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.

Xanthan, the main exopolysaccharide (EPS) synthesized by Xanthomonas spp., contributes to bacterial stress tolerance and enhances attachment to plant surfaces by helping in biofilm formation. Therefore, xanthan is essential for successful colonization and growth in planta and has also been proposed to be involved in the promotion of pathogenesis by calcium ion chelation and, hence, in the suppression of the plant defense responses in which this cation acts as a signal. The aim of this work was to study the relationship between xanthan structure and its role as a virulence factor. We analyzed four Xanthomonas campestris pv. campestris mutants that synthesize structural variants of xanthan. We found that the lack of acetyl groups that decorate the internal mannose residues, ketal-pyruvate groups, and external mannose residues affects bacterial adhesion and biofilm architecture. In addition, the mutants that synthesized EPS without pyruvilation or without the external mannose residues did not develop disease symptoms in Arabidopsis thaliana. We also observed that the presence of the external mannose residues and, hence, pyruvilation is required for xanthan to suppress callose deposition as well as to interfere with stomatal defense. In conclusion, pyruvilation of xanthan seems to be essential for Xanthomonas campestris pv. campestris virulence.

Infant botulism is the most common form of human botulism in Argentina and the United States. BabyBIG (botulism immune globulin intravenous [human]) is the antitoxin of choice for specific treatment of infant botulism in the United States. However, its high cost limits its use in many countries. We report here the effectiveness and safety of equine botulinum antitoxin (EqBA) as an alternative treatment. We conducted an analytical, observational, retrospective, and longitudinal study on cases of infant botulism registered in Mendoza, Argentina, from 1993 to 2007. We analyzed 92 medical records of laboratory-confirmed cases and evaluated the safety and efficacy of treatment with EqBA. Forty-nine laboratory-confirmed cases of infant botulism demanding admission in intensive care units and mechanical ventilation included 31 treated with EqBA within the 5 days after the onset of signs and 18 untreated with EqBA. EqBA-treated patients had a reduction in the mean length of hospital stay of 23.9 days (P ‫؍‬ 0.0007). For infants treated with EqBA, the intensive care unit stay was shortened by 11.2 days (P ‫؍‬ 0.0036), mechanical ventilation was reduced by 11.1 days (P ‫؍‬ 0.0155), and tube feeding was reduced by 24.4 days (P ‫؍‬ 0.0001). The incidence of sepsis in EqBA-treated patients was 47.3% lower (P ‫؍‬ 0.0017) than in the untreated ones. Neither sequelae nor adverse effects attributable to EqBA were noticed, except for one infant who developed a transient erythematous rash. These results suggest that prompt treatment of infant botulism with EqBA is safe and effective and that EqBA could be considered an alternative specific treatment for infant botulism when BabyBIG is not available.

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