The green tea polyphenol EGCG inhibits E. coli biofilm formation by impairing amyloid curli fibre assembly and downregulating the biofilm regulator CsgD via the σ(E) -dependent sRNA RybB.

In bacterial biofilms, which are often involved in chronic infections, cells are surrounded by a self-produced extracellular matrix that contains amyloid fibres, exopolysaccharides and other biopolymers. The matrix contributes to the pronounced resistance of biofilms against antibiotics and host immune systems. Being highly inflammatory, matrix amyloids such as curli fibres of Escherichia coli can also play a role in pathogenicity. Using macrocolony biofilms of commensal and pathogenic E. coli as a model system, we demonstrate here that the green tea polyphenol epigallocatachin gallate (EGCG) is a potent antibiofilm agent. EGCG virtually eliminates the biofilm matrix by directly interfering with the assembly of curli subunits into amyloid fibres, and by triggering the σ(E) cell envelope stress response and thereby reducing the expression of CsgD - a crucial activator of curli and cellulose biosynthesis - due to csgD mRNA targeting by the σ(E) -dependent sRNA RybB. These findings highlight EGCG as a potential adjuvant for antibiotic therapy of biofilm-associated infections. Moreover, EGCG may support therapies against pathogenic E. coli that produce inflammatory curli fibres along with Shigatoxin.

Small RNA binding to 5' mRNA coding region inhibits translational initiation.

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.

Small RNAs in the control of RpoS, CsgD, and biofilm architecture of Escherichia coli.

Amyloid curli fibers and cellulose are extracellular matrix components produced in the stationary phase top layer of E. coli macrocolonies, which confer physical protection, strong cohesion, elasticity, and wrinkled morphology to these biofilms. Curli and cellulose synthesis is controlled by a three-level transcription factor (TF) cascade with the RpoS sigma subunit of RNA polymerase at the top, the MerR-like TF MlrA, and the biofilm regulator CsgD, with two c-di-GMP control modules acting as key switching devices. Additional signal input and fine-tuning is provided by an entire series of small RNAs-ArcZ, DsrA, RprA, McaS, OmrA/OmrB, GcvB, and RydC--that differentially control all three TF modules by direct mRNA interaction. This review not only summarizes the mechanisms of action of these sRNAs, but also addresses the question of how these sRNAs and the regulators they target contribute to building the intriguing three-dimensional microarchitecture and macromorphology of these biofilms.

Targeting of csgD by the small regulatory RNA RprA links stationary phase, biofilm formation and cell envelope stress in Escherichia coli.

RprA is a small regulatory RNA known to weakly affect the translation of σ(S) (RpoS) in Escherichia coli. Here we demonstrate that csgD, which encodes a stationary phase-induced biofilm regulator, as well as ydaM, which encodes a diguanylate cyclase involved in activating csgD transcription, are novel negatively controlled RprA targets. As shown by extensive mutational analysis, direct binding of RprA to the 5'-untranslated and translational initiation regions of csgD mRNA inhibits translation and reduces csgD mRNA levels. In the case of ydaM mRNA, RprA base-pairs directly downstream of the translational start codon. In a feedforward loop, RprA can thus downregulate > 30 YdaM/CsgD-activated genes including those for adhesive curli fimbriae. However, during early stationary phase, when csgD transcription is strongly activated, the synthesis of csgD mRNA exceeds that of RprA, which allows the accumulation of CsgD protein. This situation is reversed when csgD transcription is shut off - for instance, later in stationary phase or during biofilm formation - or by conditions that further activate RprA expression via the Rcs two-component system. Thus, antagonistic regulation of csgD and RprA at the mRNA level integrates cell envelope stress signals with global gene expression during stationary phase and biofilm formation.

Evidence for an autonomous 5' target recognition domain in an Hfq-associated small RNA.

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.

Small Regulatory RNAs in the Control of Motility and Biofilm Formation in E. coli and Salmonella.

Biofilm formation in Escherichia coli and other enteric bacteria involves the inverse regulation of the synthesis of flagella and biofilm matrix components such as amyloid curli fibres, cellulose, colanic acid and poly-N-acetylglucosamine (PGA). Physiologically, these processes reflect the transition from growth to stationary phase. At the molecular level, they are tightly controlled by various sigma factors competing for RNA polymerase, a series of transcription factors acting in hierarchical regulatory cascades and several nucleotide messengers, including cyclic-di-GMP. In addition, a surprisingly large number of small regulatory RNAs (sRNAs) have been shown to directly or indirectly modulate motility and/or biofilm formation. This review aims at giving an overview of these sRNA regulators and their impact in biofilm formation in E. coli and Salmonella. Special emphasis will be put on sRNAs, that have known targets such as the mRNAs of the flagellar master regulator FlhDC, the stationary phase sigma factor σS (RpoS) and the key biofilm regulator CsgD that have recently been shown to act as major hubs for regulation by multiple sRNAs.

