Differential regulation of CsrC and CsrB by CRP-cAMP in Salmonella enterica

Post-transcriptional regulation mediated by regulatory small RNAs (sRNAs) has risen as a key player in fine-tuning gene expression in response to environmental stimuli. Here, we show that, in Salmonella enterica, the central metabolic regulator CRP-cAMP differentially regulates the sRNAs CsrB and CsrC in a growth phase-dependent manner. While CsrB expression remains unchanged during growth, CsrC displays a growth phase-dependent expression profile, being weakly expressed at the logarithmic growth phase and induced upon entry into stationary phase. We show that CRP-cAMP contributes to the expression pattern of CsrC by repressing its expression during the logarithmic growth phase. The CRP-cAMP mediated repression of CsrC is independent of SirA, a known transcriptional CsrB/CsrC activator. We further show that the sRNA Spot 42, which is derepressed in a Δcrp strain, upregulates CsrC during logarithmic growth. We propose a model where the growth-dependent regulation of CsrC is sustained by the CRP-cAMP-mediated repression of Spot 42. Together, our data point toward a differential regulation of the sRNAs CsrB and CsrC in response to environmental stimuli, leading to fine-tuning of gene expression via the sequestration of the RNA-binding protein CsrA

CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level

Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3’UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3’UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3’UTR as a hub for post-transcriptional control of Salmonella invasion gene expression.Spanish Ministry of Economy and Competitiveness BIO2010-15417 BIO2013-44220-R AGL2013-45339-RRecerCaixa program 2012/ACUP/00048Catalonian government 2017SGR49

Gre factors-mediated control of hilD transcription is essential for the invasion of epithelial cells by Salmonella enterica serovar Typhimurium

The invasion of epithelial cells by Salmonella enterica serovar Typhimurium is a very tightly regulated process. Signaling cascades triggered by different environmental and physiological signals converge to control HilD, an AraC regulator that coordinates the expression of several virulence factors. The expression of hilD is modulated at several steps of the expression process. Here, we report that the invasion of epithelial cells by S. Typhimurium strains lacking the Gre factors, GreA and GreB, is impaired. By interacting with the RNA polymerase secondary channel, the Gre factors prevent backtracking of paused complexes to avoid arrest during transcriptional elongation. Our results indicate that the Gre factors are required for the expression of the bacterial factors needed for epithelial cell invasion by modulating expression of HilD. This regulation does not occur at transcription initiation and depends on the capacity of the Gre factors to prevent backtracking of the RNA polymerase. Remarkably, genetic analyses indicate that the 3'-untranslated region (UTR) of hilD is required for Gre-mediated regulation of hilD expression. Our data provide new insight into the complex regulation of S. Typhimurium virulence and highlight the role of the hilD 3'-UTR as a regulatory motif

Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease

The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. While the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the amino-terminal FinO domain and the variable carboxy-terminal region that are required for ProQ activity. Two carboxy-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. In contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of nonfunctional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria

Gre factors are required fot biofilm formation in Salmonella enterica serovar Typhimurium by targeting transcription of the cgsD gene

Rdar biofilm formation of Salmonella typhimurium and Escherichia coli is a common ancient multicellular behavior relevant in cell-cell and inter-organism interactions equally, as in interaction with biotic and abiotic surfaces. With the expression of the characteristic extracellular matrix components amyloid curli fimbriae and the exopolysaccharide cellulose, the central hub for the delicate regulation of rdar morphotype expression is the orphan transcriptional regulator CsgD. Gre factors are ubiquitously interacting with RNA polymerase to selectively overcome transcriptional pausing. In this work, we found that GreA/GreB are required for expression of the csgD operon and consequently the rdar morphotype. The ability of the Gre factors to suppress transcriptional pausing and the 147 bp 5′-UTR of csgD are required for the stimulatory effect of the Gre factors on csgD expression. These novel mechanism(s) of regulation for the csgD operon might be relevant under specific stress conditions

