Dataset Information

Transcription-induced barriers to supercoil diffusion in the Salmonella typhimurium chromosome.

ABSTRACT: Transcription and replication both influence and are influenced by superhelical changes in DNA. Explaining how supercoil movement is channeled in living chromosomes has been a major problem for 30 years. Transcription of membrane-associated proteins leads to localized hypersupercoiling of plasmid DNA, and this behavior indicates the presence of aberrant supercoil diffusion. Using the lambda Red recombination system, we constructed model domains in the Salmonella typhimurium chromosome to analyze supercoiling dynamics of regions encoding membrane proteins. Regulation of Tn10-derived tetracycline resistance involves a repressor, TetR, and a membrane-bound export pump, TetA. Strains deficient in TetR activity had 60-fold higher transcription levels (from P(A)) than TetR-positive strains. High tetA transcription caused a 10- to 80-fold decrease in the gammadelta resolution efficiency for the domain that includes the Tet module. Replacing tetA with genes encoding cytosolic proteins LacZ and Kan also caused the appearance of supercoil diffusion barriers in a defined region of the chromosome. In strains containing a functional TetR located next to a regulated lacZ reporter (P(R)tetR-P(A)lacZ), induction of transcription with chlortetracycline caused a 5-fold drop in resolution efficiency in the test domain interval. A short half-life resolvase showed that barriers appeared and disappeared over a 10- to 20-min span. These studies demonstrate the importance of transcription in chromosome structure and the plasticity of supercoil domains in bacterial chromosomes.

SUBMITTER: Deng S

PROVIDER: S-EPMC373473 | biostudies-literature | 2004 Mar

REPOSITORIES: biostudies-literature

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Transcription-induced barriers to supercoil diffusion in the Salmonella typhimurium chromosome.

Deng Shuang S Stein Richard A RA Higgins N Patrick NP

Proceedings of the National Academy of Sciences of the United States of America 20040301 10

Transcription and replication both influence and are influenced by superhelical changes in DNA. Explaining how supercoil movement is channeled in living chromosomes has been a major problem for 30 years. Transcription of membrane-associated proteins leads to localized hypersupercoiling of plasmid DNA, and this behavior indicates the presence of aberrant supercoil diffusion. Using the lambda Red recombination system, we constructed model domains in the Salmonella typhimurium chromosome to analyze ...[more]

PMID: 14993611

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Project description:Gyrase catalyzes negative supercoiling of DNA in an ATP-dependent reaction that helps condense bacterial chromosomes into a compact interwound "nucleoid." The supercoil density (σ) of prokaryotic DNA occurs in two forms. Diffusible supercoil density (σ(D)) moves freely around the chromosome in 10 kb domains, and constrained supercoil density (σ(C)) results from binding abundant proteins that bend, loop, or unwind DNA at many sites. Diffusible and constrained supercoils contribute roughly equally to the total in vivo negative supercoil density of WT cells, so σ = σ(C)+σ(D). Unexpectedly, Escherichia coli chromosomes have a 15% higher level of σ compared to Salmonella enterica. To decipher critical mechanisms that can change diffusible supercoil density of chromosomes, we analyzed strains of Salmonella using a 9 kb "supercoil sensor" inserted at ten positions around the genome. The sensor contains a complete Lac operon flanked by directly repeated resolvase binding sites, and the sensor can monitor both supercoil density and transcription elongation rates in WT and mutant strains. RNA transcription caused (-) supercoiling to increase upstream and decrease downstream of highly expressed genes. Excess upstream supercoiling was relaxed by Topo I, and gyrase replenished downstream supercoil losses to maintain an equilibrium state. Strains with TS gyrase mutations growing at permissive temperature exhibited significant supercoil losses varying from 30% of WT levels to a total loss of σ(D) at most chromosome locations. Supercoil losses were influenced by transcription because addition of rifampicin (Rif) caused supercoil density to rebound throughout the chromosome. Gyrase mutants that caused dramatic supercoil losses also reduced the transcription elongation rates throughout the genome. The observed link between RNA polymerase elongation speed and gyrase turnover suggests that bacteria with fast growth rates may generate higher supercoil densities than slow growing species.

