title: Applying DNA affinity chromatography to specifically screen for sucrose-related DNA-binding transcriptional regulators of Xanthomonas campestris.
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.
authors.affiliation: Proteome and Metabolome Research, Faculty of Biology, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany.
authors.forename: Lennart
authors.initials: L
authors.lastname: Leßmeier
authors.affiliation: Proteome and Metabolome Research, Faculty of Biology, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany; Center for Biotechnology, Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany.
authors.forename: Rabeaa S
authors.initials: RS
authors.lastname: Alkhateeb
authors.affiliation: Proteome and Metabolome Research, Faculty of Biology, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany; Center for Biotechnology, Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Schulte
authors.affiliation: Proteome and Metabolome Research, Faculty of Biology, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany; Center for Biotechnology, Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Steffens
authors.affiliation: Center for Biotechnology, Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany.
authors.forename: Tobias Pascal
authors.initials: TP
authors.lastname: Loka
authors.affiliation: Center for Biotechnology, Universität Bielefeld, Sequenz 1, 33615 Bielefeld, Germany.
authors.forename: Alfred
authors.initials: A
authors.lastname: Pühler
authors.affiliation: Proteome and Metabolome Research, Faculty of Biology, Universität Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Niehaus
authors.affiliation: 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.
authors.forename: Frank-Jörg
authors.initials: FJ
authors.lastname: Vorhölter
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D020869:Gene Expression Profiling; D013395:Sucrose / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D037521:Virulence Factors / Q000235:genetics; D016959:Xanthomonas campestris / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D014157:Transcription Factors; D037521:Virulence Factors; D013395:Sucrose
keywords: DNA affinity purification; Promoter fishing; Pull-down assay; Regulation; Virulence genes; Xanthomonas campestris pathovar campestris