NasT-Mediated Antitermination Plays an Essential Role in the Regulation of the Assimilatory Nitrate Reductase Operon in Azotobacter vinelandii

Abstract

Azotobacter vinelandii is a well-studied model system for nitrogen fixation in bacteria. Regulation of nitrogen fixation in A. vinelandii is independent of NtrB/NtrC, a conserved nitrogen regulatory system in proteobacteria. Previous work showed that an ntrC mutation in A. vinelandii resulted in a loss of induction of assimilatory nitrate and nitrite reductases encoded by the nasAB operon. In addition to NtrC, several other proteins, including NasT, a protein containing a potential RNA-binding domain ANTAR (AmiR and NasR transcription antitermination regulators), have been implicated in nasAB regulation. In this work, we characterize the sequence upstream of nasA and identify several DNA sequence elements, including two potential NtrC binding sites and a putative intrinsic transcriptional terminator upstream of nasA that are potentially involved in nasAB regulation. Our analyses confirm that the nasAB promoter, P(nasA), is under NtrC control. However, unlike NtrC-regulated promoters in enteric bacteria, P(nasA) shows high activity in the presence of ammonium; in addition, the P(nasA) activity is altered in the nifA gene mutation background. We discuss the implication of these results on NtrC-mediated regulation in A. vinelandii. Our study provides direct evidence that induction of nasAB is regulated by NasT-mediated antitermination, which occurs within the leader region of the operon. The results also support the hypothesis that NasT binds the promoter proximal hairpin of nasAB for its regulatory function, which contributes to the understanding of the regulatory mechanism of ANTAR-containing antiterminators.