A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during <italic toggle="yes">Streptomyces</italic> 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 retardati...

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Autores principales:	Lina Thoma, Hyazinth Dobrowinski, Constanze Finger, Jamil Guezguez, Dirk Linke, Edgardo Sepulveda, Günther Muth
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2015
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/c58f90b693ba42b1af82d4084285922c
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spelling	oai:doaj.org-article:c58f90b693ba42b1af82d4084285922c2021-11-15T15:49:02ZA Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during <italic toggle="yes">Streptomyces</italic> Conjugation10.1128/mBio.02559-142150-7511https://doaj.org/article/c58f90b693ba42b1af82d4084285922c2015-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02559-14https://doaj.org/toc/2150-7511ABSTRACT 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. IMPORTANCE 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.Lina ThomaHyazinth DobrowinskiConstanze FingerJamil GuezguezDirk LinkeEdgardo SepulvedaGünther MuthAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 6, Iss 3 (2015)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Lina Thoma Hyazinth Dobrowinski Constanze Finger Jamil Guezguez Dirk Linke Edgardo Sepulveda Günther Muth A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during <italic toggle="yes">Streptomyces</italic> Conjugation
description	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. IMPORTANCE 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.
format	article
author	Lina Thoma Hyazinth Dobrowinski Constanze Finger Jamil Guezguez Dirk Linke Edgardo Sepulveda Günther Muth
author_facet	Lina Thoma Hyazinth Dobrowinski Constanze Finger Jamil Guezguez Dirk Linke Edgardo Sepulveda Günther Muth
author_sort	Lina Thoma
title	A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during <italic toggle="yes">Streptomyces</italic> Conjugation
title_short	A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during <italic toggle="yes">Streptomyces</italic> Conjugation
title_full	A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during <italic toggle="yes">Streptomyces</italic> Conjugation
title_fullStr	A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during <italic toggle="yes">Streptomyces</italic> Conjugation
title_full_unstemmed	A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during <italic toggle="yes">Streptomyces</italic> Conjugation
title_sort	multiprotein dna translocation complex directs intramycelial plasmid spreading during <italic toggle="yes">streptomyces</italic> conjugation
publisher	American Society for Microbiology
publishDate	2015
url	https://doaj.org/article/c58f90b693ba42b1af82d4084285922c
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A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during <italic toggle="yes">Streptomyces</italic> Conjugation

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