Signal transduction and bacterial conjugation: characterization of the role of ArcA in regulating conjugative transfer of the resistance plasmid R1

J Mol Biol. 1998 Mar 27;277(2):309-16. doi: 10.1006/jmbi.1997.1598.

Abstract

The role of the two-component response regulator ArcA protein in the transfer of the conjugative resistance plasmid R1 was investigated using 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 reduced by four orders of magnitude in an Escherichia coli host with a mutation in the arcA gene. Measurements of mRNAs transcribed from key plasmid transfer genes revealed that the abundance of each of the mRNA species investigated was reduced significantly in an arcA background. Gene fusion studies with the R1 PY promoter, the major promoter of the transfer operon, and a lacZ reporter gene, indicated that arcA is required for maximal expression from this promoter. However, a stimulating effect of arcA could only be detected when the plasmid-specified positive regulator of the transfer genes, traJ, was present. Electrophoretic mobility shift assays were used to demonstrate specific binding of purified ArcA protein and a purified and phosphorylated oligohistidine-tagged ArcA (His6-ArcA) to a DNA fragment containing the PY promoter region. The binding of phosphorylated His6-ArcA to the PY promoter was further characterized by DNase I footprinting. The observed protection pattern was characteristic for ArcA acting as a transcriptional activator.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Outer Membrane Proteins / genetics
  • Bacterial Outer Membrane Proteins / metabolism*
  • Base Sequence
  • Conjugation, Genetic*
  • DNA Footprinting
  • DNA, Bacterial
  • Deoxyribonuclease I / metabolism
  • Escherichia coli / genetics*
  • Escherichia coli Proteins
  • Genes, Bacterial
  • Histidine / metabolism
  • Molecular Sequence Data
  • Mutation
  • Operon
  • Phosphorylation
  • Promoter Regions, Genetic
  • R Factors / genetics*
  • Repressor Proteins*
  • Signal Transduction*
  • Transcription, Genetic

Substances

  • Bacterial Outer Membrane Proteins
  • DNA, Bacterial
  • Escherichia coli Proteins
  • Repressor Proteins
  • TraJ protein, E coli
  • TraJ protein, bacteria
  • arcA protein, E coli
  • Histidine
  • Deoxyribonuclease I