Iron starvation increases the production of the Pseudomonas aeruginosa RsmY and RsmZ sRNAs in static conditions.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that induces virulence gene expression in response to host-mediated iron starvation. Recently, our laboratory showed that some virulence factors are responsive to iron limitation in static but not shaking growth conditions. One of these is the HSI-2-type six secretion system (T6SS), which is also induced during chronic infection. Iron regulation of T6SS was partially impacted by the iron-responsive PrrF sRNA and completely dependent upon the Pseudomonas quinolone signal (PQS) biosynthetic gene pqsA. Here, we analyzed the impact of iron on the expression of two small regulatory RNAs (sRNAs), RsmY and RsmZ, that activate the expression of T6SS by sequestering the RsmA translation inhibitor. Our results demonstrate that iron starvation induces the expression of RsmY and RsmZ in static but not shaking cultures. We further show that this induction occurs through the rsmY and rsmZ promoters and is dependent upon PqsA. Disruption of either the pqsR gene also eliminated iron-dependent regulation of rsmY and rsmZ promoter activity. Taken together, our results show novel targets of iron regulation that are specific to static growth, highlighting the importance of studying regulatory mechanisms in static communities that may be more representative of growth during chronic infection.IMPORTANCEIron is a central component of various bacterial metabolic pathways making it an important host-acquired nutrient for pathogens to establish infection. Previous iron regulatory studies primarily relied on shaking bacterial cultures; while these ensure cultural homogeneity, they do not reflect growth conditions during infection. We recently showed that static growth of Pseudomonas aeruginosa promotes iron-dependent regulation of a type six secretion system (T6SS), a virulence factor that is induced during chronic infections. In the current study, we found that static growth also promotes iron-dependent regulation of the RsmY and RsmZ sRNAs, which are global regulators that affect T6SS during chronic P. aeruginosa lung infection. Hence, our work demonstrates the Rsm sRNAs as potential effectors of iron regulation during static growth that may also be relevant in chronic infection.

Dihydrolipoamide Acetyltransferase AceF Influences the Type III Secretion System and Resistance to Oxidative Stresses through RsmY/Z in Pseudomonas aeruginosa.

Carbon metabolism plays an important role in bacterial physiology and pathogenesis. The type III secretion system (T3SS) of Pseudomonas aeruginosa is a virulence factor that contributes to acute infections. It has been demonstrated that bacterial metabolism affects the T3SS. Meanwhile, expression of T3SS genes is negatively regulated by the small RNAs RsmY and RsmZ. In this study, we studied the relationship between the dihydrolipoamide acetyltransferase gene aceF and the T3SS. Our results reveal an upregulation of RsmY and RsmZ in the aceF mutant, which represses the expression of the T3SS genes. Meanwhile, the aceF mutant is more tolerant to hydrogen peroxide. We demonstrate that the expression levels of the catalase KatB and the alkyl hydroperoxide reductase AhpB are increased in the aceF mutant. The simultaneous deletion of rsmY and rsmZ in the aceF mutant restored the expression levels of katB and ahpB, as well as bacterial susceptibility to hydrogen peroxide. Thus, we identify a novel role of AceF in the virulence and oxidative response of P. aeruginosa.

NrtR Mediated Regulation of H1-T6SS in Pseudomonas aeruginosa.

