Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis.
The ParA family of proteins is involved in partition of a variety of plasmid and bacterial chromosomes. P1 ParA plays two roles in partition: it acts as a repressor of the par operon and has an undefined yet indispensable role in P1 plasmid localization. We constructed seven mutations in three putative ATP-binding motifs of ParA. Three classes of phenotypes resulted, each represented by mutations in more than one motif. Three mutations created 'super-repressors', in which repressor activity was much stronger than in wild-type ParA, while the remainder damaged repressor activity. All mutations eliminated partition activities, but two showed a plasmid stability defect that was worse than that of a null mutation. Four mutant ParAs, two super-repressors and two weak repressors, were analyzed biochemically, and all exhibited damaged ATPase activity. The super-repressors bound site-specifically to the par operator sequence, and this activity was strongly stimulated by ATP and ADP. These results support the proposal that ATP binding is essential but hydrolysis is inhibitory for ParA's repressor activity and suggest that ATP hydrolysis is essential for plasmid localization.
Fung
E
E
Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Bouet
J Y
JY
Funnell
B E
BE
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics*; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D001618:beta-Lactamases / Q000235:genetics
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012097:Repressor Proteins; C052883:chromosome partition proteins, bacterial; D000255:Adenosine Triphosphate; D001618:beta-Lactamases