Transcription factors that belong to the PadR family play an essential role in the transcriptional regulation of diverse biological processes by recognizing their cognate palindromic DNA sequences. Bacillus cereus harbors a gene that encodes a PadR-like protein (bcPLP; BC1756). bcPLP has not been structurally characterized, and it remains unelucidated how bcPLP interacts with a specific DNA sequence to function as a transcription factor. To provide structural insights into DNA recognition by bcPLP, we performed a structural study and a DNA-binding analysis of bcPLP. The crystal structure of bcPLP was determined at 1.92 angstrom resolution. bcPLP consists of two domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), and forms a homodimer mainly using the CTD. In the structure, bcPLP contains a highly positively charged elongated patch in the NTD that serves as a putative DNA-binding site. Indeed, an electrophoresis mobility shift assay and a fluorescence polarization assay showed that bcPLP specifically recognizes a palindromic DNA sequence upstream of the bcPLP-encoding region. Moreover, based on our mutagenesis and modeling studies, we demonstrate that bcPLP interacts with dsDNA primarily using the Y19, Y41, P64, and K66 residues in the NTD. (C) 2019 Elsevier Inc. All rights reserved.