The influence of a modified lipopolysaccharide O-antigen on the biosynthesis of xanthan in Xanthomonas campestris pv. campestris B100

Abstract

BackgroundThe exopolysaccharide xanthan is a natural product which is extensively used in industry. It is a thickening agent in many fields, from oil recovery to the food sector. Xanthan is produced by the Gram negative bacterium Xanthomonas campestris pv. campestris (Xcc). We analyzed the lipopolysaccharide (LPS) of three mutant strains of the Xcc wild type B100 to distinguish if the xanthan production can be increased when LPS biosynthesis is affected.ResultsThe Xcc B100 O-antigen (OA) is composed of a linear main chain of rhamnose residues with N-acetylfucosamine (FucNAc) side branches at every second rhamnose. It is the major LPS constituent. The O-antigen was missing completely in the mutant strain H21012 (deficient in wxcB), since neither rhamnose nor FucNAc could be detected as part of the LPS by MALDI-TOF-MS, and only a slight amount of rhamnose and no FucNAc was found by GC analysis. The LPS of two other mutants was analyzed, Xcc H28110 (deficient in wxcK) and H20110 (wxcN). In both of them no FucNAc could be detected in the LPS fraction, while the rhamnose moieties were more abundant than in wild type LPS. The measurements were carried out by GC and confirmed by MALDI-TOF-MS analyses that indicated an altered OA in which the branches are missing, while the rhamnan main chain seemed longer than in the wild type. Quantification of xanthan confirmed our hypothesis that a missing OA can lead to an increased production of the extracellular polysaccharide. About 6.3 g xanthan per g biomass were produced by the Xcc mutant H21012 (wxcB), as compared to the wild type production of approximately 5 g xanthan per g biomass. In the two mutant strains with modified OA however, Xcc H28110 (wxcK) and Xcc H20110 (wxcN), the xanthan production of 5.5 g and 5.3 g, respectively, was not significantly increased.ConclusionsMutations affecting LPS biosynthesis can be beneficial for the production of the extracellular polysaccharide xanthan. However, only complete inhibition of the OA resulted in increased xanthan production. The inhibition of the FucNAc side branches did not lead to increased production, but provoked a novel LPS phenotype. The data suggests an elongation of the linear rhamnan main chain of the LPS OA in both the Xcc H28110 (wxcK) and Xcc H20110 (wxcN) mutant strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0710-y) contains supplementary material, which is available to authorized users.

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“…campestris strain 8004. YipR is a transcriptional regulator carrying CheY-homologous receiver (REC) and DNA-binding domains, which governs virulence gene expression [18]. YipR homologues are present in the genome of most Xanthomonas species, but their regulons remain ill defined.…”

Mechanistic insights into host adaptation, virulence and epidemiology of the phytopathogenXanthomonas

“…In X. euvesicatoria, a wxcB knock-out mutant had reduced virulence while effects on the LPS were minimal (Park, Jung and Han 2014). In contrast in an Xcc mutant deficient in wxcB the Oantigen was missing completely (Steffens et al 2016). Another unusual gene of region 2 was wxcD, where a mutant strain (Vorhölter, Niehaus and Pühler 2001) produced LPS with fragmentary O-antigen (Braun et al 2005), relating this gene possibly to O-antigen primer or adapter biosynthesis.…”

“…Another unusual gene of region 2 was wxcD, where a mutant strain (Vorhölter, Niehaus and Pühler 2001) produced LPS with fragmentary O-antigen (Braun et al 2005), relating this gene possibly to O-antigen primer or adapter biosynthesis. Mutagenesis of the genes wxcK and wxcN (Steffens et al 2016) in region 3 of the wxc gene cluster indicates a distinct biosynthetic mechanism for the synthesis of the O-antigen side chain.…”

“…In contrast in an Xcc mutant deficient in wxcB the O-antigen was missing completely (Steffens et al . 2016 ). Another unusual gene of region 2 was wxcD , where a mutant strain (Vorhölter, Niehaus and Pühler 2001 ) produced LPS with fragmentary O-antigen (Braun et al .…”

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“…In addition, for the GC-MS analysis the supernatant containing carbohydrates were derivatized into sugar monomers by methanolysis and peracetylation according to Steffens et al and analysed via GC-MS [ 43 ]. Briefly, 100 μg of freeze dried culture supernatant were used for methanolysis in 0.5 M HCl / MeOH for 45 min at 85°C.…”