Abstract
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.
Highlights
The ability of bacteria to flourish under diverse environmental conditions requires their physiology and metabolism to be regulated by complex transcriptional and posttranscriptional circuitries
To probe for UvrY/SirA DNA binding sites in E. coli and Salmonella, we used ChIP-exo, a comprehensive genomic DNA-protein interaction assay with single nucleotide resolution and high specificity [34]
These results indicated that the csrB and csrC genes represent the strongest targets of UvrY/SirA binding in these species
Summary
The ability of bacteria to flourish under diverse environmental conditions requires their physiology and metabolism to be regulated by complex transcriptional and posttranscriptional circuitries. The stringent response describes a regulatory network of eubacteria that responds to amino acid starvation and other stresses [2] Activation of this system is characterized by a rapid downshift in synthesis of stable RNAs, such as rRNA and tRNA and stimulation of the expression of genes involved in amino acid biosynthesis and transport, these processes represent a fraction of its global regulatory role [36]. Bioinformatics analyses revealed that BarA-UvrY orthologs are strongly anti-correlated with the fliW gene, which encodes a protein that binds to and antagonizes CsrA of B. subtilis [41], indicating that few if any species use both FliW and BarA-UvrY transcribed sRNAs as CsrA antagonists These studies advance our understanding of this global regulatory circuitry and highlight the importance of including post-transcriptional regulation along with transcriptional control when modeling bacterial regulatory networks
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call