Summary The bacterial envelope stress response (ESR) is triggered by the accumulation of misfolded outer membrane proteins (OMPs) upon envelope damage or excessive OMP synthesis, and is mediated by the alternative sigma factor, σE. Activation of the σE pathway causes a rapid downregulation of major omp mRNAs, which prevents further build‐up of unassembled OMPs and liberates the translocation and folding apparatus under conditions that require envelope remodelling. The factors that facilitate the rapid removal of the unusually stable omp mRNAs in the ESR were previously unknown. We report that in Salmonella the ESR relies upon two highly conserved, σE‐controlled small non‐coding RNAs, RybB and MicA. By using a transcriptomic approach and kinetic analyses of target mRNA decay in vivo, RybB was identified as the factor that selectively accelerates the decay of multiple major omp mRNAs upon induction of the ESR, while MicA is proposed to facilitate rapid decay of the single ompA mRNA. In unstressed bacterial cells, the two σE‐dependent small RNAs function within a surveillance loop to maintain envelope homeostasis and to achieve autoregulation of σE.

Bacterial biofilms are highly structured multicellular communities whose formation involves flagella and an extracellular matrix of adhesins, amyloid fibers, and exopolysaccharides. Flagella are produced by still-dividing rod-shaped Escherichia coli cells during postexponential growth when nutrients become suboptimal. Upon entry into stationary phase, however, cells stop producing flagella, become ovoid, and generate amyloid curli fibers. These morphological changes, as well as accompanying global changes in gene expression and cellular physiology, depend on the induction of the stationary-phase sigma subunit of RNA polymerase, σS (RpoS), the nucleotide second messengers cyclic AMP (cAMP), ppGpp, and cyclic-di-GMP, and a biofilm-controlling transcription factor, CsgD. Using flagella, curli fibers, a CsgD::GFP reporter, and cell morphology as “anatomical” hallmarks in fluorescence and scanning electron microscopy, different physiological zones in macrocolony biofilms of E. coli K-12 can be distinguished at cellular resolution. Small ovoid cells encased in a network of curli fibers form the outer biofilm layer. Inner regions are characterized by heterogeneous CsgD::GFP and curli expression. The bottom zone of the macrocolonies features elongated dividing cells and a tight mesh of entangled flagella, the formation of which requires flagellar motor function. Also, the cells in the outer-rim growth zone produce flagella, which wrap around and tether cells together. Adjacent to this growth zone, small chains and patches of shorter curli-surrounded cells appear side by side with flagellated curli-free cells before curli coverage finally becomes confluent, with essentially all cells in the surface layer being encased in “curli baskets.”

Small noncoding RNAs (sRNAs) have predominantly been shown to repress bacterial mRNAs by masking the Shine-Dalgarno (SD) or AUG start codon sequence, thereby preventing 30S ribosome entry and, consequently, translation initiation. However, many recently identified sRNAs lack obvious SD and AUG complementarity, indicating that sRNA-mediated translational control could also take place at other mRNA sites. We report that Salmonella RybB sRNA represses ompN mRNA translation by pairing with the 5' coding region. Results of systematic antisense interference with 30S binding to ompN and unrelated mRNAs suggest that sRNAs can act as translational repressors by sequestering sequences within the mRNA down to the fifth codon, even without SD and AUG start codon pairing. This "five codon window" for translational control in the 5' coding region of mRNA not only has implications for sRNA target predictions but might also apply to cis-regulatory systems such as RNA thermosensors and riboswitches.

The abundant class of bacterial Hfq-associated small regulatory RNAs (sRNAs) parallels animal microRNAs in their ability to control multiple genes at the posttranscriptional level by short and imperfect base pairing. In contrast to the universal length and seed pairing mechanism of microRNAs, the sRNAs are heterogeneous in size and structure, and how they regulate multiple targets is not well understood. This paper provides evidence that a 5′ located sRNA domain is a critical element for the control of a large posttranscriptional regulon. We show that the conserved 5′ end of RybB sRNA recognizes multiple mRNAs of Salmonella outer membrane proteins by ≥7-bp Watson-Crick pairing. When fused to an unrelated sRNA, the 5′ domain is sufficient to guide target mRNA degradation and maintain σ E -dependent envelope homeostasis. RybB sites in mRNAs are often conserved and flanked by 3′ adenosine. They are found in a wide sequence window ranging from the upstream untranslated region to the deep coding sequence, indicating that some targets might be repressed at the level of translation, whereas others are repressed primarily by mRNA destabilization. Autonomous 5′ domains seem more common in sRNAs than appreciated and might improve the design of synthetic RNA regulators.envelope stress | multiple targeting | noncoding RNA | porin S mall RNAs that act on trans-encoded target mRNAs by short base pairing are important posttranscriptional regulators in many organisms. The two most abundant classes to date are the microRNAs of eukaryotes and the Hfq-associated small regulatory RNAs (sRNAs) of gram-negative bacteria such as Escherichia coli and Salmonella (1). The ∼22-nt microRNAs use wellestablished mechanisms and machinery to repress mRNAs by short seed pairing within the 3′ UTR, and a single microRNA might regulate hundreds of genes in parallel (2).The bacterial sRNAs are also increasingly found to control multiple targets, although by binding the 5′ region of bacterial mRNAs (3). In fact, some sRNAs have an impact on dozens of genes under stress or altered growth conditions (4-7), with target sites being known for a subset of the regulated mRNAs. The Sm-like protein, Hfq, is required for intracellular stability and target annealing of the sRNAs (1, 8). Global analyses of Hfq-bound transcripts suggest an excess of potential targets over regulators (9, 10), further arguing that multiple targeting might be the general mode of sRNA action.Unlike the universal length and seed pairing of microRNAs, there are few common denominators for Hfq-dependent regulators. The sRNAs dramatically vary in size (50-250 nt) and secondary structure (11), and sRNA-mRNA interactions range from >30-bp duplexes in MicF-ompF or Spot42-galK (12, 13) to only 6 bps that are critical in . Most of the sRNAs analyzed to date inhibit translational initiation of targets by sequestering the Shine-Dalgarno (SD) or start codon (AUG) sequences of the ribosome binding site (RBS); how the recognition of these conserved RBS elements would ensure highly specific target...