Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the σ54 RpoN regulon of Salmonella Typhimurium LT2Report as inadecuate




Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the σ54 RpoN regulon of Salmonella Typhimurium LT2 - Download this document for free, or read online. Document in PDF available to download.

BMC Genomics

, 14:602

Prokaryote microbial genomics

Abstract

BackgroundSigma54, or RpoN, is an alternative σ factor found widely in eubacteria. A significant complication in analysis of the global σ regulon in a bacterium is that the σ RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein bEBP to initiate transcription at a σ-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 σ-dependent promoters for the characterization of the σ regulon of Salmonella Typhimurium LT2.

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

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

KeywordsSigma54 RpoN Bacterial enhancer-binding protein Regulon Sigma factor Salmonella AbbreviationsAmpAmpicillin

bEBPBacterial enhancer-binding protein

ChIPChromatin immunoprecipitation

EσRNA polymerase holoenzyme

LBLuria-bertani media

LM-PCRLigation-mediated PCR

NBNutrient broth

RNAPRNA polymerase

SpcSpectinomycin

StrStreptomycin

UASUpstream activation sequence.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2164-14-602 contains supplementary material, which is available to authorized users.

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Author: David J Samuels - Jonathan G Frye - Steffen Porwollik - Michael McClelland - Jan Mrázek - Timothy R Hoover - Anna C Ka

Source: https://link.springer.com/



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