title: Conjugative DNA transfer in Streptomyces: SpdB2 involved in the intramycelial spreading of plasmid pSVH1 is an oligomeric integral membrane protein that binds to dsDNA.
abstract: In the current model of conjugal plasmid transfer in mycelium-forming streptomycetes, plasmid transfer by the FtsK-like TraB protein is followed by the subsequent spreading of the newly transferred plasmid within the neighbouring mycelial compartments. Several plasmid-encoded Spd proteins are involved in the plasmid spreading by an unknown mechanism. spdB2 of the conjugative pSVH1 plasmid of Streptomyces venezuelae was heterologously expressed in Escherichia coli and Streptomyces lividans, with a C-terminal His-tag-encoding sequence. Induction of spdB2-His expression affected viability in both species. The integral membrane protein SpdB2-His was eluted from the membrane fraction of S. lividans with Triton X-100, and purified as a soluble protein by Ni-NTA affinity chromatography. Cross-linking experiments with glutaraldehyde showed that SpdB2-His formed oligomers. SpdB2-His had a nonspecific DNA-binding activity: while all types of dsDNA were bound, single-stranded M13-DNA was not recognized. The spd genes of the spdB3-spd79-spdB2 operon of pSVH1 were simultaneously expressed in E. coli with different affinity tags. While expression of StrepII-SpdB3 was not detected, Spd79-flag and SpdB2-His were localized in the membrane fraction of E. coli. In the absence of SpdB2, most of the Spd79-flag protein was found in the cytoplasmic fraction, indicating that SpdB2 affects localization of Spd79. Pulldown assays with StrepII-TraB protein of pSVH1 demonstrated that TraB interacted with SpdB2, suggesting that the septal DNA translocator TraB is also involved in intramycelial plasmid spreading.
authors.affiliation: Mikrobiologie/Biotechnologie, Mikrobiologisches Institut, Fakultät für Biologie, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Tiffert
authors.affiliation: Mikrobiologie/Biotechnologie, Mikrobiologisches Institut, Fakultät für Biologie, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
authors.forename: Birke
authors.initials: B
authors.lastname: Götz
authors.affiliation: Mikrobiologie/Biotechnologie, Mikrobiologisches Institut, Fakultät für Biologie, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Reuther
authors.affiliation: Mikrobiologie/Biotechnologie, Mikrobiologisches Institut, Fakultät für Biologie, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wohlleben
authors.affiliation: Mikrobiologie/Biotechnologie, Mikrobiologisches Institut, Fakultät für Biologie, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
authors.forename: Günther
authors.initials: G
authors.lastname: Muth
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003227:Conjugation, Genetic; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D025282:Mycelium / Q000235:genetics* / Q000254:growth & development; D010957:Plasmids / Q000235:genetics*; D013302:Streptomyces / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008565:Membrane Proteins; D004247:DNA