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...

Synopsis Visual selection during successive generations of inbreeding in corn resulted in significant improvement in yield, ear appearance and plant appearance by 57%, 60% and 41%, respectively, of the 134 specific hybrid combinations. The visual selection for yield and ear appearance was more effective in introduced than in local, well‐adapted lines. For plant appaarance the visual selection was more eftective in local, well‐adapted lines.