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Microbiology 151 (2005), 259-268; DOI  10.1099/mic.0.27442-0
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Microbiology 151 (2005), 259-268; DOI  10.1099/mic.0.27442-0
© 2005 Society for General Microbiology

Detailed studies of the binding mechanism of the Sinorhizobium meliloti transcriptional activator ExpG to DNA

Birgit Baumgarth1,{dagger}, Frank Wilco Bartels2,{dagger}, Dario Anselmetti2, Anke Becker1 and Robert Ros2

1 Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, 33615 Bielefeld, Germany
2 Experimentelle Biophysik, Fakultät für Physik, Universität Bielefeld, 33615 Bielefeld, Germany

Correspondence
Anke Becker
Anke.Becker{at}genetik.uni-bielefeld.de

The exopolysaccharide galactoglucan promotes the establishment of symbiosis between the nitrogen-fixing Gram-negative soil bacterium Sinorhizobium meliloti 2011 and its host plant alfalfa. The transcriptional regulator ExpG activates expression of galactoglucan biosynthesis genes by direct binding to the expA1, expG/expD1 and expE1 promoter regions. ExpG is a member of the MarR family of regulatory proteins. Analysis of target sequences of an ExpG(His)6 fusion protein in the exp promoter regions resulted in the identification of a binding site composed of a conserved palindromic region and two associated sequence motifs. Association and dissociation kinetics of the specific binding of ExpG(His)6 to this binding site were characterized by standard biochemical methods and by single-molecule spectroscopy based on the atomic force microscope (AFM). Dynamic force spectroscopy indicated a distinct difference in the kinetics between the wild-type binding sequence and two mutated binding sites, leading to a closer understanding of the ExpG–DNA interaction.

Abbreviations: AFM, atomic force microscopy; EMSA, electrophoretic mobility shift assay; EPS, exopolysaccharide; EPS I, succinoglycan; EPS II, galactoglucan; HTH, helix–turn–helix; kon, on-rate; koff, off-rate; Kd, dissociation constant

{dagger}B. B. and F. W. B. contributed equally to this work.




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