Exploring the expression and functionality of the rsm sRNAs in Pseudomonas syringae pv. tomato DC3000.

The Gac-rsm pathway is a global regulatory network that governs mayor lifestyle and metabolic changes in gamma-proteobacteria. In a previous study, we uncovered the role of CsrA proteins promoting growth and repressing motility, alginate production and virulence in the model phytopathogen Pseudomonas syringae pv. tomato (Pto) DC3000. Here, we focus on the expression and regulation of the rsm regulatory sRNAs, since Pto DC3000 exceptionally has seven variants (rsmX1-5, rsmY and rsmZ). The presented results offer further insights into the functioning of the complex Gac-rsm pathway and the interplay among its components. Overall, rsm expressions reach maximum levels at high cell densities, are unaffected by surface detection, and require GacA for full expression. The rsm levels of expression and GacA-dependence are determined by the sequences found in their -35/-10 promoter regions and GacA binding boxes, respectively. rsmX5 stands out for being the only rsm in Pto DC3000 whose high expression does not require GacA, constituting the main component of the total rsm pool in a gacA mutant. The deletion of rsmY and rsmZ had minor effects on Pto DC3000 motility and virulence phenotypes, indicating that rsmX1-5 can functionally replace them. On the other hand, rsmY or rsmZ overexpression in a gacA mutant did not revert its phenotype. Additionally, a negative feedback regulatory loop in which the CsrA3 protein promotes its own titration by increasing the levels of several rsm RNAs in a GacA-dependent manner has been disclosed as part of this work.

RNA pentaloop structures as effective targets of regulators belonging to the RsmA/CsrA protein family.

In the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens CHA0, the dimeric RNA-binding proteins RsmA and RsmE, which belong to the vast bacterial RsmA/CsrA family, effectively repress translation of target mRNAs containing a typical recognition sequence near the translation start site. Three small RNAs (RsmX, RsmY, RsmZ) with clustered recognition sequences can sequester RsmA and RsmE and thereby relieve translational repression. According to a previously established structural model, the RsmE protein makes optimal contacts with an RNA sequence 5'- (A)/(U)CANGGANG(U)/(A)-3', in which the central ribonucleotides form a hexaloop. Here, we questioned the relevance of the hexaloop structure in target RNAs. We found that two predicted pentaloop structures, AGGGA (in pltA mRNA encoding a pyoluteorin biosynthetic enzyme) and AAGGA (in mutated pltA mRNA), allowed effective interaction with the RsmE protein in vivo. By contrast, ACGGA and AUGGA were poor targets. Isothermal titration calorimetry measurements confirmed the strong binding of RsmE to the AGGGA pentaloop structure in an RNA oligomer. Modeling studies highlighted the crucial role of the second ribonucleotide in the loop structure. In conclusion, a refined structural model of RsmE-RNA interaction accommodates certain pentaloop RNAs among the preferred hexaloop RNAs.

Little reason to call them small noncoding RNAs.

Hundreds of different species of small RNAs can populate a bacterial cell. This small transcriptome contains important information for the adaptation of cellular physiology to environmental changes. Underlying cellular networks involving small RNAs are RNA-RNA and RNA-protein interactions, which are often intertwined. In addition, small RNAs can function as mRNAs. In general, small RNAs are referred to as noncoding because very few are known to contain translated open reading frames. In this article, we intend to highlight that the number of small RNAs that fall within the set of translated RNAs is bound to increase. In addition, we aim to emphasize that the dynamics of the small transcriptome involve different functional codes, not just the genetic code. Therefore, since the role of small RNAs is always code-driven, we believe that there is little reason to continue calling them small noncoding RNAs.

CsrA selectively modulates sRNA-mRNA regulator outcomes.

Post-transcriptional regulation, by small RNAs (sRNAs) as well as the global Carbon Storage Regulator A (CsrA) protein, play critical roles in bacterial metabolic control and stress responses. The CsrA protein affects selective sRNA-mRNA networks, in addition to regulating transcription factors and sigma factors, providing additional avenues of cross talk between other stress-response regulators. Here, we expand the known set of sRNA-CsrA interactions and study their regulatory effects. In vitro binding assays confirm novel CsrA interactions with ten sRNAs, many of which are previously recognized as key regulatory nodes. Of those 10 sRNA, we identify that McaS, FnrS, SgrS, MicL, and Spot42 interact directly with CsrA in vivo. We find that the presence of CsrA impacts the downstream regulation of mRNA targets of the respective sRNA. In vivo evidence supports enhanced CsrA McaS-csgD mRNA repression and showcases CsrA-dependent repression of the fucP mRNA via the Spot42 sRNA. We additionally identify SgrS and FnrS as potential new sRNA sponges of CsrA. Overall, our results further support the expanding impact of the Csr system on cellular physiology via CsrA impact on the regulatory roles of these sRNAs.

