Part:BBa_K5246009

CB2/CB2A HfsI Polysaccharide polymerase

Usage and Biology

Gene from Caulobacter crescentus that codes a paralogous protein of 440 amino acids of HfsC polysaccharide polymerase. HfsI that polymerases repeats of monomers into a mature holdfast polymer, deletion of polysaccharide polymerase gene hfsI in C.Crescentus didn't cause holdfast synthesis defects, because of its paralogue - HfsC. Double mutants of HfsI and HfsC had severe holdfast synthesis defects.

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 1034
  Illegal XhoI site found at 448
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 513
  Illegal AgeI site found at 228
  
• 1000
  COMPATIBLE WITH RFC[1000]

Experimental characterization

Bioinformatic analysis

CDD analysis showed that HfsI, analogous to HfsC, has a domain similar to that of O-antigen ligase family proteins. Proteins of this family are responsible for outer membrane lipopolysaccharide synthesis in E. Coli.

Protein BLAST also showed partial similarities with E.Coli O-antigen ligases suggested by the CDD analysis.

DeepTMHMM predicted that the protein is embedded in the membrane, crossing it approximately 12 times. This prediction is supported by structural evidence from AlphaFold3, 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 (Fig.1).

Considering our findings with information present in the literature. We predict that HfsI is a paralogous gene to HfsC with the same functionality in the organism. [1][2][3]

Fig. 1. AlphaFold 3 structure showing

References

1. 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.
2. 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.
3. Chepkwony, N.K., Berne, C. and Brun, Y.V. (2019b) ‘Comparative analysis of ionic strength tolerance between freshwater and marine Caulobacterales adhesins’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00061-19.