P22-based challenge phage constructs to study protein-protein interactions between the σ⁵⁴-dependent promoter, dctA, and its transcriptional regulators

To study interactions between C₄-dicarboxylic acid transport protein D and Eσ⁵⁴ in the dctA promoter regulatory region, we used the challenge phage system. An ant'-'lac fusion was recombined onto the challenge phage, and this ant'-'lac fusion along with Pant and the R. meliloti dctA promoter regulatory region were cloned onto a plasmid. The plasmid bearing the ant'-'lac fusion was used as a reporter plasmid in a coupled transcription-translation system. Addition of purified σ⁵⁴ to the coupled system specifically repressed transcription of the plasmid-borne ant'-'lac fusion. When DCTD was added along with σ⁵⁴ to the coupled system, transcription of the ant'-'lac fusion was even further repressed, suggesting that DCTD may stabilize closed complexes between Eσ⁵⁴ and the dctA promoter.