Applying DNA affinity chromatography to specifically screen for sucrose-related DNA-binding transcriptional regulators of Xanthomonas campestris.
| Title: | Applying DNA affinity chromatography to specifically screen for sucrose-related DNA-binding transcriptional regulators of Xanthomonas campestris. |
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| Authors: | Leßmeier L; Proteome and Metabolome Research, Faculty of Biology, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany.; Alkhateeb RS; Proteome and Metabolome Research, Faculty of Biology, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany; Center for Biotechnology, Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany.; Schulte F; Proteome and Metabolome Research, Faculty of Biology, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany; Center for Biotechnology, Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany.; Steffens T; Proteome and Metabolome Research, Faculty of Biology, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany; Center for Biotechnology, Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany.; Loka TP; Center for Biotechnology, Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany.; Pühler A; Center for Biotechnology, Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany.; Niehaus K; Proteome and Metabolome Research, Faculty of Biology, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany.; Vorhölter FJ; Proteome and Metabolome Research, Faculty of Biology, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany; Center for Biotechnology, Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany. Electronic address: frank@cebitec.uni-bielefeld.de. |
| Source: | Journal of biotechnology [J Biotechnol] 2016 Aug 20; Vol. 232, pp. 89-98. Date of Electronic Publication: 2016 Apr 07. |
| Publication Type: | Journal Article |
| Language: | English |
| Journal Info: | Publisher: Elsevier Science Publishers Country of Publication: Netherlands NLM ID: 8411927 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1873-4863 (Electronic) Linking ISSN: 01681656 NLM ISO Abbreviation: J Biotechnol Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Amsterdam : Elsevier Science Publishers, c1984- |
| MeSH Terms: | Bacterial Proteins/*isolation & purification ; Chromatography, Affinity/*methods ; DNA, Bacterial/*metabolism ; Sucrose/*metabolism ; Transcription Factors/*isolation & purification ; Xanthomonas campestris/*genetics; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; DNA, Bacterial/analysis ; DNA, Bacterial/chemistry ; Transcription Factors/genetics ; Transcription Factors/metabolism ; Virulence Factors/genetics ; Gene Expression Profiling |
| Abstract: | At a molecular level, the regulation of many important cellular processes is still obscure in xanthomonads, a bacterial group of outstanding relevance as world-wide plant pathogens and important for biotechnology as producers of the polysaccharide xanthan. Transcriptome analysis indicated a sucrose-dependent regulation of 18 genes in Xanthomonas campestris pv. campestris (Xcc) B100. The expression of 12 of these genes was clearly increased in the presence of sucrose. Only part of these genes was obviously involved in sucrose utilization. To identify regulatory proteins involved in transcriptional regulation, a DNA fragment-specific pull-down approach was established for Xcc. Putative promoter regions were identified and used to isolate DNA-binding proteins, which were separated by SDS PAGE and identified by MALDI-TOF mass spectrometry. This led to the identification of four transcriptional regulators, among them the global transcriptional regulator Clp and a previously identified regulator of sucrose utilization, SuxR, plus a third DNA-binding transcriptional regulator encoded by xcc-b100_2861 and recently shown to interact with a cyclic di-GMP-binding protein. The fourth regulatory protein was encoded by xcc-b100_2791. These results indicate DNA fragment-specific pull-down experiments as promising approaches to screen for specific DNA-binding regulatory proteins in Xcc.; (Copyright © 2016 Elsevier B.V. All rights reserved.) |
| Contributed Indexing: | Keywords: DNA affinity purification; Promoter fishing; Pull-down assay; Regulation; Virulence genes; Xanthomonas campestris pathovar campestris |
| Substance Nomenclature: | 0 (Bacterial Proteins); 0 (DNA, Bacterial); 0 (Transcription Factors); 0 (Virulence Factors); 57-50-1 (Sucrose) |
| Entry Date(s): | Date Created: 20160410 Date Completed: 20170227 Latest Revision: 20170227 |
| Update Code: | 20260130 |
| DOI: | 10.1016/j.jbiotec.2016.04.007 |
| PMID: | 27060555 |
| Database: | MEDLINE |
Journal Article