Different phenotypes of Walker-like A box mutants of ParA homolog IncC of broad-host-range IncP plasmids

作者：Siddique Azeem; Figurski David H^*

来源：Plasmid, 2012, 68(2): 93-104.

DOI：10.1016/j.plasmid.2012.04.003

• KorB
• Partition
• Segregation
• RK2
• R995
• ATPase

The promiscuous IncPc alpha plasmids RK2 and R995 encode a broad-host-range partition system, whose essential components include the incC and korB genes and a DNA site (OB) to which the korB product binds. IncC2, the smaller of the two incC products, is sufficient for stabilization of R995 Delta incC. It is a member of the type la ParA family of partition ATPases. To better understand the role of ATP in partition, we constructed three alanine-substitution mutants of IncC2. Each mutation changed a different residue of the Walker-like ATP-binding and hydrolysis motif, including a lysine (K10) conserved solely among members of the ParA and MinD families. All three IncC2 mutants were defective in plasmid partition, but they differed from one another in other respects. The IncC2 T16A mutant, predicted to be defective in Mg2+ coordination, was severely impaired in all activities tested. IncC2 K10A, predicted to be defective in ATP hydrolysis, mediated enhanced incompatibility with R995 derivatives. IncC2 Kl5A, predicted to be defective in ATP binding, exhibited two distinct incompatibility properties depending on the genotype of the target plasmid. When in trans to plasmids carrying a complementable incC deletion, IncC2 K1 5A caused dramatic plasmid loss, even at low levels of expression. In trans to wild-type R995 or to R995 Delta incC carrying a functional P1 partition system, IncC2 K15A-mediated incompatibility was significantly less than that caused by wild-type IncC2. All three Walker-like A box mutants were also defective for the host toxicity that normally results from co-over-expression of incC and korB. The phenotypes of the mutants support a model in which nucleotide hydrolysis is required for separation of paired plasmid complexes and possible interaction with a host factor.

• 出版日期2012-9