SIGNAL-TRANSDUCTION AND BACTERIAL CONJUGATION - CHARACTERIZATION OF THE ROLE OF ARCA IN REGULATING CONJUGATIVE TRANSFER OF THE RESISTANCE PLASMID R1

The role of the two-component response regulator ArcA protein in the t ransfer of the conjugative resistance plasmid R1 was investigated usin g a variety of in vivo and in vitro assays. The frequency of conjugal DNA transfer of plasmid R1-16, a derepressed variant of R1, was reduce d by four orders of magnitude in an Escherichia coli host with a mutat ion in the arcA gene. Measurements of mRNAs transcribed from key plasm id transfer genes revealed that the abundance of each of the mRNA spec ies investigated was reduced significantly in an arcA background. Gene fusion studies with the R1 P-y promoter, the major promoter of the tr ansfer operon, and a lacZ reporter gene, indicated that arcA is requir ed for maximal expression from this promoter. However, a stimulating e ffect of arcA could only be detected when the plasmid-specified positi ve regulator of the transfer genes, traJ, was present. Electrophoretic mobility shift assays were used to demonstrate specific binding of pu rified ArcA protein and a purified and phosphorylated oligohistidine-t agged ArcA (HisG-ArcA) to a DNA fragment containing the P-y promoter r egion. The binding of phosphorylated HisG-ArcA to the P-y promoter was further characterized by DNase I footprinting. The observed protectio n pattern was characteristic for ArcA acting as a transcriptional acti vator. (C) 1998 Academic Press Limited.