The N-terminal domain of the repressor of Staphylococcus aureus phage Φ11 possesses an unusual dimerization ability and DNA binding affinity.

Anindya Biswas,Sukhendu Mandal,Subrata Sau,Fenfei Leng

doi:10.1371/journal.pone.0095012

Abstract

Bacteriophage Φ11 uses Staphylococcus aureus as its host and, like lambdoid phages, harbors three homologous operators in between its two divergently oriented repressor genes. None of the repressors of Φ11, however, showed binding to all three operators, even at high concentrations. To understand why the DNA binding mechanism of Φ11 repressors does not match that of lambdoid phage repressors, we studied the N-terminal domain of the Φ11 lysogenic repressor, as it harbors a putative helix-turn-helix motif. Our data revealed that the secondary and tertiary structures of the N-terminal domain were different from those of the full-length repressor. Nonetheless, the N-terminal domain was able to dimerize and bind to the operators similar to the intact repressor. In addition, the operator base specificity, binding stoichiometry, and binding mechanism of this domain were nearly identical to those of the whole repressor. The binding affinities of the repressor and its N-terminal domain were reduced to a similar extent when the temperature was increased to 42°C. Both proteins also adequately dislodged a RNA polymerase from a Φ11 DNA fragment carrying two operators and a promoter. Unlike the intact repressor, the binding of the N-terminal domain to two adjacent operator sites was not cooperative in nature. Taken together, we suggest that the dimerization and DNA binding abilities of the N-terminal domain of the Φ11 repressor are distinct from those of the DNA binding domains of other phage repressors.

Highlights

The two alternate life cycles of temperate bacteriophages are typically regulated by phage-encoded repressor proteins and cognate operator DNAs [1,2,3,4,5]
To obtain information on the structure and DNA-binding activity of the N-terminal domain (NTD) of W11 CI, we purified recombinant NTD by affinity column chromatography (Figure 2A). rNTD is composed of amino acid residues 1-118 of W11 CI and an additional eight amino acid residues at its C-terminus
To properly compare the structure and function of rNTD with those of intact W11 CI, W11 CI with a C-terminal histidine tag was designed and purified in similar manner (Figure 2A). Both rNTD and rCI reacted with an anti-his antibody (Figure 2B), and their molecular masses nearly matched with their calculated molecular masses

Summary

Introduction

The two alternate life cycles (lytic and lysogenic) of temperate bacteriophages are typically regulated by phage-encoded repressor proteins and cognate operator DNAs [1,2,3,4,5]. These regulatory proteins typically block the transcription of phage genes from their early promoters by binding to partially overlapped operator DNA sequences. Studies on phage repressors have greatly enriched the fields of both applied and basic molecular biology. Despite their usefulness, most phage repressors have not yet been investigated in detail

Methods

Results

Conclusion