title: Unique conjugation mechanism in mycelial streptomycetes: a DNA-binding ATPase translocates unprocessed plasmid DNA at the hyphal tip.
abstract: A single plasmid-encoded protein, the septal DNA translocator TraB, is sufficient to promote conjugal plasmid transfer in mycelial streptomycetes. To analyse the molecular mechanism of conjugation the closely related TraB proteins from plasmids pSG5 of Streptomyces ghanaensis and pSVH1 of Streptomyces venezuelae were characterized. TraB of pSG5 was expressed as a fusion protein with eGFP and found to be localized at the hyphal tips of Streptomyces lividans by fluorescence microscopy, which strongly indicates that conjugation takes place at the tips of the mating mycelium. The TraB protein of pSVH1 was heterologously expressed in S. lividans with an N-terminal strep-tagII and purified as a soluble protein to near homogeneity. The purified protein was shown to hydrolyse ATP and to bind to a 50 bp non-coding pSVH1 sequence containing a 14 bp direct repeat. The protein-DNA complex was too large to enter an agarose gel, indicating that multimers of TraB were bound to the DNA. Denaturation of the protein-DNA complex released unprocessed plasmid DNA demonstrating that the TraB protein does not possess nicking activity. Our experimental data provide evidence that conjugal DNA transfer in streptomycetes is mediated by the septal DNA translocator TraB, an plasmid-encoded ATPase that interacts non-covalently with DNA and translocates an unprocessed double-stranded DNA molecule at the hyphal tip into the recipient.
authors.affiliation: Mikrobiologie/Biotechnologie, Mikrobiologisches Institut, Fakultaet für Biologie, Eberhard Karls Universitaet Tuebingen, Auf der Morgenstelle 28, 72076 Tuebingen, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Reuther
authors.forename: Cordula
authors.initials: C
authors.lastname: Gekeler
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Tiffert
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wohlleben
authors.forename: Günther
authors.initials: G
authors.lastname: Muth
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D003227:Conjugation, Genetic; D004247:DNA / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D021901:DNA, Intergenic; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D008969:Molecular Sequence Data; D012091:Repetitive Sequences, Nucleic Acid; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*; D048170:Streptomyces lividans / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D021901:DNA, Intergenic; C082576:traB protein, Bacteria; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases