Sigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ⁵⁴ regulon in a bacterium is that the σ⁵⁴ RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a σ⁵⁴-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 σ⁵⁴-dependent promoters for the characterization of the σ⁵⁴ regulon of Salmonella Typhimurium LT2.

The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted σ⁵⁴-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 σ⁵⁴-dependent promoter upstream of STM2939, a homolog of the cas1 component of a CRISPR system. ChIP-chip analysis revealed at least 70 σ⁵⁴ binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic σ⁵⁴ binding sites suggest that many of these sites are capable of functioning as σ⁵⁴-dependent promoters.

Since the DctD AAA+ ATPase domain proved effective in activating transcription from the diverse σ⁵⁴-dependent promoters of the S. Typhimurium LT2 σ⁵⁴ regulon under a single growth condition, this approach is likely to be valuable for examining σ⁵⁴ regulons in other bacterial species. The S. Typhimurium σ⁵⁴ regulon included a high number of intragenic σ⁵⁴ binding sites/promoters, suggesting that σ⁵⁴ may have multiple regulatory roles beyond the initiation of transcription at the start of an operon.