Abstract
The locus of enterocyte effacement-encoded regulator (Ler) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) functions to activate transcription of virulence genes silenced by the histone-like nucleoid-structuring protein (H-NS). Despite its important role in the bacterial gene regulation, the binding mode of Ler to DNA and its mechanism in alleviating genes repressed by H-NS are largely unknown. In this study, we use magnetic tweezers to demonstrate that Ler binds extended DNA through a largely noncooperative process, which results in DNA stiffening and DNA folding depending on protein concentration. We also show that Ler can replace prebound H-NS on DNA over a range of potassium and magnesium concentrations. Our findings reveal the DNA binding properties of Ler and shed light to further understand the anti-silencing activity of Ler.
Highlights
In enteropathogenic Escherichia coli and enterohemorrhagic E. coli, the genes responsible for their pathogenic activity are encoded in the locus of enterocyte effacement (LEE) pathogenicity island, which consists of five major operons, LEE1 to LEE5
In contrast to DNA stiffening by histone-like nucleoid-structuring protein (H-NS), which is caused by formation of nucleoprotein filaments, we found that the DNA stiffening induced by locus of enterocyte effacement-encoded regulator (Ler) proteins is not caused by a cooperative polymerization process
The dissociation constant (Kd) of Ler and DNA has been reported to be in the range of 200 nM based on binding to proU and LEE5 operons, which is lower than the Kd of H-NS binding to the same operons [20]
Summary
In enteropathogenic Escherichia coli and enterohemorrhagic E. coli, the genes responsible for their pathogenic activity are encoded in the locus of enterocyte effacement (LEE) pathogenicity island, which consists of five major operons, LEE1 to LEE5 Under normal conditions, these operons are silenced by a histone-like nucleoid-structuring protein (H-NS), a global. Ler and H-NS are important transcriptional regulators of the LEE pathogenicity islands Both proteins bind overlapping DNA regions, where H-NS serves to repress the gene expression and Ler counters the H-NS-mediated gene silencing. In our in vitro transcription assays, we did not see stimulation of LEE5 by Ler in the absence of H-NS.5 This suggests that Ler relieves the H-NS-mediated gene silencing likely by inhibiting H-NS binding to DNA.
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