| Autor: |
Zere TR; Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, United States of America., Vakulskas CA; Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, United States of America., Leng Y; Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, United States of America., Pannuri A; Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, United States of America., Potts AH; Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, United States of America., Dias R; Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, United States of America., Tang D; Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, United States of America., Kolaczkowski B; Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, United States of America., Georgellis D; Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México D.F., México., Ahmer BM; Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, United States of America., Romeo T; Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, United States of America. |
| Abstrakt: |
The two-component signal transduction system BarA-UvrY of Escherichia coli and its orthologs globally regulate metabolism, motility, biofilm formation, stress resistance, virulence of pathogens and quorum sensing by activating the transcription of genes for regulatory sRNAs, e.g. CsrB and CsrC in E. coli. These sRNAs act by sequestering the RNA binding protein CsrA (RsmA) away from lower affinity mRNA targets. In this study, we used ChIP-exo to identify, at single nucleotide resolution, genomic sites for UvrY (SirA) binding in E. coli and Salmonella enterica. The csrB and csrC genes were the strongest targets of crosslinking, which required UvrY phosphorylation by the BarA sensor kinase. Crosslinking occurred at two sites, an inverted repeat sequence far upstream of the promoter and a site near the -35 sequence. DNAse I footprinting revealed specific binding of UvrY in vitro only to the upstream site, indicative of additional binding requirements and/or indirect binding to the downstream site. Additional genes, including cspA, encoding the cold-shock RNA-binding protein CspA, showed weaker crosslinking and modest or negligible regulation by UvrY. We conclude that the global effects of UvrY/SirA on gene expression are primarily mediated by activating csrB and csrC transcription. We also used in vivo crosslinking and other experimental approaches to reveal new features of csrB/csrC regulation by the DeaD and SrmB RNA helicases, IHF, ppGpp and DksA. Finally, the phylogenetic distribution of BarA-UvrY was analyzed and found to be uniquely characteristic of γ-Proteobacteria and strongly anti-correlated with fliW, which encodes a protein that binds to CsrA and antagonizes its activity in Bacillus subtilis. We propose that BarA-UvrY and orthologous TCS transcribe sRNA antagonists of CsrA throughout the γ-Proteobacteria, but rarely or never perform this function in other species. |
