Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the sigma(54) (RpoN) regulon of Salmonella Typhimurium LT2

作者：Samuels David J; Frye Jonathan G; Porwollik Steffen; McClelland Michael; Mrazek Jan; Hoover Timothy R; Karls Anna C^*

来源：BMC Genomics, 2013, 14(1): 602.

DOI：10.1186/1471-2164-14-602

• Sigma54
• RpoN
• Bacterial enhancer-binding protein
• Regulon
• Sigma factor
• Salmonella

Background: Sigma54, or RpoN, is an alternative sigma factor found widely in eubacteria. A significant complication in analysis of the global sigma(54) regulon in a bacterium is that the sigma(54) RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a sigma(54)-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 sigma(54)-dependent promoters for the characterization of the sigma(54) regulon of Salmonella Typhimurium LT2. %26lt;br%26gt;Results: The AAA+ ATPase domain of DctD was able to drive transcription from nearly all previously characterized or predicted sigma(54)-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 sigma(54)-dependent promoter upstream of STM2939, a homolog of the cas1 component of a CRISPR system. ChIP-chip analysis revealed at least 70 sigma(54) binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic sigma(54) binding sites suggest that many of these sites are capable of functioning as sigma(54)-dependent promoters. %26lt;br%26gt;Conclusion: Since the DctD AAA+ ATPase domain proved effective in activating transcription from the diverse sigma(54)-dependent promoters of the S. Typhimurium LT2 sigma(54) regulon under a single growth condition, this approach is likely to be valuable for examining sigma(54) regulons in other bacterial species. The S. Typhimurium sigma(54) regulon included a high number of intragenic sigma(54) binding sites/promoters, suggesting that sigma(54) may have multiple regulatory roles beyond the initiation of transcription at the start of an operon.

• 出版日期2013-9-5