Abstract
During bacterial conjugation, genetic material from one cell is transferred to another as single-stranded DNA. The introduction of single-stranded DNA into the recipient cell would ordinarily trigger a potentially deleterious transcriptional response called SOS, which is initiated by RecA protein filaments formed on the DNA. During F plasmid conjugation, however, the SOS response is suppressed by PsiB, an F-plasmid-encoded protein that binds and sequesters free RecA to prevent filament formation. Among the many characterized RecA modulator proteins, PsiB is unique in using sequestration as an inhibitory mechanism. We describe the crystal structure of PsiB from the Escherichia coli F plasmid. The stucture of PsiB is surprisingly similar to CapZ, a eukaryotic actin filament capping protein. Structure-directed neutralization of electronegative surfaces on PsiB abrogates RecA inhibition whereas neutralization of an electropositive surface element enhances PsiB inhibition of RecA. Together, these studies provide a first molecular view of PsiB and highlight its use as a reagent in studies of RecA activity.
Highlights
The discovery of conjugation, the transfer of genetically diverse DNA between bacteria, was a foundational achievement in molecular genetic studies
We describe the crystal structure of PsiB from the Escherichia coli F plasmid
The E. coli F plasmid undergoes rolling circle replication to transfer single-stranded DNA2 from a donor to a recipient cell [3, 6]. This is an efficient transfer mechanism, introduction of ssDNA into the recipient cell presents a potential problem since ssDNA is the primary trigger for the SOS transcriptional response in bacterial cells
Summary
Proteins—E. coli F plasmid PsiB protein and PsiB variants were purified as previously described [20]. RecA ATPase Assays—M13mp ssDNA (3 M, nucleotides) was incubated with 0.5 M E. coli SSB (monomer), 0 or 10 M PsiB (or 10 M PsiB variant) (monomer) and 3 mM ATP in 1xRecA buffer (25 mM Tris acetate (80% cation, pH 7.58), 3 mM potassium glutamate, 10 mM magnesium acetate, 5% glycerol, 1 mM DTT) in the presence of an ATP regeneration system [30] for 10 min at 37 °C. LexA Cleavage Assays—M13mp ssDNA (3 M, nucleotides) was incubated for 10 min at 37 °C with 0.5 M E. coli SSB (monomer), 10 M PsiB variant (monomer), and 3 mM ATP in 25 mM Tris acetate 80% cation (pH 7.62), 3 mM potassium glutamate, 3 mM magnesium acetate, 5% glycerol, 1 mM DTT) in the presence of an ATP regeneration system. Data points are the mean of three independent experiments, and error bars represent one standard error of the mean
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