Difference between revisions of "Part:BBa K5246008"
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The main strain used in this study was Caulobacter crescentus CB15 ΔhfaB (YB4251),29 a mutant strain from C. crescentus CB15 wild-type | 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. [3] | (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. [3] | ||
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| + | This part also has a non his-tagged variant <a href="https://parts.igem.org/Part:BBa_K5246026">BBa_K5246026</a>. | ||
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===Sequence and Features=== | ===Sequence and Features=== | ||
Revision as of 12:55, 28 September 2024
CB2/CB2A HfsH Deacetylase
Introduction
Usage and Biology
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.30 C. crescentus ΔhfsH produces smaller holdfasts compared to the wild-type and the ΔhfaB strains. These fully acetylated holdfasts are not anchored properly to the cell envelope and are shed in the medium. [2] 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. [3]
This part also has a non his-tagged variant BBa_K5246026.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 10
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 10
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 10
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 10
- 25INCOMPATIBLE 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 - 1000COMPATIBLE 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. [1][2][3]
References
1. Wan, Z. et al. (2013a) ‘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.
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.
3. 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.