Xanthan chain length is modulated by increasing the availability of the polysaccharide copolymerase protein GumC and the outer membrane polysaccharide export protein GumB

Abstract

Xanthan is a polysaccharide secreted by Xanthomonas campestris that contains pentameric repeat units. The biosynthesis of xanthan involves an operon composed of 12 genes (gumB to gumM). In this study, we analyzed the proteins encoded by gumB and gumC. Membrane fractionation showed that GumB was mainly associated with the outer membrane, whereas GumC was an inner membrane protein. By in silico analysis and specific globomycin inhibition, GumB was characterized as a lipoprotein. By reporter enzyme assays, GumC was shown to contain two transmembrane segments flanking a large periplasmic domain. We confirmed that gumB and gumC mutant strains uncoupled the synthesis of the lipid-linked repeat unit from the polymerization process. We studied the effects of gumB and gumC gene amplification on the production, composition and viscosity of xanthan. Overexpression of GumB, GumC or GumB and GumC simultaneously did not affect the total amount or the chemical composition of the polymer. GumB overexpression did not affect xanthan viscosity; however, a moderate increase in xanthan viscosity was achieved when GumC protein levels were increased 5-fold. Partial degradation of GumC was observed when only that protein was overexpressed; but co-expression of GumB and GumC diminished GumC degradation and resulted in higher xanthan viscosity than individual GumB or GumC overexpression. Compared with xanthan from the wild-type strain, longer polymer chains from the strain that simultaneously overexpressed GumB and GumC were observed by atomic force microscopy. Our results suggest that GumB-GumC protein levels modulate xanthan chain length, which results in altered polymer viscosity.

Bypassing the need for subcellular localization of a polysaccharide export‐anchor complex by overexpressing its protein subunits

“…Since homologues of HfsA/B are predicted to have a role in CPS and EPS polymerization as well as its export, probably involving interaction with members of the biosynthesis machinery (Whitfield and Larue, ; Cuthbertson et al ., ; Galván et al ., ), we hypothesized that there might be a requirement of certain holdfast synthesis proteins for HfsD localization. We analysed HfsD–mCherry localization in deletion mutants of each known holdfast synthesis gene and found that its subcellular localization pattern was unaffected (Fig.…”

“…Overexpression of any of the holdfast export proteins in a wild‐type background did not increase holdfast synthesis (http://onlinelibrary.wiley.com/doi/10.1111/mmi.12281/suppinfo). However recent studies of xanthan gum synthesis suggest that overexpressing HfsDA, HfsAB or HfsDAB may produce a level of polysaccharide that exceeds that of wild‐type (Galván et al ., ).…”

Xanthomonas citri ssp. citri requires the outer membrane porin OprB for maximal virulence and biofilm formation

“…The xanthan from wild‐type Xcc also gave a distinct peak for pyruvate, indicating that the Xcc xanthan has a similar composition to that in X. campestris pv . campestris (Galvan et al ., ). However, almost no pyruvate was detected in the xanthan from the mutant strain (Fig.…”

Enzymatic Transformations Involved in the Biosynthesis of Microbial Exo‐polysaccharides Based on the Assembly of Repeat Units

“…There are currently three X-ray structures availablet hat show differences in the interfaces andt he oligomeric state. [41,42,61,62] In xanthan biosynthesis, GumC represents the PCP protein, belonging to the PCP-2a family,b ut hasn o kinase domain as would be expected for PCP proteinsinvolved in the production of high-molecular-weight EPS. [61] Recently more structurali nformation about GumC was obtained, proving its oligomeric conformation andt he influence of the transmembrane segmentso ns tabilitya nd expression.…”

“…[41,42,61,62] In xanthan biosynthesis, GumC represents the PCP protein, belonging to the PCP-2a family,b ut hasn o kinase domain as would be expected for PCP proteinsinvolved in the production of high-molecular-weight EPS. [61] Recently more structurali nformation about GumC was obtained, proving its oligomeric conformation andt he influence of the transmembrane segmentso ns tabilitya nd expression. [63] For welan gum, WelC and WelE seem to be involved in chain-length regulation and act as PCP proteins;t hey include kinase domains in their sequences.…”