title: A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation.
abstract: Conjugative DNA transfer in mycelial Streptomyces is a unique process involving the transfer of a double-stranded plasmid from the donor into the recipient and the subsequent spreading of the transferred plasmid within the recipient mycelium. This process is associated with growth retardation of the recipient and manifested by the formation of circular inhibition zones, named pocks. To characterize the unique Streptomyces DNA transfer machinery, we replaced each gene of the conjugative 12.1-kbp Streptomyces venezuelae plasmid pSVH1, with the exception of the rep gene required for plasmid replication, with a hexanucleotide sequence. Only deletion of traB, encoding the FtsK-like DNA translocase, affected efficiency of the transfer dramatically and abolished pock formation. Deletion of spdB3, spd79, or spdB2 had a minor effect on transfer but prevented pock formation and intramycelial plasmid spreading. Biochemical characterization of the encoded proteins revealed that the GntR-type regulator TraR recognizes a specific sequence upstream of spdB3, while Orf108, SpdB2, and TraR bind to peptidoglycan. SpdB2 promoted spheroplast formation by T7 lysozyme and formed pores in artificial membranes. Bacterial two-hybrid analyses and chemical cross-linking revealed that most of the pSVH1-encoded proteins interacted with each other, suggesting a multiprotein DNA translocation complex of TraB and Spd proteins which directs intramycelial plasmid spreading.
  
  
  OBJECTIVE
  Mycelial soil bacteria of the genus Streptomyces evolved specific resistance genes as part of the biosynthetic gene clusters to protect themselves from their own antibiotic, making streptomycetes a huge natural reservoir of antibiotic resistance genes for dissemination by horizontal gene transfer. Streptomyces conjugation is a unique process, visible on agar plates with the mere eye by the formation of circular inhibition zones, called pocks. To understand the Streptomyces conjugative DNA transfer machinery, which does not involve a type IV secretion system (T4SS), we made a thorough investigation of almost all genes/proteins of the model plasmid pSVH1. We identified all genes involved in transfer and intramycelial plasmid spreading and showed that the FtsK-like DNA translocase TraB interacts with multiple plasmid-encoded proteins. Our results suggest the existence of a macromolecular DNA translocation complex that directs intramycelial plasmid spreading.
authors.affiliation: Interfakultaeres Institut für Mikrobiologie und Infektionsmedizin Tuebingen IMIT, Mikrobiologie, Biotechnologie, Eberhard Karls Universitaet Tuebingen, Tuebingen, Germany.
authors.forename: Lina
authors.initials: L
authors.lastname: Thoma
authors.affiliation: Interfakultaeres Institut für Mikrobiologie und Infektionsmedizin Tuebingen IMIT, Mikrobiologie, Biotechnologie, Eberhard Karls Universitaet Tuebingen, Tuebingen, Germany.
authors.forename: Hyazinth
authors.initials: H
authors.lastname: Dobrowinski
authors.affiliation: Interfakultaeres Institut für Mikrobiologie und Infektionsmedizin Tuebingen IMIT, Mikrobiologie, Biotechnologie, Eberhard Karls Universitaet Tuebingen, Tuebingen, Germany.
authors.forename: Constanze
authors.initials: C
authors.lastname: Finger
authors.affiliation: Interfakultaeres Institut für Mikrobiologie und Infektionsmedizin Tuebingen IMIT, Mikrobiologie, Biotechnologie, Eberhard Karls Universitaet Tuebingen, Tuebingen, Germany.
authors.forename: Jamil
authors.initials: J
authors.lastname: Guezguez
authors.affiliation: Max-Planck-Institut für Entwicklungsbiologie, Tuebingen, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Linke
authors.affiliation: Interfakultaeres Institut für Mikrobiologie und Infektionsmedizin Tuebingen IMIT, Mikrobiologie, Biotechnologie, Eberhard Karls Universitaet Tuebingen, Tuebingen, Germany.
authors.forename: Edgardo
authors.identifier: http://orcid.org/0000-0002-2413-8261
authors.initials: E
authors.lastname: Sepulveda
authors.affiliation: Interfakultaeres Institut für Mikrobiologie und Infektionsmedizin Tuebingen IMIT, Mikrobiologie, Biotechnologie, Eberhard Karls Universitaet Tuebingen, Tuebingen, Germany gmuth@biotech.uni-tuebingen.de.
authors.forename: Günther
authors.initials: G
authors.lastname: Muth
mesh_terms: D001692:Biological Transport; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000378:metabolism*; D017353:Gene Deletion; D046912:Multiprotein Complexes / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D004269:DNA, Bacterial; D046912:Multiprotein Complexes