Abstract
Bacterial low-copy-number plasmids require partition (par) systems to ensure their stable inheritance by daughter cells. In general, these systems consist of three components: a centromeric DNA sequence, a centromere-binding protein and a nucleotide hydrolase that polymerizes and functions as a motor. Type III systems, however, segregate plasmids using three proteins: the FtsZ/tubulin-like GTPase TubZ, the centromere-binding protein TubR and the MerR-like transcriptional regulator TubY. Although the TubZ filament is sufficient to transport the TubR-centromere complex in vitro, TubY is still necessary for the stable maintenance of the plasmid. TubY contains an N-terminal DNA-binding helix-turn-helix motif and a C-terminal coiled-coil followed by a cluster of lysine residues. This study determined the crystal structure of the C-terminal domain of TubY from the Bacillus cereus pXO1-like plasmid and showed that it forms a tetrameric parallel four-helix bundle that differs from the typical MerR family proteins with a dimeric anti-parallel coiled-coil. Biochemical analyses revealed that the C-terminal tail with the conserved lysine cluster helps TubY to stably associate with the TubR-centromere complex as well as to nonspecifically bind DNA. Furthermore, this C-terminal tail forms an amphipathic helix in the presence of lipids but must oligomerize to localize the protein to the membrane in vivo. Taken together, these data suggest that TubY is a component of the nucleoprotein complex within the partitioning machinery, and that lipid membranes act as mediators of type III systems.
Highlights
Accurate DNA segregation is essential for transmission of genetic information to daughter cells
TubY modulates the interaction between the segrosome and the TubZ filaments [16]; it remains unclear how TubY is involved in plasmid segregation
In analysis of DNA binding by BcYN and BcYCD with the tail (BcYC) using electrophoretic mobility shift assays (EMSA), only BcY bound pro12 (65171–65577 nt), indicating that both domains are required for DNA binding (Fig. 1C)
Summary
Accurate DNA segregation is essential for transmission of genetic information to daughter cells. TubY is a DNA-binding protein with a putative helix-turn-helix (HTH) motif that functions as a transcriptional activator of the tubRZ operon [15]. I found that the amphipathic C-terminal tail of TubY contributes to nonspecific DNA binding and to association with lipid membranes.
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