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Expression, purification and crystallization of the outer membrane lipoprotein GumB from Xanthomonas campestris.

ABSTRACT: GumB is a predicted outer membrane lipoprotein that is involved in the synthesis and/or secretion of xanthan gum. This exopolysaccharide, produced by Xanthomonas campestris, is valuable in industry because of its important rheological properties. Solution of the GumB structure will provide insight into the polymerization and/or secretion mechanisms of xanthan gum. GumB was overexpressed and purified and diffraction-quality crystals of native GumB were obtained. A complete data set was collected to 2.54?Å resolution with an R(p.i.m.) of 0.034. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.4, b = 90.5, c = 120.7?Å.

SUBMITTER: Jacobs M

PROVIDER: S-EPMC3497991 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Expression, purification and crystallization of the outer membrane lipoprotein GumB from Xanthomonas campestris.

Jacobs Melisa M Salinas Silvina R SR Bianco María I MI Ielpi Luis L

Acta crystallographica. Section F, Structural biology and crystallization communications 20120929 Pt 10

GumB is a predicted outer membrane lipoprotein that is involved in the synthesis and/or secretion of xanthan gum. This exopolysaccharide, produced by Xanthomonas campestris, is valuable in industry because of its important rheological properties. Solution of the GumB structure will provide insight into the polymerization and/or secretion mechanisms of xanthan gum. GumB was overexpressed and purified and diffraction-quality crystals of native GumB were obtained. A complete data set was collected ...[more]

PMID: 23027761

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Project description:Many plant-pathogenic bacteria utilize type II secretion (T2S) systems to secrete degradative enzymes into the extracellular milieu. T2S substrates presumably mediate the degradation of plant cell wall components during the host-pathogen interaction and thus promote bacterial virulence. Previously, the Xps-T2S system from Xanthomonas campestris pv. vesicatoria was shown to contribute to extracellular protease activity and the secretion of a virulence-associated xylanase. The identities and functions of additional T2S substrates from X. campestris pv. vesicatoria, however, are still unknown. In the present study, the analysis of 25 candidate proteins from X. campestris pv. vesicatoria led to the identification of two type II secreted predicted xylanases, a putative protease and a lipase which was previously identified as a virulence factor of X. campestris pv. vesicatoria. Studies with mutant strains revealed that the identified xylanases and the protease contribute to virulence and in planta growth of X. campestris pv. vesicatoria. When analyzed in the related pathogen X. campestris pv. campestris, several T2S substrates from X. campestris pv. vesicatoria were secreted independently of the T2S systems, presumably because of differences in the T2S substrate specificities of the two pathogens. Furthermore, in X. campestris pv. vesicatoria T2S mutants, secretion of T2S substrates was not completely absent, suggesting the contribution of additional transport systems to protein secretion. In line with this hypothesis, T2S substrates were detected in outer membrane vesicles, which were frequently observed for X. campestris pv. vesicatoria. We, therefore, propose that extracellular virulence-associated enzymes from X. campestris pv. vesicatoria are targeted to the Xps-T2S system and to outer membrane vesicles.The virulence of plant-pathogenic bacteria often depends on TS2 systems, which secrete degradative enzymes into the extracellular milieu. T2S substrates are being studied in several plant-pathogenic bacteria, including Xanthomonas campestris pv. vesicatoria, which causes bacterial spot disease in tomato and pepper. Here, we show that the T2S system from X. campestris pv. vesicatoria secretes virulence-associated xylanases, a predicted protease, and a lipase. Secretion assays with the related pathogen X. campestris pv. campestris revealed important differences in the T2S substrate specificities of the two pathogens. Furthermore, electron microscopy showed that T2S substrates from X. campestris pv. vesicatoria are targeted to outer membrane vesicles (OMVs). Our results, therefore, suggest that OMVs provide an alternative transport route for type II secreted extracellular enzymes.

Dataset Information

Expression, purification and crystallization of the outer membrane lipoprotein GumB from Xanthomonas campestris.

Publications

Expression, purification and crystallization of the outer membrane lipoprotein GumB from Xanthomonas campestris.

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