Dual role of CsrA in regulating the hemolytic activity of Escherichia coli O157:H7.

Post-transcriptional global carbon storage regulator A (CsrA) is a sequence-specific RNA-binding protein involved in the regulation of multiple bacterial processes. Hemolysin is an important virulence factor in the enterohemorrhagic Escherichia coli O157:H7 (EHEC). Here, we show that CsrA plays a dual role in the regulation of hemolysis in EHEC. CsrA significantly represses plasmid-borne enterohemolysin (EhxA)-mediated hemolysis and activates chromosome-borne hemolysin E (HlyE)-mediated hemolysis through different mechanisms. RNA structure prediction revealed a well-matched stem-loop structure with two potential CsrA binding sites located on the 5' untranslated region (UTR) of ehxB, which encodes a translocator required for EhxA secretion. CsrA inhibits EhxA secretion by directly binding to the RNA leader sequence of ehxB to repress its expression in two different ways: CsrA either binds to the Shine-Dalgarno sequence of ehxB to block ribosome access or to ehxB transcript to promote its mRNA decay. The predicted CsrA-binding site 1 of ehxB is essential for its regulation. There is a single potential CsrA-binding site at the 5'-end of the hlyE transcript, and its mutation completely abolishes CsrA-dependent activation. CsrA can also stabilize hlyE mRNA by directly binding to its 5' UTR. Overall, our results indicate that CsrA acts as a hemolysis modulator to regulate pathogenicity under certain conditions.

Lon protease modulates virulence traits in Erwinia amylovora by direct monitoring of major regulators and indirectly through the Rcs and Gac-Csr regulatory systems.

Lon, an ATP-dependent protease in bacteria, influences diverse cellular processes by degrading damaged, misfolded and short-lived regulatory proteins. In this study, we characterized the effects of lon mutation and determined the molecular mechanisms underlying Lon-mediated virulence regulation in Erwinia amylovora, an enterobacterial pathogen of apple. Erwinia amylovora depends on the type III secretion system (T3SS) and the exopolysaccharide (EPS) amylovoran to cause disease. Our results showed that mutation of the lon gene led to the overproduction of amylovoran, increased T3SS gene expression and the non-motile phenotype. Western blot analyses showed that mutation in lon directly affected the accumulation and stability of HrpS/HrpA and RcsA. Mutation in lon also indirectly influenced the expression of flhD, hrpS and csrB through the accumulation of the RcsA/RcsB proteins, which bind to the promoter of these genes. In addition, lon expression is under the control of CsrA, possibly at both the transcriptional and post-transcriptional levels. Although mutation in csrA abolished both T3SS and amylovoran production, deletion of the lon gene in the csrA mutant only rescued amylovoran production, but not T3SS. These results suggest that CsrA might positively control both T3SS and amylovoran production partly by suppressing Lon, whereas CsrA may also play a critical role in T3SS by affecting unknown targets.

Discovery of the first small-molecule CsrA-RNA interaction inhibitors using biophysical screening technologies.

AIM: CsrA is a global post-transcriptional regulator protein affecting mRNA translation and/or stability. Widespread among bacteria, it is essential for their full virulence and thus represents a promising anti-infective drug target. Therefore, we aimed at the discovery of CsrA-RNA interaction inhibitors. Results & methodology: We followed two strategies: a screening of small molecules (A) and an RNA ligand-based approach (B). Using surface plasmon resonance-based binding and fluorescence polarization-based competition assays, (A) yielded seven small-molecule inhibitors, among them MM14 (IC50 of 4 µM). (B) resulted in RNA-based inhibitor GGARNA (IC50 of 113 µM). CONCLUSION: The first small-molecule inhibitors of the CsrA-RNA interaction were discovered exhibiting micromolar affinities. These hits represent tools to investigate the effects of CsrA-RNA interaction inhibition on bacterial virulence.