Biophysical characterization of the outer membrane polysaccharide export protein and the polysaccharide co-polymerase protein from Xanthomonas campestris

Protein Expr Purif. 2014 Sep;101:42-53. doi: 10.1016/j.pep.2014.06.002. Epub 2014 Jun 10.

Abstract

This study investigated the structural and biophysical characteristics of GumB and GumC, two Xanthomonas campestris membrane proteins that are involved in xanthan biosynthesis. Xanthan is an exopolysaccharide that is thought to be a virulence factor that contributes to bacterial in planta growth. It also is one of the most important industrial biopolymers. The first steps of xanthan biosynthesis are well understood, but the polymerization and export mechanisms remain unclear. For this reason, the key proteins must be characterized to better understand these processes. Here we characterized, by biochemical and biophysical techniques, GumB, the outer membrane polysaccharide export protein, and GumC, the polysaccharide co-polymerase protein of the xanthan biosynthesis system. Our results suggested that recombinant GumB is a tetrameric protein in solution. On the other hand, we observed that both native and recombinant GumC present oligomeric conformation consistent with dimers and higher-order oligomers. The transmembrane segments of GumC are required for GumC expression and/or stability. These initial results provide a starting point for additional studies that will clarify the roles of GumB and GumC in the xanthan polymerization and export processes and further elucidate their functions and mechanisms of action.

Keywords: Exopolysaccharide; Membrane proteins; Xanthan; Xanthomonas campestris.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Outer Membrane Proteins / analysis
  • Bacterial Outer Membrane Proteins / chemistry
  • Bacterial Outer Membrane Proteins / genetics*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Carrier Proteins / analysis
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics*
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism*
  • Polysaccharides, Bacterial / biosynthesis
  • Proteolysis
  • Recombinant Fusion Proteins / analysis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Xanthomonas campestris / enzymology*
  • Xanthomonas campestris / genetics
  • Xanthomonas campestris / metabolism

Substances

  • Bacterial Outer Membrane Proteins
  • Bacterial Proteins
  • Carrier Proteins
  • Membrane Transport Proteins
  • Polysaccharides, Bacterial
  • Recombinant Fusion Proteins
  • xanthan gum