Mutational analysis of the transcriptional activator VirG of Agrobacterium tumefaciens

• Published in: Journal of Bacteriology Vol. 176; no. 21; pp. 6418 - 6426
• Main Authors: Scheeren-Groot, E.P. (Leiden University, Leiden, The Netherlands), Rodenburg, K.W, Dulk-Ras, A. den, Turk, S.C.H.J, Hooykaas, P.J.J
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01-11-1994
• Subjects: Acetophenones - pharmacology; ADN; AGROBACTERIUM TUMEFACIENS; Agrobacterium tumefaciens - genetics; Agrobacterium tumefaciens - pathogenicity; Amino Acid Sequence; Bacterial Proteins - genetics; Bacteriology; Base Sequence; COMPOSE PHENOLIQUE; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; COMPUESTOS FENOLICOS; DNA Mutational Analysis; DNA, Single-Stranded - genetics; DNA-Binding Proteins - genetics; EXPRESION GENICA; EXPRESSION DES GENES; GENE; Gene Expression Regulation, Bacterial; GENES; GENETICA; GENETIQUE; KALANCHOE; Molecular Sequence Data; MUTACION; MUTACION INDUCIDA; Mutagenesis; Mutagens; MUTATION; MUTATION PROVOQUEE; NEOPLASMAS; NEOPLASME; Open Reading Frames - genetics; Plant Tumors - microbiology; Plants - microbiology; PODER PATOGENO; Point Mutation; POUVOIR PATHOGENE; PROTEINAS; PROTEINE; SECUENCIA NUCLEICA; Selection, Genetic; SEQUENCE NUCLEIQUE; SINTOMAS; SYMPTOME; Transcription Factors - genetics; Transcription, Genetic
• ISSN: 0021-9193; 1098-5530; 1067-8832
• DOI: 10.1128/jb.176.21.6418-6426.1994

To find VirG proteins with altered properties, the virG gene was mutagenized. Random chemical mutagenesis of single-stranded DNA containing the Agrobacterium tumefaciens virG gene led with high frequency to the inactivation of the gene. Sequence analysis showed that 29% of the mutants contained a virG gene with one single-base-pair substitution somewhere in the open reading frame. Thirty-nine different mutations that rendered the VirG protein inactive were mapped. Besides these inactive mutants, two mutants in which the vir genes were active even in the absence of acetosyringone were found on indicator plates. A VirG protein with an N54D substitution turned out to be able to induce a virB-lacZ reporter gene to a high level even in the absence of the inducer acetosyringone. A VirG protein with an I77V substitution exhibited almost no induction in the absence of acetosyringone but showed a maximum induction level already at low concentrations of acetosyringone