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

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Streptomyces conjugation is a unique DNA transfer process in mycelial bacteria.
• This process involves plasmid spreading within the recipient and causes growth retardation, forming visible pocks.
• Streptomyces are a reservoir for antibiotic resistance genes, disseminated via horizontal gene transfer.

Purpose of the Study:

• To characterize the DNA transfer machinery of the Streptomyces venezuelae plasmid pSVH1.
• To identify genes essential for conjugative DNA transfer and intramycelial plasmid spreading.
• To elucidate the protein interactions involved in this unique bacterial conjugation.

Main Methods:

• Systematic gene deletion of the pSVH1 plasmid, excluding the essential rep gene.
• Biochemical characterization of plasmid-encoded proteins, including DNA binding and membrane interaction assays.
• Bacterial two-hybrid analyses and chemical cross-linking to study protein-protein interactions.

Main Results:

• Deletion of traB, encoding an FtsK-like DNA translocase, abolished plasmid transfer and pock formation.
• Deletion of spdB3, spd79, or spdB2 impaired plasmid spreading and pock formation but had minor effects on initial transfer.
• Proteins TraR, Orf108, and SpdB2 bind to peptidoglycan; SpdB2 forms pores in membranes.
• Bacterial two-hybrid and cross-linking data suggest a multiprotein complex involving TraB and Spd proteins.

Conclusions:

• The FtsK-like protein TraB is crucial for efficient conjugative DNA transfer in Streptomyces.
• Specific Spd proteins are involved in intramycelial plasmid spreading and pock formation.
• A macromolecular DNA translocation complex, including TraB and Spd proteins, likely directs plasmid spreading within the Streptomyces mycelium.