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

Samuels DJ, Frye JG, Porwollik S, McClelland M, Mrázek J, Hoover TR, Karls AC
BMC Genomics. 2013 14: 602

PMID: 24007446 · PMCID: PMC3844500 · DOI:10.1186/1471-2164-14-602

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.

MeSH Terms (12)

Binding Sites Chromatin Immunoprecipitation DNA-Binding Proteins Gene Expression Regulation, Bacterial Oligonucleotide Array Sequence Analysis Open Reading Frames Operon Promoter Regions, Genetic Regulon RNA Polymerase Sigma 54 Salmonella typhimurium Transcriptional Activation

Connections (1)

This publication is referenced by other Labnodes entities:

Links