CRP-cAMP mediated silencing of virulence expression in Salmonella enterica serovar Typhimurium

[eng] The regulation of the expression of virulence genes in Salmonella enterica serovar Typhimurium is an intensively studied feature of Salmonella lifestyle. How Salmonella integrates environmental signals to activate virulence-related genes within the host has been explored. The genes encoded in Salmonella pathogenicity island I (SPI-1) required for the invasion of epithelial cells are well characterized, and many regulators involved in the activation of SPI-1 genes have been described. However, little is known about mechanism involved in the repression of SPI-1 under conditions were Salmonella does not require the expression of virulence-related genes. The expression of SPI-1 encoded genes have been reported to be a burden for Salmonella physiology and therefore mechanism to control shut down of SPI-1 under non permissive conditions might play a crucial role in Salmonella physiology. Here we describe that at exponential phase, CRP-cAMP, which acts as an activator at stationary phase, represses the expression of SPI-1 genes. The overall objective of this thesis was to characterize how CRP-cAMP silences SPI-1 expression under non-permissive conditions and to describe the molecular mechanism behind this phenotypic observation. In this thesis we i) define the target gene for the CRP-mediated regulation of SPI-1 ii) elucidate at which level of regulation CRP-cAMP modulates the expression of the SPI-1 genes master regulator hilD iii) characterize the involvement of CRP-cAMP dependent sRNA in hilD regulation iv) characterize the interaction of the CRP-cAMP dependent sRNA Spot 42 with the hilD 3’UTR region to regulate SPI-1 and v) explore the role of CRP-cAMP in the modulation of the SPI-1 repressor CsrA through the regulation of the long non-coding RNA csrB and csrC. CRP-cAMP represses hilA expression at exponential phase (non-permissive conditions for SPI-1 expression) and acts as an activator at stationary phase (permissive conditions for SPI-1 expression). CRP-cAMP mediated repression of hilA causes a concomitant attenuation in the expression level of SPI-1 encoded effector proteins. The regulation of SPI-1 during logarithmic growth phase occurs upstream of HilA by repressing hilD, hilC and rtsA expression and is mediated by the regulation of hilD expression at the post transcriptional level through the hilD 3’UTR. CRP-cAMP mediated regulation of hilD requires, in addition to the hilD 3’UTR, the sRNA chaperone Hfq and the major endonuclease RNAse E. CRP-cAMP represses the expression of the sRNA Spot 42 at exponential phase. We show that Spot 42 positively regulates hilD expression at exponential growth phase and requires of the presence of the hilD 3’UTR, the sRNA chaperone Hfq and the major endonuclease RNAse E. Interestingly, Spot 42 and the hilD 3’UTR region physically bind to Hfq. Spot 42 physically interacts with the last 150 nt of the hilD 3’UTR and unstructured region III of Spot 42 is required for the regulation of hilD. CRP-cAMP represses csrC but not csrB expression at exponential phase to regulate the expression of hilD. Remarkably, the CRP-cAMP dependent sRNA Spot 42 positively regulates the expression of csrC.[spa] La regulación de la expresión de genes de virulencia en Salmonella enterica serovar Typhimurium es una característica intensamente estudiada en Salmonella. La forma en que Salmonella integra señales ambientales para activar los genes relacionados con la virulencia dentro del huésped ha sido explorada. Los genes codificados en la isla de patogenicidad I de Salmonella (SPI-1) son necesarios para la invasión de células epiteliales, están bien caracterizados y se han descrito muchos reguladores implicados en su activación. Sin embargo, poco se sabe sobre el mecanismo implicado en la represión de SPI-1 en condiciones en las que Salmonella no requiere la expresión de genes relacionados con la virulencia. Es sabido que la expresión de genes codificados por SPI-1 son una carga para la fisiología de Salmonella y por lo tanto el mecanismo para controlar la represión de SPI-1 en condiciones no permisivas podría desempeñar un papel crucial en la fisiología de Salmonella. Aquí se describe que en fase exponencial, CRP-cAMP, que actúa como un activador en fase estacionaria, reprime la expresión de los genes SPI-1. El objetivo general de esta tesis fue caracterizar cómo el CRP-cAMP silencia la expresión de SPI-1 en condiciones no permisivas y describir el mecanismo molecular detrás de esta observación fenotípica. El CRP-cAMP reprime la expresión de hilA en la fase exponencial (condiciones no permisivas para la expresión de SPI-1) y actúa como un activador en fase estacionaria (condiciones permisivas para la expresión de SPI-1). La represión mediada por CRP-cAMP de hilA provoca una atenuación concomitante en el nivel de expresión de proteínas efectoras codificadas por SPI-1. La regulación de SPI-1 durante la fase de crecimiento logarítmico se produce aguas arriba de HilA mediante la represión hilD, hilC y rtsA expresión y está mediada por la regulación de hilD a nivel post transcripcional a través de la hilD 3'UTR. La regulación mediada por CRP-cAMP de hilD requiere, además de la hilD 3'UTR, la chaperona Hfq y la endonucleasa RNAsa E. CRP-cAMP reprime la expresión del sRNA Spot 42 en la fase exponencial. Mostramos que Spot 42 regula positivamente la expresión hilD en la fase de crecimiento exponencial, Spot 42 interacciona físicamente con los últimos 150 nt de la hilD 3'UTR

