Abstract
The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein—VirE2—enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated—using single-molecule techniques—that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes.
Highlights
Agrobacterium tumefaciens is a Gram-negative pathogenic bacterium able to transfer and integrate up to 150,000bases-long single-stranded DNA into the infected cell nuclear genome [1]
The importation of genetic material into cells is a common and fundamental mechanism occurring in bacterial conjugation, DNA uptake, and Agrobacterium plant infection and is, for instance, responsible for antibiotic resistance spread
Previous studies suggested that this process relied only on the activity of complex molecular machines pumping the single-stranded DNA into the recipient cell
Summary
Agrobacterium tumefaciens is a Gram-negative pathogenic bacterium able to transfer and integrate up to 150,000bases-long single-stranded DNA (ssDNA) into the infected cell nuclear genome [1]. T4SS exports T-DNA and effector proteins out of the bacterium [3,4,5]. VirE2 was shown to form channels, which transport ssDNA, and VirE2 was proposed to mediate transfer of T-DNA through the eukaryotic plasma membrane [8,9,10]. As evidenced by scanning transmission electron microscopy (STEM), the VirE2–ssDNA complex consists of a helical structure in which 19 nucleotides are bound per VirE2 monomer [12]. This structure prevents exonuclease degradation in vitro [13]. Recent in vitro experiments demonstrate the microtubule-guided transport of such DNA–VirE2 complexes [14]
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