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

Conjugative DNA-transfer in mycelial streptomycetes is a unique process, manifested on agar plates by the formation of circular growth retardation zones called pocks. Because pock size correlates with the extent of the transconjugant zone, it was suggested that pocks reflect the spreading of the transferred plasmid in the recipient mycelium. However, this concept has not been experimentally proven yet. The use of an eGFP-encoding derivative of the conjugative pIJ303 plasmid and Streptomyces lividans T7-mCherry as recipient enabled us to differentiate donor, recipient and transconjugant hyphae in mating experiments by fluorescence microscopy. Microscopic observation of the conjugation process suggested DNA-transfer via the lateral walls. At the contact sites mCherry was never observed in the donor, indicating that the conjugative DNA-transfer does not involve interfusion of cytoplasms of donor and recipient. The spreading of the transferred plasmid to the older parts of the recipient mycelium was demonstrated. This spreading was impaired when plasmid-encoded spd genes were inactivated. Deletion of the FtsK-like DNA-translocase encoding tra gene from the plasmid and mating experiments with strains containing chromosomal copies of tra either in the donor and/or in the recipient revealed that Tra had an essential role in intramycelial plasmid spreading.

A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation.

UNLABELLED: 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.

The conjugative DNA-transfer apparatus of Streptomyces.

Conjugation is a major route of horizontal gene transfer, an important driving force in the evolution of bacterial genomes. Since antibiotic producing streptomycetes represent a natural reservoir of antibiotic resistance genes, the Streptomyces conjugation system might have a particular role in the dissemination of the resistance genes. Streptomycetes transfer DNA in a unique process, clearly distinguished from the well-known DNA-transfer by type IV secretion systems. A single plasmid-encoded DNA-translocase, TraB, transfers a double-stranded DNA-molecule to the recipient. Elucidation of the structure, pore forming ability and DNA binding characteristics of TraB indicated that the TraB conjugation system is derived from an FtsK-like ancestor protein suggesting that Streptomyces adapted the FtsK/SpoIIIE chromosome segregation system to transfer DNA between two distinct Streptomyces cells. Following the primary transfer, a multi-protein DNA-translocation apparatus consisting of TraB and several Spd-proteins spreads the newly transferred DNA to the neighbouring mycelial compartments resulting in the rapid colonization of the recipient mycelium by the donor DNA.

The stability region of the Streptomyces lividans plasmid pIJ101 encodes a DNA-binding protein recognizing a highly conserved short palindromic sequence motif.

Conjugation is a driving force in the evolution and shaping of bacterial genomes. In antibiotic producing streptomycetes even small plasmids replicating via the rolling-circle mechanism are conjugative. Although they encode only genes involved in replication and transfer, the molecular function of most plasmid encoded proteins is unknown. In this work we show that the conjugative plasmid pIJ101 encodes an overlooked protein, SpdA2. We show that SpdA2 is a DNA binding protein which specifically recognizes a palindromic DNA sequence (sps). sps is localized within the spdA2 coding region and highly conserved in many Streptomyces plasmids. Elimination of the palindrome or deletion of spdA2 in plasmid pIJ303 did not interfere with conjugative plasmid transfer or pock formation, but affected segregational stability.

Conjugative DNA transfer in Streptomyces by TraB: is one protein enough?

Antibiotic-producing soil bacteria of the genus Streptomyces form a huge natural reservoir of antibiotic resistance genes for the dissemination within the soil community. Streptomyces plasmids encode a unique conjugative DNA transfer system clearly distinguished from classical conjugation involving a single-stranded DNA molecule and a type IV protein secretion system. Only a single plasmid-encoded protein, TraB, is sufficient to translocate a double-stranded DNA molecule into the recipient in Streptomyces matings. TraB is a hexameric pore-forming ATPase that resembles the chromosome segregator protein FtsK and translocates DNA by recognizing specific 8-bp repeats present in the plasmid clt locus. Mobilization of chromosomal genes does not require integration of the plasmid, because TraB also recognizes clt-like sequences distributed all over the chromosome.