3' untranslated regions: regulation at the end of the road

Post-transcriptional gene regulation in bacteria plays a major role in the adaptation of bacterial cells to the changing conditions encountered in the environment. In bacteria, most of the regulation at the level of mRNA seems to be targeting the 5′untranslated regions where accessibility to the ribosome-binding site can be modulated to alter gene expression. In recent years, the role of 3′untranslated regions has gained attention also as a site for post-transcriptional regulation. In addition to be a source of trans-encoded small RNAs, the 3′untranslated regions can be targets to modulate gene expression. Taking recent findings in the post-transcriptional regulation of the hilD gene, encoding for the main regulator of virulence in Salmonella enterica serovar Typhimurium, we highlight the role of 3′untranslated regions as targets of post-transcriptional regulation mediated by small RNAs and discuss the implications of transcriptional elongation in the 3′UTR-mediated regulation in bacteria

In vivo targets of Salmonella FinO include a FinP-like small RNA controlling copy number of a cohabitating plasmid.

FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/traJ) for the plasmid-encoded FinO protein, to transcriptome-wide activity as documented for chromosomally encoded ProQ proteins. Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level

Global discovery of bacterial RNA-binding proteins by RNase-sensitive gradient profiles reports a new FinO domain protein.

RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq, and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinO-domain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalog of microbial RBPs

Global RNA interactome of Salmonella discovers a 5' UTR sponge for the MicF small RNA that connects membrane permeability to transport capacity.

The envelope of Gram-negative bacteria is a vital barrier that must balance protection and nutrient uptake. Small RNAs are crucial regulators of the envelope composition and function. Here, using RIL-seq to capture the Hfq-mediated RNA-RNA interactome in Salmonella enterica, we discover envelope-related riboregulators, including OppX. We show that OppX acts as an RNA sponge of MicF sRNA, a prototypical porin repressor. OppX originates from the 5' UTR of oppABCDF, encoding the major inner-membrane oligopeptide transporter, and sequesters MicF's seed region to derepress the synthesis of the porin OmpF. Intriguingly, OppX operates as a true sponge, storing MicF in an inactive complex without affecting its levels or stability. Conservation of the opp-OppX-MicF-ompF axis in related bacteria suggests that it serves an important mechanism, adjusting envelope porosity to specific transport capacity. These data also highlight the resource value of this Salmonella RNA interactome, which will aid in unraveling RNA-centric regulation in enteric pathogens