Circuitry Linking the Global Csr- and σ^E-Dependent Cell Envelope Stress Response Systems

Bacterial cells respond to environmental stress through complex regulatory networks. While much is known about how sigma factors like σE coordinate extracytoplasmic stress responses, the extent of RNA-based regulation in these systems remains unclear. Prior research has shown that CsrA, an RNA-binding protein in Escherichia coli, influences diverse processes like motility and biofilm formation. However, the direct role of CsrA in regulating σE-dependent pathways had not been fully established. This gap motivated the investigation into whether CsrA interacts with rpoE mRNA. No prior work had resolved how CsrA and σE systems might influence each other. Understanding these interactions could clarify how bacteria balance stress responses with metabolic needs. Existing knowledge suggested that CsrA regulates translation by binding to target mRNAs. Yet, the specific mechanism by which CsrA affects rpoE remained unknown. This study aimed to address these uncertainties by examining the molecular details of CsrA and σE interplay.

This study aimed to explore the regulatory relationship between CsrA and σE in Escherichia coli. The researchers sought to determine whether CsrA directly represses rpoE translation and how σE might influence CsrA activity. They focused on identifying the binding sites of CsrA on rpoE mRNA and evaluating the functional consequences of these interactions. The motivation stemmed from prior findings that rpoE is a CsrA target, but the exact mechanism was unclear. The team also aimed to investigate whether σE could regulate csrB and csrC, which encode sRNAs that sequester CsrA. Another goal was to examine the role of an upstream ORF in rpoE translation. The study aimed to clarify how these interactions contribute to the cell envelope stress response. By addressing these questions, the researchers hoped to reveal how CsrA and σE systems coordinate to manage bacterial stress.

The researchers used gel mobility shift assays to test CsrA binding to rpoE mRNA. They performed footprinting experiments to identify specific CsrA binding sites. Toeprint assays were employed to assess translation initiation at rpoE. Coupled transcription-translation experiments were conducted to evaluate the effect of CsrA on rpoE translation. The team analyzed the overlap of CsrA binding sites with the Shine-Dalgarno sequence and translation initiation codon. They also examined the effect of σE on csrB and csrC transcription using RNA-seq data. The study included functional analysis of an upstream ORF, ORF51, and its coupling with rpoE. The researchers named ORF51 as rseD based on its proximity to rpoE and its role in translation.

CsrA was found to repress rpoE translation by binding to three sites in the rpoE leader transcript. One binding site overlaps the Shine-Dalgarno sequence, while another overlaps the translation initiation codon. Coupled transcription-translation experiments confirmed that CsrA binding inhibits rpoE translation. The study showed that σE indirectly activates csrB and csrC transcription, increasing CsrA sequestration. This sequestration reduces CsrA-mediated repression of rpoE. The researchers identified an upstream ORF, rseD, whose stop codon overlaps the rpoE start codon. Loss of coupling between rseD and rpoE reduced rpoE translation by over 50%. The operon arrangement rseD-rpoE-rseA-rseB-rseC was established. These findings suggest that rseD may play a role in the cell envelope stress response.

The authors propose that the Csr system fine-tunes the σE-dependent cell envelope stress response. CsrA represses rpoE translation by binding to specific sites in the mRNA. σE indirectly activates csrB and csrC transcription, reducing CsrA-mediated repression. This reciprocal regulation helps reset RpoE abundance after envelope damage is repaired. The study suggests that extracytoplasmic stress leads to derepression of rpoE translation. The discovery of rseD adds complexity to translational control of rpoE. The findings indicate that CsrA and σE systems interact to manage bacterial stress responses. The authors emphasize that these interactions may help bacteria balance stress responses with metabolic needs.