamine in the holdfast synthesis pathway; previous research supports our conclusions. [7][8][9]

hfsh.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. Wan, Z. et al. (2013) ‘The adhesive and cohesive properties of a bacterial polysaccharide adhesin are modulated by a deacetylase’, Molecular Microbiology, 88(3), pp. 486–500. doi:10.1111/mmi.12199.
8. 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. Liu, Q. et al. (2022) ‘The screening and expression of polysaccharide deacetylase from caulobacter crescentus and its function analysis’, Biotechnology and Applied Biochemistry, 70(2), pp. 688–696. doi:10.1002/bab.2390.