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. 2014 Sep;196(17):3082-90.
doi: 10.1128/JB.01496-14. Epub 2014 Jun 16.

Unique Helicase Determinants in the Essential Conjugative TraI Factor From Salmonella Enterica Serovar Typhimurium Plasmid pCU1

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Free PMC article

Unique Helicase Determinants in the Essential Conjugative TraI Factor From Salmonella Enterica Serovar Typhimurium Plasmid pCU1

Krystle J McLaughlin et al. J Bacteriol. .
Free PMC article

Abstract

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.

Figures

FIG 1
FIG 1
pCU1 TraI helicase sequence alignment. C termini of pCU1 TraI (GenBank accession number AAD27542.1), R388 TrwC (GenBank accession number CAA44853.2), and F TraI (GenBank accession number BAA97974.1) were aligned to show conserved SF1 helicase motifs (boxed). Identical residues are shaded black, and similar residues are shaded gray.
FIG 2
FIG 2
pCU1 TraI domain organization and protein constructs used in this study. Wild-type TraI has 1,078 amino acids (WT_1078). The N-terminal region contains the relaxase domain between residues 1 and 299, while the C-terminal region harbors a predicted helicase domain. Canonical helicase motifs (white boxes) span a region between residues 498 and 894. Residue ranges for each TraI deletion construct used are indicated. Filled triangles represent the location of point mutations in conserved helicase motifs investigated in full-length TraI.
FIG 3
FIG 3
pCU1 TraI DNA binding activity. Representative fluorescence anisotropy curves with standard error bars for wild-type pCU1 TraI (WT_1078) and TraI helicase (WT_311-1078) constructs binding to 35oriT and 10-mer DNA substrates. The DNA binding curves generated by WT_1078 and WT_311-1078 were best fit by sigmoidal and hyperbolic fits, respectively.
FIG 4
FIG 4
pCU1 TraI NTPase activity. (A) NTP hydrolysis by TraI. Hydrolysis is indicated by a reduction in absorbance at 350 nm and was observed only in the presence of ATP. Little or no activity was observed in the presence of TTP, GTP, and CTP. All reaction mixtures contained 100 nM 60-mer ssDNA. (B) Influence of ssDNA length on the ATPase activity of TraI. TraI exhibits low background ATPase activity in the absence of DNA. An increase in activity was seen as the ssDNA length increased from 37 to 60 nucleotides, whereas a shorter substrate reduced activity. Similar activity was seen for the 60-nucleotide substrate and the shorter 35oriTc (Table 1), indicating some sequence specificity. All DNA substrate concentrations were 500 nM. (For clarity, averages of three experiments each are shown and error bars are omitted from panels A and B.) (C) ATPase activity of full-length TraI, deletion mutants, and ATPase motif mutants. Deletion mutant constructs containing at least residues 311 to 932 retained ATPase activity. Point mutations in the conserved helicase motif residues K507 and E569 eliminated ATPase activity, while constructs with substitutions at residue D568 retained all or some ATPase activity. Error bars represent standard errors between at least two independent experiments. ND, not detected.
FIG 5
FIG 5
pCU1 TraI dsDNA separation activity. (A) Kinetics of the unwinding reaction at different TraI concentrations. The time course of the pCU1 TraI unwinding reaction using the partial duplex 17-mer–80-mer dsDNA substrate is shown. Helicase assays were performed at 37°C as described in Materials and Methods with a starting TraI concentration of 3.5 μM (●), which was then increased to 7 μM (○). Standard error between multiple experiments is shown. (B) Representative electrophoretic mobility shift assay. dsDNA strand separation by TraI constructs with or without the extreme C terminus (residues 933 to 1078) was measured with EMSA. The TraI construct used is indicated, and each reaction was run in duplicate. Helicase assays were performed at 37°C for 30 min with 8 μM TraI and 0.5 μM dsDNA substrate as described in Materials and Methods. (C) Bar graph of normalized unwinding activity for each TraI construct tested, with standard error shown. Percent dsDNA fragment unwound was normalized to wild-type levels. TraI constructs lacking the extreme C terminus have significantly reduced ability to unwind the 17-mer–80-mer dsDNA substrate (see Table 1 for sequence information).
FIG 6
FIG 6
Dissection of functional domains of pCU1 TraI. All pCU1 constructs have DNA binding ability. Those lacking the N-terminal relaxase retain both ATPase and DNA unwinding activities (+). However, constructs lacking the extreme C terminus (residues 933 to 1078) are unable to unwind dsDNA substrates efficiently (−).

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