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Updated: Oct 19, 2025

An Assay for Quantifying Protein-RNA Binding in Bacteria
Published on: June 12, 2019
The low-nanomolar 4-nitrobenzoate-responsive repressor PnbX negatively regulates the actinomycete-derived
Minggen Cheng1, Yingying Qian1, Ziyu Xing1
1Department of Microbiology, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu, China.
Abstract:
Nitroaromatic compounds pose severe threats to public health and environmental safety. Nitro group removal via ammonia release is an important strategy for bacterial detoxification of nitroaromatic compounds, such as the conversion of 4-nitrobenzoate (4-NBA) to protocatechuate by the bacterial pnb operon. In contrast to the LysR-family transcriptional regulator PnbR in proteobacteria, the actinomycete-derived pnb locus (4-NBA degradation structural genes) formed an operon with the TetR-family transcriptional regulator gene pnbX, implying that it has a distinct regulatory mechanism. Here, pnbBA from the actinomycete Nocardioides sp. strain LMS-CY was biochemically confirmed to express 4-NBA degradation enzymes, and pnbX was essential for inducible degradation of 4-NBA. Purified PnbX-6His could bind the promoter probe of the pnb locus in vitro, and 4-NBA prevented this binding. 4-NBA could bind PnbX at a 1:1 molar ratio with KD = 26.7 ± 4.2 nM. Low-nanomolar levels of 4-NBA induced the transcription of the pnb operon in strain LMS-CY. PnbX bound a palindromic sequence motif (5'-TTACGTTACA-N8 -TGTAACGTAA-3') that encompasses the pnb promoter. This study identified a TetR-family repressor for the actinomycete-derived pnb operon that recognizes 10-8 M 4-NBA as its ligand, implying that nitro group removal of nitroaromatic compounds may be especially important for actinomycetes.
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