Abstract
Untreatable bacterial infections caused by a perpetual increase of antibiotic resistant strains represent a serious threat to human healthcare in the 21st century. Conjugative DNA transfer is the most important mechanism for antibiotic resistance and virulence gene dissemination among bacteria and is mediated by a protein complex, known as type IV secretion system (T4SS). The core of the T4SS is a multiprotein complex that spans the bacterial envelope as a channel for macromolecular secretion. We report the NMR structure and functional characterization of the transfer protein TraH encoded by the conjugative Gram-positive broad-host range plasmid pIP501. The structure exhibits a striking similarity to VirB8 proteins of Gram-negative secretion systems where they play an essential role in the scaffold of the secretion machinery. Considering TraM as the first VirB8-like protein discovered in pIP501, TraH represents the second protein affiliated with this family in the respective transfer operon. A markerless traH deletion in pIP501 resulted in a total loss of transfer in Enterococcus faecalis as compared with the pIP501 wild type (wt) plasmid, demonstrating that TraH is essential for pIP501 mediated conjugation. Moreover, oligomerization state and topology of TraH in the native membrane were determined providing insights in molecular organization of a Gram-positive T4SS.
Highlights
Prokaryotic genome plasticity and evolutionary success greatly relies on a variety of independent mechanisms congruously summarized under the term of horizontal gene transfer (HGT)[1,2]
NMR secondary chemical shift analysis indicated that the N-terminal residues 28–58 adopt a random coil conformation, followed by three α -helices, which are connected by short loops
T4SSs function as elaborated transport machines dedicated to the trafficking of proteins or DNA-protein complexes across the bacterial envelope into eukaryotic host cells or bacterial recipients
Summary
Prokaryotic genome plasticity and evolutionary success greatly relies on a variety of independent mechanisms congruously summarized under the term of horizontal gene transfer (HGT)[1,2]. Structural and functional studies on distinct transfer proteins from the conjugative plasmid pIP501 have contributed to a better understanding of pIP501 mediated conjugation. The extracellular C-terminal domain of the bitopic transmembrane (TM) protein TraM was identified as VirB8-like, while the 3.0 Å structure of TraK exhibits a novel fold[26,27]. We present the NMR solution structure of the soluble domain of the essential 21.2 kDa transfer protein TraH (formerly ORF8; GenBank CAD44388.1) along with a detailed biophysical and biochemical characterization of its putative role in the conjugative conduit. N-terminally truncated TraH always appears as monomer in solution, size exclusion chromatography, in vitro and in vivo crosslinking studies and semi-native PAGE analysis suggested oligomerization of the full-length protein. Deletion of traH in pIP501 leads to a complete loss of transfer efficiency, implying that TraH is an essential player in G+ bacterial conjugation
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