Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the σ⁵⁴ (RpoN) regulon of Salmonella Typhimurium LT2

Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ&sup5;&sup4; regulon in a bacterium is that the σ&sup5;&sup4; RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a σ&sup5;&sup4;-dependent promoter. Many bacteria possess multiple bEBPs, which are activated by diverse environmental stimuli. In this work, we assess the ability of a promiscuous, constitutively-active bEBP-the AAA+ ATPase domain of DctD from Sinorhizobium meliloti-to activate transcription from all σ&sup5;&sup4;-dependent promoters for the characterization of the σ&sup5;&sup4; regulon of Salmonella Typhimurium LT2.

Since the DctD AAA + ATPase domain proved effective in activating transcription from the diverse σ&sup5;&sup4;-dependent promoters of the S. Typhimurium LT2 σ&sup5;&sup4; regulon under a single growth condition, this approach is likely to be valuable for examining σ&sup5;&sup4; regulons in other bacterial species. The S. Typhimurium σ&sup5;&sup4; regulon included a high number of intragenic σ&sup5;&sup4; binding sites/promoters, suggesting that σ&sup5;&sup4; may have multiple regulatory roles beyond the initiation of transcription at the start of an operon.

The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted σ&sup5;&sup4;-dependent promoters in Salmonella under a single condition. These promoters are controlled by a variety of native activators and, under the condition tested, are not transcribed in the absence of the DctD AAA+ ATPase domain. We also identified a novel σ&sup5;&sup4;-dependent promoter upstream of STM2939, a homolog of the cas1 component of a CRISPR system. ChIP-chip analysis revealed at least 70 σ&sup5;&sup4; binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic σ&sup5;&sup4; binding sites suggest that many of these sites are capable of functioning as σ&sup5;&sup4;-dependent promoters.