General Mutagenesis of F Plasmid TraI Reveals Its Role in Conjugative Regulation

Abstract

Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI that can tolerate insertions of a moderate size. We also demonstrate that wild-type TraI, when overexpressed, plays a dominant-negative regulatory role in conjugation, repressing plasmid transfer frequencies ϳ100-fold. Mutant TraI proteins with insertions in a region of approximately 400 residues between the consensus relaxase and helicase sequences did not cause conjugative repression. These unrestrictive TraI variants have normal relaxase activity in vivo, and several have wild-type conjugative functions when expressed at normal levels. We postulate that TraI negatively regulates conjugation by interacting with and sequestering some component of the conjugative apparatus. Our data indicate that the domain responsible for conjugative repression resides in the central region of TraI between the protein's catalytic domains.Much of the lateral gene transfer between bacteria occurs through the action of conjugative plasmids that encode all of the functions necessary for their hosts to transmit them to recipient cells. Plasmid transfer is achieved through direct cell contact and active transport of DNA by the donor. In gram-negative conjugation systems, typified by the F plasmid of Escherichia coli, only one strand of DNA is translocated, so single-strand cleavage and unwinding of the substrate DNA must occur prior to transfer (9). Strand scission is performed by plasmid-encoded "relaxases" that cleave their cognate plasmid at a specific site called nic within the origin-of-transfer region (oriT). In the case of the F and related plasmids, the accessory proteins TraM, TraY, and integration host factor also bind at oriT as part of the relaxosome complex (15,17,19,32). DNA unwinding is usually performed by a separate helicase, though in some systems, such as the F and R388 plasmids of E. coli, relaxase and helicase activities are both present in the relaxase (11,27,42). After making a single-stranded break at nic, the relaxase is thought to deliver the DNA to a type IV secretory apparatus that can translocate it across the recipient cell membrane. The relaxase remains covalently bound to the nic site and religates the scission once DNA transfer is complete. The replication of single-stranded plasmid DNA in the donor and recipient regenerates the double-stranded DNA plasmid in both cells.The F plasmid relaxase TraI is a well-studied model of structure and function for relaxases. The single-stranded DNA-cleaving activity of TraI is present in the first ϳ310 residues of this 1,756-residue protein, while the helicase motifs are locat...

Interbacterial Macromolecular Transfer by the Campylobacter fetus subsp. venerealis Type IV Secretion System

“…To evaluate the principle of how imbalanced expression of substrate and receptor genes might affect T4 secretion, we instead returned to the well-characterized F system. Overexpression of the F traI gene in trans to the wild-type conjugation genes causes a negative dominant phenotype for conjugation (30). That negative effect is also visible in our experiments where pOX38 transfer was reduced by 66-fold in the presence of a plasmid expressing Cre-TraI F compared to Cre alone (Fig.…”

Common Requirement for the Relaxosome of Plasmid R1 in Multiple Activities of the Conjugative Type IV Secretion System

“…High levels of TraD led to lower ColE1 mobilization frequencies with the pOX38⌬oriT⌬traD derivative than were observed without overexpression. Inhibition of the pOX38 transfer system by an imbalance of traD and traI expression has been reported earlier (66,67). Importantly, however, increased TraD concentrations eliminated the requirement for oriT R1 in ColE1 transfer such that wild-type frequencies of mobilization by the R1-16⌬oriT⌬traD derivative were observed regardless of the source of traD.…”

Autoregulation of the Synthesis of the MobM Relaxase Encoded by the Promiscuous Plasmid pMV158

“…These are the cases of the TraY of F plasmid (34), TrwA of R388 (33), MbeC of ColE1 (49), and MobC of pC221 (42). In other cases, like TraI of plasmid F, TraA of pIP501, and Mob of pBBR1 (19,22,47), it has been shown that expression of the relaxase gene is negatively regulated at the transcriptional level by the activity of the relaxase protein itself. However, the interactions between the relaxase and the host RNA polymerase (RNAP) at the plasmid origin of transfer (oriT) remain to be investigated.…”