Project description:We have shown that the ligand-responsive MarR family member SlyA plays an important role in transcription activation of multiple virulence genes in Salmonella enterica serovar Typhimurium by responding to guanosine tetraphosphate (ppGpp). Here, we demonstrate that another MarR family member, EmrR, is required for virulence of S. Typhimurium and another enteric bacterium, Yersinia pestis EmrR is found to activate transcription of an array of virulence determinants, including Salmonella pathogenicity island 2 (SPI-2) genes and several divergent operons, which have been shown to be activated by SlyA and the PhoP/PhoQ two-component system. We studied the regulatory effect of EmrR on one of these genetic loci, i.e., the pagC-pagD divergent operon, and characterized a catecholamine neurotransmitter, dopamine, as an EmrR-sensed signal. Dopamine acts on EmrR to reduce its ability to bind to the target promoters, thus functioning as a negative signal to downregulate this EmrR-activated transcription. EmrR can bind to AT-rich sequences, which particularly overlap the SlyA and PhoP binding sites in the pagC-pagD divergent promoter. EmrR is a priming transcription regulator that binds its target promoters prior to successive transcription activators, by which it displaces universal silencer H-NS from these promoters and facilitates successive regulators to bind these regions. Regulation of the Salmonella-specific gene in Escherichia coli and Y. pestis reveals that EmrR-dependent regulation is conserved in enteric bacteria. These observations suggest that EmrR is a transcription activator to control the expression of virulence genes, including the SPI-2 genes. Dopamine can act on the EmrR-mediated signal transduction, thus downregulating expression of these virulence factors.IMPORTANCE In this study, MarR family regulator EmrR is identified as a novel virulence factor of enteric bacteria, here exemplified by Salmonella enterica serovar Typhimurium and Yersinia pestis EmrR exerts an essential effect as a transcription activator for expression of virulence determinants, including Salmonella pathogenicity island 2 genes and a set of horizontally acquired genetic loci that formed divergent operons. EmrR senses the neurotransmitter dopamine and is subsequently released from target promoters, resulting in downregulation of the virulence gene expression. Through this action on EmrR, dopamine can weaken Salmonella resistance against host defense mechanisms. This provides an explanation for the previous observation that dopamine inhibits bacterial infection in animal gastrointestinal tracts. Our findings provide evidence that this neurotransmitter can modulate bacterial gene expression through interaction with virulence regulator EmrR.

Project description:BackgroundSalmonella are able to modulate host cell functions facilitating both uptake and resistance to cellular host defence mechanisms. While interactions between bacterial modulators and cellular proteins have been the main focus of Salmonella research, relatively little is known about mammalian gene regulation in response to Salmonella infection. A major class of mammalian gene modulators consists of microRNAs. For our study we examined interactions of microRNAs and regulated mRNAs in mammalian intestinal Salmonella infections using a piglet model.Methodology/principal findingsAfter performing microRNA as well as mRNA specific microarray analysis of ileal samples from Salmonella infected as well as control piglets, we integrated expression analysis with target prediction identifying microRNAs that mainly regulate focal adhesion as well as actin cytoskeleton pathways. Particular attention was given to miR-29a, which was involved in most interactions including Caveolin 2. RT-qPCR experiments verified up-regulation of miR-29a after infection while its predicted target Caveolin 2 was significantly down-regulated as examined by transcript and protein detection. Reporter gene assays as well as RNAi experiments confirmed Caveolin 2 to be a miR-29a target. Knock-down of Caveolin 2 in intestinal epithelial cells resulted in retarded proliferation as well as increased bacterial uptake. In addition, our experiments showed that Caveolin 2 regulates the activation of the small Rho GTPase CDC42 but apparently not RAC1 in human intestinal cells.Conclusions/significanceOur study outlines for the first time important regulation pathways in intestinal Salmonella infection pointing out that focal adhesion and organisation of actin cytoskeleton are regulated by microRNAs. Functional relevance is shown by miR-29a mediated Caveolin 2 regulation, modulating the activation state of CDC42. Further analysis of examined interactions may support the discovery of novel strategies impairing the uptake of intracellular pathogens.

Dataset Information

Transcription-induced barriers to supercoil diffusion in the Salmonella typhimurium chromosome.

Publications

Transcription-induced barriers to supercoil diffusion in the Salmonella typhimurium chromosome.

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