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      A Fundamental Regulatory Mechanism Operating through OmpR and DNA Topology Controls Expression of Salmonella Pathogenicity Islands SPI-1 and SPI-2

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      PLoS Genetics

      Public Library of Science

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          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          DNA topology has fundamental control over the ability of transcription factors to access their target DNA sites at gene promoters. However, the influence of DNA topology on protein–DNA and protein–protein interactions is poorly understood. For example, relaxation of DNA supercoiling strongly induces the well-studied pathogenicity gene ssrA (also called spiR) in Salmonella enterica, but neither the mechanism nor the proteins involved are known. We have found that relaxation of DNA supercoiling induces expression of the Salmonella pathogenicity island (SPI)-2 regulator ssrA as well as the SPI-1 regulator hilC through a mechanism that requires the two-component regulator OmpR-EnvZ. Additionally, the ompR promoter is autoregulated in the same fashion. Conversely, the SPI-1 regulator hilD is induced by DNA relaxation but is repressed by OmpR. Relaxation of DNA supercoiling caused an increase in OmpR binding to DNA and a concomitant decrease in binding by the nucleoid-associated protein FIS. The reciprocal occupancy of DNA by OmpR and FIS was not due to antagonism between these transcription factors, but was instead a more intrinsic response to altered DNA topology. Surprisingly, DNA relaxation had no detectable effect on the binding of the global repressor H-NS. These results reveal the underlying molecular mechanism that primes SPI genes for rapid induction at the onset of host invasion. Additionally, our results reveal novel features of the archetypal two-component regulator OmpR. OmpR binding to relaxed DNA appears to generate a locally supercoiled state, which may assist promoter activation by relocating supercoiling stress-induced destabilization of DNA strands. Much has been made of the mechanisms that have evolved to regulate horizontally-acquired genes such as SPIs, but parallels among the ssrA, hilC, and ompR promoters illustrate that a fundamental form of regulation based on DNA topology coordinates the expression of these genes regardless of their origins.

          Author Summary

          DNA is often considered to be a passive carrier of genetic information, but in fact DNA is an active participant in coordinating the expression of the genes it carries. This is because DNA is a dynamic molecule that can assume a wide range of topologies, and this has a direct impact on the formation of the protein–DNA complexes that drive gene expression. In a bacterium, the chromosome is supercoiled to variable levels according to environmental conditions, and supercoiling in turn governs the topology of gene promoters. Thus DNA supercoiling is able to transduce environmental signals to regulate promoter output. A previous study found that the intestinal pathogen Salmonella enterica may use changes in DNA supercoiling to detect when it has entered host immune cells, allowing the bacterium to induce the pathogenicity genes it requires to evade killing by macrophage. In dissecting the underlying molecular mechanisms, we have found that changes in DNA supercoiling also upregulate other key pathogenicity genes, and we have identified the proteins involved in this gene regulatory process. These findings indicate that a fundamental level of gene control arising from the interplay between protein transcription factors and DNA topology regulates Salmonella pathogenicity.

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          Most cited references 52

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          One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.

          We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.
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            Salmonellae interplay with host cells.

            Salmonellae are important causes of enteric diseases in all vertebrates. Characterization of the molecular mechanisms that underpin the interactions of salmonellae with their animal hosts has advanced greatly over the past decade, mainly through the study of Salmonella enterica serovar Typhimurium in tissue culture and animal models of infection. Knowledge of these bacterial processes and host responses has painted a dynamic and complex picture of the interaction between salmonellae and animal cells. This Review focuses on the molecular mechanisms of these host-pathogen interactions, in terms of their context, significance and future perspectives.
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              Epitope tagging of chromosomal genes in Salmonella.

              We have developed a simple and efficient procedure for adding an epitope-encoding tail to one or more genes of interest in the bacterial chromosome. The procedure is a modification of the gene replacement method of Datsenko and Wanner [Datsenko, K. A. & Wanner, B. L. (2000) Proc. Natl. Acad. Sci. USA 97, 6640-6645]. A DNA module that begins with the epitope-encoding sequence and includes a selectable marker is amplified by PCR with primers that carry extensions (as short as 36 nt) homologous to the last portion of the targeted gene and to a region downstream from it. Transformation of a strain expressing bacteriophage lambda red functions yields recombinants carrying the targeted gene fused to the epitope-encoding sequence. The resulting C-terminal-tagged protein can be identified by standard immuno-detection techniques. In an initial application of the method, we have added the sequences encoding the FLAG and 3xFLAG and influenza virus hemagglutinin epitopes to various genes of Salmonella enterica serovar Typhimurium, including putative and established pathogenic determinants present in prophage genomes. Epitope fusion proteins were detected in bacteria growing in vitro, tissue culture cells, and infected mouse tissues. This work identified a prophage locus specifically expressed in bacteria growing intracellularly. The procedure described here should be applicable to a wide variety of Gram-negative bacteria and is particularly suited for the study of intracellular pathogens.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                March 2012
                March 2012
                22 March 2012
                : 8
                : 3
                Affiliations
                Department of Microbiology, Moyne Institute of Preventive Medicine, School of Genetics and Microbiology, Trinity College Dublin, Dublin, Ireland
                Universidad de Sevilla, Spain
                Author notes

                ¤: Current address: Department of Biology, University of Regina, Regina, Canada

                Conceived and designed the experiments: ADSC CJD. Performed the experiments: ADSC. Analyzed the data: ADSC CJD. Contributed reagents/materials/analysis tools: CJD. Wrote the paper: ADSC CJD.

                PGENETICS-D-11-02759
                10.1371/journal.pgen.1002615
                3310775
                22457642
                Cameron, Dorman. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Counts
                Pages: 10
                Categories
                Research Article
                Biology
                Biochemistry
                Nucleic Acids
                DNA
                Proteins
                Genetics
                Gene Expression
                Molecular Genetics
                Microbiology
                Bacterial Pathogens

                Genetics

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