1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI of plasmid R1

Bhattiprolu Krishna,Klaus Zangger,Gabriel E Wagner,Sandra Raffl,Ellen Zechner,Walter Becker,Evelyne Schrank,Nina Gubensäk

doi:10.1007/s12104-018-9863-y

Bhattiprolu Krishna, Klaus Zangger + Show 6 more

Open Access

https://doi.org/10.1007/s12104-018-9863-y

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Abstract

Transfer of genetic material is the main mechanism underlying the spread of antibiotic resistance and virulence factors within the bacterial community. Conjugation is one such process by which the genetic material is shared from one bacterium to another. The DNA substrate is processed and prepared for transfer by a multi-protein complex called the relaxosome .The relaxosome of plasmid R1 possesses the most crucial enzyme TraI which, both nicks and unwinds the dsDNA substrate. TraI comprises 1765 residues and multiple functional domains, including those catalyzing the DNA trans-esterase (relaxase) on the dsDNA designated for a conjugative transfer and DNA helicase activities. Structural and functional studies have been reported for most of the TraI except the C-terminal domain spanning from residue 1630 to 1765. This region is the least understood part of TraI and is thought to be highly disordered and flexible. This region, being intrinsically disordered, is hypothesized to be serving as an interacting platform for other proteins involved in this DNA transfer initiation mechanism. In this work, we report the 1H, 13C, 15N resonance assignment of this region as well as the secondary structure information based on the backbone chemical shifts.

Highlights

Bacterial conjugation is a major mechanism driving rapid dissemination of genetic information among the bacterial community, leading to the spread of antibiotic resistance and virulence factors
TraI of plasmid R1 is a multi-functional enzyme of the relaxosome that is essential for the conjugative transfer of F like plasmids (Wong et al 2012; Lang et al 2010, 2011; Haft et al 2006)
To initiate the process of DNA transfer, R1 TraI first nicks a unique phosphodiester bond at nic site within the plasmid origin of transfer, binds covalently to the 5′ end of the nicked plasmid stand through a tyrosine residue and unwinds the nicked strand of plasmid DNA with the help of its functional helicase domain (Matson and Ragonese 2005)

Summary

Biological background

Bacterial conjugation is a major mechanism driving rapid dissemination of genetic information among the bacterial community, leading to the spread of antibiotic resistance and virulence factors. Restriction digestion was carried out with NcoI and KpnI to create sticky ends for both the amplified R1 TraIC126 gene fragment and the template for subsequent ligation This pET Z2 R1 TraIC126 construct was transformed into BL21 (DE3) cells (Thermofisher, Waltham, US) and expressed in M9 media (containing 1 g/l 15N ammonium chloride and 3 g/l 13C glucose) for isotope labelling of proteins for NMR experiments (M9 minimal medium (standard) 2010). TEV protease (1:50 TEV protease to protein ratio), 4 mM β-mercaptoethanol was added to the sample and dialyzed against 2 L of dialysis buffer (50 mM Na2HPO4, 10 mM imidazole, 300 mM NaCl, 4 mM β-mercaptoethanol, pH 8.0) for 16 h This step removes the 6xHIS and Z-domain. 1H, 13C, 15N resonance assignment of the C-terminal domain of the bifunctional enzyme TraI

NMR experiments

Findings

Assignments and secondary structure information