title: Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the pyochelin biosynthesis gene cluster.
abstract: Acarbose is an α-glucosidase inhibitor produced by Actinoplanes sp. SE50/110 that is medically important due to its application in the treatment of type2 diabetes. In this work, a comprehensive proteome analysis of Actinoplanes sp. SE50/110 was carried out to determine the location of proteins of the acarbose (acb) and the putative pyochelin (pch) biosynthesis gene cluster. Therefore, a comprehensive state-of-the-art proteomics approach combining subcellular fractionation, shotgun proteomics and spectral counting to assess the relative abundance of proteins within fractions was applied. The analysis of four different proteome fractions (cytosolic, enriched membrane, membrane shaving and extracellular fraction) resulted in the identification of 1582 of the 8270 predicted proteins. All 22 Acb-proteins and 21 of the 23 Pch-proteins were detected. Predicted membrane-associated, integral membrane or extracellular proteins of the pch and the acb gene cluster were found among the most abundant proteins in corresponding fractions. Intracellular biosynthetic proteins of both gene clusters were not only detected in the cytosolic, but also in the enriched membrane fraction, indicating that the biosynthesis of acarbose and putative pyochelin metabolites takes place at the inner membrane.
  
  
  CONCLUSIONS
  Actinoplanes sp. SE50/110 is a natural producer of the α-glucosidase inhibitor acarbose, a bacterial secondary metabolite that is used as a drug for the treatment of type 2 diabetes, a disease which is a global pandemic that currently affects 387 million people and accounts for 11% of worldwide healthcare expenditures (www.idf.org). The work presented here is the first comprehensive investigation of protein localization and abundance in Actinoplanes sp. SE50/110 and provides an extensive source of information for the selection of genes for future mutational analysis and other hypothesis driven experiments. The conclusion that acarbose or pyochelin family siderophores are synthesized at the inner side of the cytoplasmic membrane determined from this work, indicates that studying corresponding intermediates will be challenging. In addition to previous studies on the genome and transcriptome, the work presented here demonstrates that the next omic level, the proteome, is now accessible for detailed physiological analysis of Actinoplanes sp. SE50/110, as well as mutants derived from this and related species.
authors.affiliation: Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany. Electronic address: Wendler@CeBiTec.Uni-Bielefeld.DE.
authors.forename: Sergej
authors.initials: S
authors.lastname: Wendler
authors.affiliation: Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald, F.-L. Jahnstrasse 15, 17489 Greifswald, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Otto
authors.affiliation: Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany; Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Ortseifen
authors.affiliation: Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald, F.-L. Jahnstrasse 15, 17489 Greifswald, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Bonn
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Armin
authors.initials: A
authors.lastname: Neshat
authors.affiliation: Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Schneiker-Bekel
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany; Department of Proteome and Metabolome Research, Faculty of Biology, Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, Germany.
authors.forename: Frederik
authors.initials: F
authors.lastname: Walter
authors.affiliation: Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany; Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Timo
authors.initials: T
authors.lastname: Wolf
authors.affiliation: Product Supply, Bayer Pharma AG, Friedrich Ebert Str. 217-475, 42117 Wuppertal, Germany.
authors.forename: Till
authors.initials: T
authors.lastname: Zemke
authors.affiliation: Department for Sportsmedicine, University of Wuppertal, Pauluskirchstr. 7, 42285 Wuppertal, Germany.
authors.forename: Udo F
authors.initials: UF
authors.lastname: Wehmeier
authors.affiliation: Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald, F.-L. Jahnstrasse 15, 17489 Greifswald, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hecker
authors.affiliation: Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Jörn
authors.initials: J
authors.lastname: Kalinowski
authors.affiliation: Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald, F.-L. Jahnstrasse 15, 17489 Greifswald, Germany.
authors.forename: Dörte
authors.initials: D
authors.lastname: Becher
authors.affiliation: Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
authors.forename: Alfred
authors.initials: A
authors.lastname: Pühler
mesh_terms: D020909:Acarbose / Q000378:metabolism*; D039903:Actinobacteria / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005810:Multigene Family; D010636:Phenols / Q000378:metabolism*; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D013844:Thiazoles / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D010636:Phenols; D020543:Proteome; D013844:Thiazoles; C025316:pyochelin; D020909:Acarbose
keywords: Actinoplanes; Comprehensive proteomics; Membrane proteome acarbose; Pyochelin; Subcellular fractionation