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- Title
Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the σ<sup>54</sup> (RpoN) regulon of Salmonella Typhimurium LT2.
- Authors
Samuels, David J.; Frye, Jonathan G.; Porwollik, Steffen; McClelland, Michael; Mrázek, Jan; Hoover, Timothy R.; Karls, Anna C.
- Abstract
Background: 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. Results: 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. Conclusion: 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.
- Subjects
FOODBORNE diseases; ADENOSINE triphosphatase; SALMONELLA typhimurium; NUCLEIC acids; RNA polymerases
- Publication
BMC Genomics, 2013, Vol 14, Issue 1, p1
- ISSN
1471-2164
- Publication type
Article
- DOI
10.1186/1471-2164-14-602