A new Vibrio cholerae sRNA modulates colonization and affects release of outer membrane vesicles.

We discovered a new small non-coding RNA (sRNA) gene, vrrA of Vibrio cholerae O1 strain A1552. A vrrA mutant overproduces OmpA porin, and we demonstrate that the 140 nt VrrA RNA represses ompA translation by base-pairing with the 5' region of the mRNA. The RNA chaperone Hfq is not stringently required for VrrA action, but expression of the vrrA gene requires the membrane stress sigma factor, sigma(E), suggesting that VrrA acts on ompA in response to periplasmic protein folding stress. We also observed that OmpA levels inversely correlated with the number of outer membrane vesicles (OMVs), and that VrrA increased OMV production comparable to loss of OmpA. VrrA is the first sRNA known to control OMV formation. Moreover, a vrrA mutant showed a fivefold increased ability to colonize the intestines of infant mice as compared with the wild type. There was increased expression of the main colonization factor of V. cholerae, the toxin co-regulated pili, in the vrrA mutant as monitored by immunoblot detection of the TcpA protein. VrrA overproduction caused a distinct reduction in the TcpA protein level. Our findings suggest that VrrA contributes to bacterial fitness in certain stressful environments, and modulates infection of the host intestinal tract.

Microanatomy at cellular resolution and spatial order of physiological differentiation in a bacterial biofilm.

UNLABELLED: 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, σ(S) (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." IMPORTANCE: Heterogeneity or cellular differentiation in biofilms is a commonly accepted concept, but direct evidence at the microscale has been difficult to obtain. Our study reveals the microanatomy and microphysiology of an Escherichia coli macrocolony biofilm at an unprecedented cellular resolution, with physiologically different zones and strata forming as a function of known global regulatory networks that respond to biofilm-intrinsic gradients of nutrient supply. In addition, this study identifies zones of heterogeneous and potentially bistable CsgD and curli expression, shows bacterial curli networks to strikingly resemble Alzheimer plaques, and suggests a new role of flagella as an architectural element in biofilms.

Poly(A)-polymerase I links transcription with mRNA degradation via sigmaS proteolysis.

Bacteria rapidly adapt to changes in growth conditions through control of transcription and specific mRNA degradation. Interplay of both mechanisms must exist in order to achieve fine-tuned regulation of gene expression. Transcription of the Escherichia coli bolA gene is mediated by the RpoS/sigmaS transcription factor in response to environmental signals. In this report it is shown that the mechanisms of bolA1p mRNA transcription and degradation are tightly connected at the onset of stationary phase and in response to sudden carbon starvation. In stationary phase, bolA1p mRNA levels were reduced 2.5-fold in a poly(A)-polymerase I (PAPI) mutant, explained by the significant threefold reduction in sigmaS protein levels in the same strain. Furthermore, fusions with the rpoS gene, analysis of the stability of sigmaS and the levels of RssB indicate that the absence of PAPI enhances RssB-mediated sigmaS proteolysis specifically in starved cells. The fact that PAPI induces higher cellular levels of a global regulator is a novel finding of wide biological significance. PAPI could work as a linker between transcription and mRNA degradation with the ultimate goal of adapting and surviving to growth-limiting conditions.

SigmaE-dependent small RNAs of Salmonella respond to membrane stress by accelerating global omp mRNA decay.

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, sigmaE. Activation of the GE 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, sigmaE-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 oE-dependent small RNAs function within a surveillance loop to maintain envelope homeostasis and to achieve autoregulation of oE.

A two-component phosphotransfer network involving ArcB, ArcA, and RssB coordinates synthesis and proteolysis of sigmaS (RpoS) in E. coli.

The general stress sigma factor sigma(S) (RpoS) in Escherichia coli is controlled at the levels of transcription, translation, and proteolysis. Here we demonstrate that the phosphorylated response regulator ArcA is a direct repressor of rpoS transcription that binds to two sites flanking the major rpoS promoter, with the upstream site overlapping an activating cAMP-CRP-binding site. The histidine sensor kinase ArcB not only phosphorylates ArcA, but also the sigma(S) proteolytic targeting factor RssB, and thereby stimulates sigma(S) proteolysis. Thus, ArcB/ArcA/RssB constitute a branched "three-component system", which coordinates rpoS transcription and sigma(S) proteolysis and thereby maintains low sigma(S) levels in rapidly growing cells. We suggest that the redox state of the quinones, which controls autophosphorylation of ArcB, not only monitors oxygen but also energy supply, and we show that the ArcB/ArcA/RssB system is involved in sigma(S) induction during entry into starvation conditions. Moreover, this induction is enhanced by a positive feedback that involves sigma(S)-dependent induction of ArcA, which further reduces sigma(S) proteolysis, probably by competing with RssB for residual phosphorylation by ArcB.