Genomic Targets and Features of BarA-UvrY (-SirA) Signal Transduction Systems
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ABSTRACT: Using ChIP-exo, we have identified at single nucleotide resolution genomic binding sites for UvrY in E. coli and its homolog SirA in S. Typhimurium.
ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S Escherichia coli str. K-12 substr. MG1655
Project description:We present the low-input ChIP-exo minimization (ChIP-mini) method, which is suitable for processing small bacterial cell numbers, as few as 4.8x10⁶ (bacterial culture volume: 10 µl). This method faithfully maintains the relevant binding profiles of Escherichia coli K-12 MG1655 RpoD, as generated by the traditional ChIP-exo method (bacterial culture volume: 50 ml). Additionally, ChIP-mini was applied in the conventional infection process of mouse macrophages-like cells (J774A.1, RAW264.7) and human epithelial cells (HeLa, INT407) with Salmonella Typhimurium 14028s to directly capture both host extracellular and intracellular bacteria (extracellular and intracellular ChIP-mini). These applications of ChIP-mini in infectious S. Typhimurium provided near-base pair resolution to measure the impact of environmental changes on the binding profiles of H-NS and RpoD.
Project description:Two component sensor-response regulator systems (TCSs) are very common in the genomes of the Streptomyces species that have been fully sequenced to date. It has been suggested that this large number is an evolutionary response to the variable environment that Streptomyces encounter in soil. Notwithstanding this, TCSs are also more common in the sequenced genomes of other Actinomycetales when these are compared to the genomes of most other eubacteria. In this study, we have used DNA/DNA genome microarray analysis to compare fourteen Streptomyces species and one closely related genus to Streptomyces coelicolor in order to identify a core group of such systems. This core group is compared to the syntenous and non-syntenous TCSs present in the genome sequences of other Actinomycetales in order to separate the systems into those present in Actinomycetales in general, the Streptomyces specific systems and the species specific systems. Horizontal transfer does not seem to play a very important role in the evolution of the TCS complement analyzed in this study. However, cognate pairs do not necessarily seem to evolve at the same pace, which may indicate the evolutionary responses to environmental variation may be reflected differently in sequence changes within the two components of the TCSs. The overall analysis allowed subclassification of the orphan TCSs and the TCS cognate pairs and identification of possible targets for further study using gene knockouts, gene overexpression, reporter genes and yeast two hybrid analysis.
Project description:Two component sensor-response regulator systems (TCSs) are very common in the genomes of the Streptomyces species that have been fully sequenced to date. It has been suggested that this large number is an evolutionary response to the variable environment that Streptomyces encounter in soil. Notwithstanding this, TCSs are also more common in the sequenced genomes of other Actinomycetales when these are compared to the genomes of most other eubacteria. In this study, we have used DNA/DNA genome microarray analysis to compare fourteen Streptomyces species and one closely related genus to Streptomyces coelicolor in order to identify a core group of such systems. This core group is compared to the syntenous and non-syntenous TCSs present in the genome sequences of other Actinomycetales in order to separate the systems into those present in Actinomycetales in general, the Streptomyces specific systems and the species specific systems. Horizontal transfer does not seem to play a very important role in the evolution of the TCS complement analyzed in this study. However, cognate pairs do not necessarily seem to evolve at the same pace, which may indicate the evolutionary responses to environmental variation may be reflected differently in sequence changes within the two components of the TCSs. The overall analysis allowed subclassification of the orphan TCSs and the TCS cognate pairs and identification of possible targets for further study using gene knockouts, gene overexpression, reporter genes and yeast two hybrid analysis. DNA/DNA comparative analysis using the University of Surrey PCR Microarray chip