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 Å 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 Å 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.

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

“…The largest increase in affinity occurred when the relaxase region was absent (WT_311-1078, WT_311-932, and WT_483-932) (Table 2), although this was maximally a 3-fold difference that was not consistent across all three DNA substrates. When removed from the context of the full-length protein, the TraI relaxase is also able to bind these DNA substrates, though at a much lower affinity (ϳ1 M [31][32][33][34][35]53]). The presence of the relaxase domain does not appear to impact significantly the overall DNA binding activity of pCU1 TraI.…”

“…The N-terminal region encodes a relaxase enzyme with transesterase activity (40,41,53), while the C-terminal region contains a predicted helicase domain ( Fig. 1 and 2).…”

“…ATPase point mutants were generated by QuikChange site-directed mutagenesis (Stratagene). All TraI constructs were cloned into the pTYB2 vector of the IMPACT system (New England BioLabs) as previously described (4)(5)(6)53).…”

“…Alternatively, equation 2 could be used to yield a value for K D as previously described (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)53):…”

“…The structure and function of the pCU1 TraI N-terminal relaxase domain have been well studied (53,54), with only limited data available for its C-terminal helicase. In this work, we establish that the C-terminal domain of pCU1 TraI contains a functional SF1B helicase capable of binding DNA, hydrolyzing ATP and unwinding double-stranded DNA (dsDNA) substrates.…”