Conjugative DNA transfer in Streptomyces: SpdB2 involved in the intramycelial spreading of plasmid pSVH1 is an oligomeric integral membrane protein that binds to dsDNA

Mikrobiologie/Biotechnologie, Mikrobiologisches Institut, Fakultät für Biologie, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany Correspondence Günther Muth gmuth{at}biotech.uni-tuebingen.de In the current model of conjugal plasmid transfer in mycelium-forming s...

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Published in:	Microbiology (Society for General Microbiology) Vol. 153; no. 9; pp. 2976 - 2983
Main Authors:	Tiffert, Yvonne, Gotz, Birke, Reuther, Jens, Wohlleben, Wolfgang, Muth, Gunther
Format:	Journal Article
Language:	English
Published:	Reading Soc General Microbiol 01-09-2007 Society for General Microbiology
Subjects:	Bacterial Proteins - genetics Bacterial Proteins - metabolism Biological and medical sciences Conjugation, Genetic Dimerization DNA - genetics DNA - metabolism DNA, Bacterial - genetics DNA, Bacterial - metabolism Escherichia coli Fundamental and applied biological sciences. Psychology Gene Expression Regulation, Bacterial Membrane Proteins - genetics Membrane Proteins - metabolism Microbiology Mycelium - genetics Mycelium - growth & development Plasmids - genetics Streptomyces Streptomyces - genetics Streptomyces - growth & development Streptomyces - metabolism Streptomyces lividans Streptomyces venezuelae Streptomycetes Actinomycetales Plasmid Membrane protein Streptomycetaceae Oligomerization DNA Streptomyces Bacteria Actinomycetes
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Summary:	Mikrobiologie/Biotechnologie, Mikrobiologisches Institut, Fakultät für Biologie, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany Correspondence Günther Muth gmuth{at}biotech.uni-tuebingen.de 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. Present address: Institute of Molecular Biology and Biophysics, ETH Zurich, HPK D14.1, Schafmattstrasse 20, 8093 Zurich, Switzerland.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1350-0872 1465-2080
DOI:	10.1099/mic.0.2006/005413-0