Mutational analysis of TraM correlates oligomerization and DNA binding with autoregulation and conjugative DNA transfer.
F plasmid TraM, an autoregulatory homotetramer, is essential for F plasmid bacterial conjugative transfer, one of the major mechanisms for horizontal gene dissemination. TraM cooperatively binds to three sites (sbmA, -B, and -C) near the origin of transfer in the F plasmid. To examine whether or not tetramerization of TraM is required for autoregulation and F conjugation, we used a two-plasmid system to screen for autoregulation-defective traM mutants generated by random PCR mutagenesis. A total of 72 missense mutations in TraM affecting autoregulation were selected, all of which also resulted in a loss of TraM function during F conjugation. Mutational analysis of TraM defined three regions important for F conjugation, including residues 3-10 (region I), 31-53 (region II), and 80-121 (region III); in addition, residues 3-47 were also important for the immunoreactivity of TraM. Biochemical analysis of mutant proteins indicated that region I defined a DNA binding domain that was not involved in tetramerization, whereas regions II and III were important for both tetramerization and efficient DNA binding. Mutations in region III affected the cooperativity of binding of TraM to sbmA, -B, and -C. Our results suggest that tetramerization is important for specific DNA binding, which, in turn, is essential for traM autoregulation and F conjugation. These findings support the hypothesis that TraM functions as a "signaling" factor that triggers DNA transport during F conjugation.
Lu
Jun
J
Department of Biological Sciences, CW405 Biological Sciences Building, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
Zhao
Wen
W
Frost
Laura S
LS
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001665:Binding Sites; D049109:Cell Proliferation; D002845:Chromatography; D003062:Codon; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004252:DNA Mutational Analysis; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005144:F Factor / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D020125:Mutation, Missense; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D003062:Codon; D009841:Oligonucleotides; C074281:TraM protein, bacterial; D004247:DNA; D001616:beta-Galactosidase