Mutational analysis of Agrobacterium tumefaciens pTiA6 virD1: identification of functionally important residues

Abstract

Mutagenesis experiments were used to identify functionally important regions of Agrobacterium tumefaciens pTiA6 VirD1. Random mutations were introduced by using Taq polymerase in a mutagenic reaction buffer containing manganese and altered nucleotide ratios to increase errors during the polymerase chain reaction (PCR). The mutants were assayed for VirD1-, VirD2-dependent border-nicking activity in Escherichia coli harbouring a border-containing substrate plasmid. Analysis of the mutants led to the identification of a region from amino acids 45-60 that is important for VirD1 activity. This region corresponds to a previously postulated potential DNA-binding domain. Deletion mutagenesis indicated that amino acids 2-16 could be deleted without affecting VirD1 function, whereas a larger deletion, amino acids 5-27, completely inactivated VirD1.