Dataset Information

Strength of a “single-tube” protocol for protein isolation from multiple cell fractions shown for the plant pathogen Xanthomonas campestris pv. campestris B100 with a focus on proteins involved in the biosynthesis of the exopolysaccharide xanthan

ABSTRACT: Xanthomonas campestris pv. campestris (Xcc) B100 is a phytopathogenic bacterium, which uses xanthan, a secreted heterogeneous exopolysaccharide in both stages of infection of Brassicaceae. Until now, proteome analysis of Xcc lacking a detailed view on the proteins involved in xanthan biosynthesis. Proteins involved in the biosynthesis of this polysaccharide are located near, in or at the cell membrane. This study aims to establish a robust and rapid protocol for a comprehensive proteome analysis of Xcc strains, without the need to isolate different cell fractions. Therefore, a method for the analysis of the whole cell proteome was compared to the isolation of specific fractions regarding the total number of identified proteins, the overlap as well as the differences between the approaches. The whole cell proteome analysis using the “single-tube” preparation protocol resulted in more than 2,000 identified proteins with a coverage of 95.11% of the cell fraction specific identifications. To prove the usability of the protocol to study natural product formation at the envelope with proteins distributed over various cell fractions, Gum proteins of Xcc B100 were identified and quantified by a label free quantification (LFQ) approach. Thus, expression profiles of 9 out of 12 Gum proteins comparing stationary phase and growth phase were measured and showed differential expression levels within the operon structure. Five out of ten identified Gum proteins show a differential regulation indicating regulatory mechanisms beyond transcription level.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Xanthomonas Campestris Pv. Campestris Str. B100

SUBMITTER: Julia Voss

LAB HEAD: Dr. Vera Ortseifen

PROVIDER: PXD027261 | Pride | 2022-02-16

REPOSITORIES: Pride

ACCESS DATA

Publications

Analysis of Gum proteins involved in xanthan biosynthesis throughout multiple cell fractions in a "single-tube".

Hahn Julia J Koch Daniel D Niehaus Karsten K Ortseifen Vera V

Journal of proteomics 20220208

Xanthomonas is a phytopathogenic bacterium and of industrial interest due to its capability to produce xanthan, used as a thickener and emulsifier in the food and non-food industry. Until now, proteome analyses of Xcc lacking a detailed view on the proteins involved in xanthan biosynthesis. The proteins involved in the biosynthesis of this polysaccharide are located near, in or at the cell membrane. This study aims to establish a robust and rapid protocol for a comprehensive proteome analysis of ...[more]

PMID: 35149254

Similar Datasets

Project description:Oxidative stress due to endogenous hydrogen peroxide production by Lactobacillus species is a well-known issue in the food industry. In this study, the transcriptional response to oxygen of Lactobacillus johnsonii, one of the H2O2-producing strains used in the food industry, was analyzed. It was found that aerobic growth conditions led to a more than two-fold downregulation of 45 genes as compared to anaerobic growth, whereas 6 genes were more than twofold upregulated. Among the upregulated genes were two genes that displayed significant homology to NADH-dependent oxidoreductase (NOX). The postulated transcriptional regulation of the nox promoter by oxygen was studied using a GUS-reporter construct, confirming a 2.1-fold upregulated GUS-expression upon aerobic growth. Exposure to sublethal levels of hydrogen peroxide did not result in significant regulation of the nox promoter. In a previous study of hydrogen peroxide production by L. johnsonii, a NADH flavin reductase (NFR) was identified to be involved in hydrogen peroxide production. An NFR-deficient derivative was strongly impaired in H2O2 production, but regained a partial H2O2 producing capacity upon prolonged oxygen exposure. The nox-promoter appeared to be 3.6-fold upregulated under aerobic conditions in the NFR-deficient background, which may imply a role of this gene in the regained H2O2 production. Indeed, deletion of the nox-gene in the NFR-deletion background, resulted in a strain that no longer produced H2O2, also during prolonged exposure to oxygen. The double-mutant (nfr, nox) displayed strongly impaired aerobic growth and oxygenation induced rapid growth stagnation that is not caused by H2O2. We conclude that H2O2 production in L. johnsonii is primarily dependent on NFR but can also involve an oxygen-inducible NADH oxidase under aerobic conditions. Moreover, our results imply that H2O2 production plays a prominent role in oxygen tolerance of L. johnsonii. loop design of the samples including two shortcuts

Dataset Information

Strength of a “single-tube” protocol for protein isolation from multiple cell fractions shown for the plant pathogen Xanthomonas campestris pv. campestris B100 with a focus on proteins involved in the biosynthesis of the exopolysaccharide xanthan

Publications

Analysis of Gum proteins involved in xanthan biosynthesis throughout multiple cell fractions in a "single-tube".

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure