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.

Fluorescence microscopy of Streptomyces conjugation suggests DNA‐transfer at the lateral walls and reveals the spreading of the plasmid in the recipient mycelium

“…Spd proteins do not have similarities to functionally characterized proteins in databases. SpdB2 of plasmid pSVH1 is an oligomeric integral membrane protein that binds non‐specifically to double‐stranded DNA (Tiffert et al ., ) and interacts with PG (Thoma and Muth, ; Thoma et al ., ). None of the Spd proteins is a predicted motor protein with ATPase activity, required for the translocation of a DNA molecule.…”

“…In vivo interaction of Tra with SpdA, Orf66 and KilB was confirmed in the bacterial two‐hybrid assays. Interaction of TraB with Spd proteins has been also reported for plasmid pSVH1 (Tiffert et al ., ; Thoma and Muth, ; Thoma et al ., ). By constructing the mobilizable pIJ303 derivative pIJ303ΔTra, we were able to split spd and tra functions.…”

“…Mutations that reduced the pock size and the transconjugant zone, but did not affect the efficiency of transfer from the original donor to the recipient revealed the existence of three to five spd genes on conjugative Streptomyces plasmids (Kieser et al ., ; Kataoka et al ., ; Servín‐González et al ., ). The predicted Spd proteins do not show sequence similarity to any functionally characterized protein and only a few Spd proteins have been biochemically characterized (Pettis et al ., ; Tiffert et al ., ; Thoma et al ., 2014; 2015; Thoma and Muth, ). Whereas the role of TraB in plasmid transfer from the donor into the recipient has been well characterized (Pettis and Cohen, 1994; 1996; 2000; Reuther et al ., ; Vogelmann et al ., ), the molecular mechanism of plasmid spreading in the recipient mycelium has not been elucidated yet and it is unclear whether the FtsK‐like DNA‐translocase TraB is involved or if the Spd proteins can promote the spreading of the plasmid by an TraB‐independent process.…”

Prevalence of Flp Pili-Encoding Plasmids in Cutibacterium acnes Isolates Obtained from Prostatic Tissue

“…Thus, taken these aspects together, it is possible that the plasmid is actually a conjugative plasmid. Many conjugative plasmids in Gram-positive bacteria have been identified; at least two different strategies for horizontal plasmid transfer in Gram-positive bacteria are employed, i.e., transfer of single-stranded plasmid DNA via a type IV secretion system, and the macromolecular DNA translocation complex in streptomycetes that transports double-stranded DNA from donor to recipient cells ( Goessweiner-Mohr et al, 2014 ; Thoma et al, 2015 ). The large plasmid of C. acnes encodes components of both systems.…”

Spreading the news about the novel conjugation mechanism in Streptomyces bacteria

“…Chemical crosslinking and bacterial two‐hybrid analyses revealed complex interactions between the TraB and Spd components of plasmid pSVH1. These data point to the existence of a multiprotein DNA translocation apparatus for plasmid spreading (Thoma et al ., ).…”