The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1

Abstract

Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 A crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that are required for DNA nicking and religation were displaced up to 14 A out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid's origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1.

Figure 1.

The 2.3 Å resolution crystal structure of the pCU1 TraI relaxase. (A) The overall structure of the pCU1 relaxase (WT_299) with secondary-structures colored as represented in Figure 2 (β-strands are represented by yellow arrows, α-helices by red helices and loop regions by gray loops). The histidine triad of the active site and the tyrosines implicated in DNA nicking are shown as sticks. The manganese ion bound by the histidine triad is shown as a grey sphere. (B) The active site of the pCU1 relaxase (WT_299) with secondary structures represented as in (A). At the center is the histidine triad and bound manganese ion. In the upper right is loop A and the four tyrosines implicated in DNA nicking, and to the left is the N-terminus, represented as a black sphere. (C) The active site of the pCU1 relaxase [WT_299, colored as described in (A)] aligned with the active sites of the F plasmid TraI relaxase (in blue) and the relaxase of plasmid R388 TrwC (in magenta). The histidine triad and the primary DNA nicking tyrosine of each protein are shown as sticks. The distances between the tyrosines are indicated. Note that the in the structure of the plasmid R388 TrwC, Tyr18 was mutated to Phe18.

Figure 2.

Amino acid sequence alignment and secondary structure of the pCU1 TraI relaxase. The initial 337 amino acids of pCU1 TraI (GenBank ID AAD27542) were aligned with R388 TrwC (GenBank ID CAA44853) and F TraI (GenBank ID BAA97974) using the Clustal X program in BioEdit. Identical residues are shaded green, similar residues are shaded blue. Orange and yellow boxes indicate the first and last residues of the relaxase domains of each protein. Two constructs of the F TraI relaxase domain have been crystallized (3,23), therefore the terminal residues of both are boxed. Red boxes indicate the location of the conserved tyrosine residues and the active site histidine triad. Red lines underline the residues forming the ‘thumb’ regions of each protein. Since this region is disordered in the pCU1 TraI relaxase crystal, the extent of this region is estimated. Above the three sequences is the secondary structure of the pCU1 relaxase. β-strands are represented by yellow arrows; α-helices by red cylinders; and loop regions by black wavy lines. The secondary structure of residues 1–225 reflects that observed in the crystal structure of WT_299; the secondary structure of residues 226–337 reflects that predicted by Jpred 3.

Figure 3.

DNA binding by the pCU1 TraI relaxase. DNA binding curves were generated by monitoring the change in fluorescence anisotropy (FA) of a fluorescein-labeled DNA substrate as a function of protein concentration and then plotting the normalized FA as a function of total protein concentration. Each data point is the average of at least three replicates, with error bars representing the standard error of these replicates. The DNA binding affinity (K_D, in nM) was calculated by fitting the data with Equation using non-linear regression. The error represents the standard error of the K_D when calculated by Equation . (A) Binding of the substrates 35oriT-hairpin and 35/7oriT-hairpin by WT_299 and the pCU1 relaxase mutant Y18,19,26,27F_299, respectively. The substrate 35/7oriT-hairpin extends 7 nt beyond the oriT nic site, while 35oriT-hairpin is truncated at the nic site. (B) Binding of the substrate 35/7oriT-hairpin by Y18,19,26,27F_299 in the absence or presence of 4 mM MnCl₂.

Figure 4.

DNA nicking and religation by the pCU1 TraI relaxase domain. (A) To determine the DNA nicking activity of the wild-type pCU1 TraI relaxase, as well as several relaxase mutants, 5 μM enzyme was incubated with 1 μM substrate (35/7oriT- or 20/7oriT-hairpin) for 1 h at 37°C, and products were then separated on denaturing polyacrylamide gels. Nicking and religation activity is represented as percent product generated. Each experiment was performed in triplicate; the error is the standard error of these replicates. For each experiment, a representative gel is provided. Below each gel, a reaction scheme is provided, where a fluorescein label is indicated by a green star and the substrate nic site by a red triangle. Additional experimental details can be found in the Materials and Methods section and the Supplementary Data. (B) A reaction scheme illustrating a DNA cross-over experiment, where a fluorescein label is indicated by a green star and the substrate nic site by a red triangle.

Figure 5.

DNA nicking by the pCU1 TraI relaxase in the presence of metals. To determine the DNA nicking activity of the pCU1 TraI relaxase (WT_299) after treatment with EDTA and in the presence of the indicated concentration of metal, 5 μM enzyme was incubated with 1 μM substrate (35/7oriT-hairpin) for 1 h at 37°C, and products were then separated on denaturing polyacrylamide gels. Nicking and religation activity is represented as percent product generated. Each experiment was performed in triplicate; the error is the standard error of these replicates. Additional details can be found in the Materials and Methods section and the Supplementary Data.

Figure 6.

Nucleic acid sequence and sub-divisions of the pCU1 oriT. The nucleic acid sequence of the plasmid pCU1 oriT is divided into four regions (hairpin in dark blue, TAG in green, pentanucleotide in red and post nic in light blue). The bases forming the inverted repeat are shaded black and the location of the predicted hairpin is indicated.