title: Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the σ⁵⁴ (RpoN) regulon of Salmonella Typhimurium LT2.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Microbiology, University of Georgia, 30602, Athens, GA, USA. akarls@uga.edu.
authors.forename: David J
authors.initials: DJ
authors.lastname: Samuels
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Frye
authors.forename: Steffen
authors.initials: S
authors.lastname: Porwollik
authors.forename: Michael
authors.initials: M
authors.lastname: McClelland
authors.forename: Jan
authors.initials: J
authors.lastname: Mrázek
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Hoover
authors.forename: Anna C
authors.initials: AC
authors.lastname: Karls
mesh_terms: D001665:Binding Sites / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D020411:Oligonucleotide Array Sequence Analysis; D016366:Open Reading Frames; D009876:Operon; D011401:Promoter Regions, Genetic; D051877:RNA Polymerase Sigma 54 / Q000235:genetics*; D018085:Regulon; D012486:Salmonella typhimurium / Q000235:genetics*; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; D051877:RNA Polymerase Sigma 54