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Breidenstein, A.

Publications and source records attributed to Breidenstein, A..

2 recordsLinked to original sources

PrgE: an OB-fold protein from plasmid pCF10 with striking differences to prototypical bacterial SSBs

A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded Type 4 Secretion Systems (T4SS). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many their function remains unclear. Here, we characterize PrgE, a proposed SSB from Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very uncharacteristic DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string, and this plasticity of PrgEs oligomerization is further confirmed by in vitro studies. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from the eukaryotic SSBs.

biochemistry↗

Structural and functional characterization of TraI from pKM101 reveals basis for DNA processing

Type 4 Secretion Systems (T4SSs) are large and versatile protein machineries that facilitate the spread of antibiotic resistance and other virulence factors via horizontal gene transfer. Conjugative T4SSs depend on relaxases to process the DNA in preparation for transport. TraI from the well-studied conjugative plasmid pKM101 is one such relaxase. Here, we report the crystal structure of the trans-esterase domain of TraI in complex with its substrate oriT DNA, highlighting the conserved DNA binding mechanism of conjugative relaxases. Additionally, we present an apo structure of the trans-esterase domain of TraI that includes most of the flexible thumb region. This allows us for the first time to visualize the large conformational change of the thumb domain upon DNA binding. We also characterize the DNA binding, nicking and religation activity of the trans-esterase domain, helicase domain and full-length TraI. Unlike previous indications in the literature, our results reveal that the TraI trans-esterase domain from pKM101 behaves in a conserved manner with its homologs from the R388 and F plasmids.

biochemistry↗