Open AccessGeneral Mutagenesis of F Plasmid TraI Reveals Its Role in Conjugative Regulation
Author(s) -
Rembrandt J. F. Haft,
Gilberto Palacios,
Tran Nguyen,
Manuela Mally,
Eliora Gachelet,
Ellen L. Zechner,
Beth Traxler
Publication year - 2006
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.00462-06
Subject(s) - bacterial conjugation , biology , plasmid , mutant , mutagenesis , genetics , psychological repression , transfer dna , gene , microbiology and biotechnology , transformation (genetics) , gene expression , agrobacterium tumefaciens
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 genetraI 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 ∼100-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.