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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR APPROACHES FOR THE IDENTIFICATION AND CHARACTERIZATION OF ANTIMICROBIAL RESISTANCE IN FOODBORNE PATHOGENS

Location: Bacterial Epidemiology and Antimicrobial Resistance

Title: Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the Sigma54 (RpoN) regulon of Salmonella Typhimurium LT2

Authors
item Samuels, David -
item Frye, Jonathan
item Porwollik, Steffen -
item Mcclelland, Michael -
item Mrazek, Jan -
item Hoover, Timothy -
item Karls, Anna -

Submitted to: Biomed Central (BMC) Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 23, 2013
Publication Date: September 5, 2013
Citation: Samuels, D.J., Frye, J.G., Porwollik, S., Mcclelland, M., Mrazek, J., Hoover, T.R., Karls, A.C. 2013. Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the Sigma54 (RpoN) regulon of Salmonella Typhimurium LT2. Biomed Central (BMC) Genomics. 14(1):602.

Interpretive Summary: To be a successful foodborne pathogen, Salmonella enterica bacteria must survive under different environmental and host conditions by regulating its gene expression (turning genes “on and off”) which enable the bacteria to persist in all types of environments and to evade the host immune cells. This gene regulation in Salmonella is complex and only partially understood. One way Salmonella regulates gene expression is through the use of sigma factors which recognize special DNA codes (sequences) near specific genes and controls their expression. One of these sigma factors, sigma54, must be activated by an enhancer protein before the genes under its control can be expressed. Sigma54 can be activated by different enhancers, each of which responds to different environmental signals. These enhancers stimulate the expression of genes controlling cellular processes that allow Salmonella to adapt to environments in the host, survive, and cause infection. To determine all the genes controlled by sigma54, a modified enhancer protein capable of activating all sigma54 controlled genes without an environmental signal was used. The genes expressed and sequences bound by sigma54 were then detected with a microarray assay. Previously unknown sigma54-dependent genes and sequences recognized by sigma54 were identified. These experiments have generated critical information about Salmonella gene expression that was previously unknown. This knowledge enables researchers to develop treatments to disrupt infection or treat disease in the host.

Technical Abstract: Background: Sigma54, or RpoN, is an alternative s factor found widely in eubacteria. A significant complication in analysis of the global sigma54 regulon in a bacterium is that the sigma54 RNA polymerase holoenzyme requires interaction with an active bacterial enhancer-binding protein (bEBP) to initiate transcription at a sigma54-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 sigma54-dependent promoters for the characterization of the sigma54 regulon of Salmonella Typhimurium LT2. Results: The AAA+ domain of DctD was able to drive transcription from nearly all previously characterized or predicted sigma54-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+ domain. We also identified a novel sigma54-dependent promoter upstream of STM2939, a homolog of cas1 from the CRISPR system. ChIP-chip analysis revealed at least 70 sigma54 binding sites in the chromosome, of which 58% are located within coding sequences. Promoter-lacZ fusions with selected intragenic sigma54 binding sites suggest that many of these sites are capable of functioning as sigma54-dependent promoters. Conclusion: Since the DctD AAA+ domain proved effective in activating transcription from the diverse sigma54-dependent promoters of the S. Typhimurium LT2 sigma54 regulon under a single growth condition, this approach is likely to be valuable for examining sigma54 regulons in other bacterial species. The S. Typhimurium sigma54 regulon characterized in the presence of the DctD AAA+ domain included a high number of intragenic sigma54 binding sites/promoters, suggesting that sigma54 has regulatory roles beyond the initiation of transcription at the start of an operon.

Last Modified: 10/1/2014
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