title: The cytosolic and extracellular proteomes of Actinoplanes sp. SE50/110 led to the identification of gene products involved in acarbose metabolism.
abstract: The pseudotetrasaccharide acarbose is a medically relevant secondary metabolite produced by strains of the genera Actinoplanes and Streptomyces. In this study gene products involved in acarbose metabolism were identified by analyzing the cytosolic and extracellular proteome of Actinoplanes sp. SE50/110 cultures grown in a high-maltose minimal medium. The analysis by 2D protein gel electrophoresis of cytosolic proteins of Actinoplanes sp. SE50/110 resulted in 318 protein spots and 162 identified proteins. Nine of those were acarbose cluster proteins (Acb-proteins), namely AcbB, AcbD, AcbE, AcbK, AcbL, AcbN, AcbR, AcbV and AcbZ. The analysis of proteins in the extracellular space of Actinoplanes sp. SE50/110 cultures resulted in about 100 protein spots and 22 identified proteins. The identifications included the three acarbose gene cluster proteins AcbD, AcbE and AcbZ. After their identification, proteins were classified into functional groups. The dominant functional groups were the carbohydrate binding, carbohydrate cleavage and carbohydrate transport proteins. The other functional groups included protein cleavage, amino acid degradation, nucleic acid cleavage and a number of functionally uncharacterized proteins. In addition, signal peptide structures of extracellularly found proteins were analyzed. Of the 22 detected proteins 19 contained signal peptides, while 2 had N-terminal transmembrane helices explaining their localization. The only protein having neither of them was enolase. Under the conditions applied, the secretome of Actinoplanes sp. SE50/110 was dominated by seven proteins involved in carbohydrate metabolism (PulA, AcbE, AcbD, MalE, AglE, CbpA and Cgt). Of special interest were the identified extracellular pullulanase PulA and the two solute-binding proteins MalE and AglE. The identifications suggest that Actinoplanes sp. SE50/110 has two maltose/maltodextrin import systems. We postulate the identified MalEFG transport system of Actinoplanes sp. SE50/100 as the missing acarbose-metabolite importer and present a model of acarbose metabolism that is extended by the newly identified gene products.
authors.affiliation: Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany. Wendler@CeBiTec.Uni-Bielefeld.de
authors.forename: Sergej
authors.initials: S
authors.lastname: Wendler
authors.forename: Daniel
authors.initials: D
authors.lastname: Hürtgen
authors.forename: Jörn
authors.initials: J
authors.lastname: Kalinowski
authors.forename: Andreas
authors.initials: A
authors.lastname: Klein
authors.forename: Karsten
authors.initials: K
authors.lastname: Niehaus
authors.forename: Fabian
authors.initials: F
authors.lastname: Schulte
authors.forename: Patrick
authors.initials: P
authors.lastname: Schwientek
authors.forename: Hermann
authors.initials: H
authors.lastname: Wehlmann
authors.forename: Udo F
authors.initials: UF
authors.lastname: Wehmeier
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; D050260:Carbohydrate Metabolism / Q000235:genetics*; D002241:Carbohydrates / Q000235:genetics; D003600:Cytosol / Q000378:metabolism*; D005798:Genes, Bacterial; D008320:Maltose / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family; D011134:Polysaccharides / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D017433:Protein Structure, Secondary; D020543:Proteome / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002241:Carbohydrates; D008565:Membrane Proteins; D011134:Polysaccharides; D021382:Protein Sorting Signals; D020543:Proteome; D008320:Maltose; C008315:maltodextrin; D020909:Acarbose