NrtR is a Nudix-related transcriptional regulator that is distributed among diverse bacteria and plays an important role in modulating bacterial intracellular NAD homeostasis. Previously, we showed that NrtR influences the T3SS expression and pathogenesis of Pseudomonas aeruginosa and demonstrated that NrtR mediates T3SS regulation through the cAMP/Vfr pathway. In the present study, we found that mutation of the nrtR gene leads to upregulation of the Hcp secretion island-I type VI secretion system (H1-T6SS). Further analysis revealed that mutation of the nrtR gene results in upregulation of regulatory RNAs (RsmY/RsmZ) that are known to control the H1-T6SS by sequestration of RsmA or RsmN. Simultaneous deletion of rsmY/rsmZ reduced the expression of H1-T6SS in the ΔnrtR mutant. In addition, overexpression of either rsmA or rsmN in ΔnrtR decreased H1-T6SS expression. Chromatin immunoprecipitation (ChIP)-Seq and electrophoretic mobility shift assay (EMSA) analyses revealed that NrtR directly binds to the promoters of rsmY, rsmZ and tssA1 (first gene of the H1-T6SS operon). Overall, the results from this study reveal the molecular details of NrtR-mediated regulation of H1-T6SS in P. aeruginosa. IMPORTANCE NrtR is a Nudix-related transcriptional regulator and controls the NAD cofactor biosynthesis in bacteria. P. aeruginosa NrtR binds to the intergenic region between nadD2 and pcnA to repress the expression of the two operons, therefore controlling the NAD biosynthesis. We have previously reported that NrtR controls T3SS expression via the cAMP/Vfr pathway in P. aeruginosa. However, the global regulatory function and direct binding targets of the NrtR remain elusive in P. aeruginosa. This study reveals novel direct regulatory targets of the NrtR in P. aeruginosa, elucidating the molecular mechanism of NrtR-mediated regulation of H1-T6SS.

Direct Inhibition of RetS Synthesis by RsmA Contributes to Homeostasis of the Pseudomonas aeruginosa Gac/Rsm Signaling System.

The Gac/Rsm system is a global regulator of Pseudomonas aeruginosa gene expression. The primary effectors are RsmA and RsmF. Both are RNA-binding proteins that interact with target mRNAs to modulate protein synthesis. RsmA/RsmF recognize GGA sequences presented in the loop portion of stem-loop structures. For repressed targets, the GGA sites usually overlap the ribosome binding site (RBS) and RsmA/RsmF binding inhibits translation initiation. RsmA/RsmF activity is controlled by several small non-coding RNAs (sRNA) that sequester RsmA/RsmF from target mRNAs. The most important sequestering sRNAs are RsmY and RsmZ. Transcription of rsmY/rsmZ is directly controlled by the GacSA two-component regulatory system. GacSA activity is antagonized by RetS, a hybrid sensor kinase. In the absence of retS, rsmY/rsmZ transcription is derepressed and RsmA/RsmF are sequestered by RsmY/RsmZ. Gac/Rsm system homeostasis is tightly controlled by at least two mechanisms. First, direct binding of RsmA to the rsmA and rsmF mRNAs inhibits further synthesis of both proteins. Second, RsmA stimulates rsmY/rsmZ transcription through an undefined mechanism. In this study we demonstrate that RsmA stimulates rsmY/rsmZ transcription by directly inhibiting RetS synthesis. RetS protein levels are elevated 2.5-fold in an rsmA mutant. Epistasis experiments demonstrate that the rsmA requirement for rsmY/rsmZ transcription is entirely suppressed in an rsmA, retS double mutant. RsmA directly interacts with the retS mRNA and requires two distinct GGA sites, one of which overlaps the RBS. We propose a model wherein RsmA inhibits RetS synthesis to promote rsmY/rsmZ transcription and that this acts as a checkpoint to limit RsmA/RsmF availability. IMPORTANCE The Pseudomonas aeruginosa Gac/Rsm system controls ∼500 genes and governs a critical lifestyle switch by inversely regulating factors that favor acute or chronic colonization. Control of gene expression by the Gac/Rsm system is mediated through RsmA and RsmF, small RNA-binding proteins that interact with target mRNAs to inhibit or promote protein synthesis and/or mRNA stability. RsmA/RsmF activity is governed by two small non-coding RNAs (RsmY and RsmZ) that sequester RsmA/RsmF from target mRNAs. The GacSA two-component regulatory system plays a pivotal role in the Gac/Rsm system by controlling rsmYZ transcription. This study provides insight into the control of homeostasis by demonstrating that RsmA directly targets and inhibits expression of RetS, an orphan sensor kinase critical for rsmYZ transcription.

Study of the sRNA RsmY involved in the genetic regulation of the synthesis of alginate and alkyl resorcinols in Azotobacter vinelandii.

Azotobacter vineladii is a Gram-negative bacterium that produces alginate and poly-hydroxybutyrate (PHB), two polymers of biotechnological interest. This bacterium has the ability to form desiccation-resistant cysts. In the cyst the membrane phospholipids are replaced with a family of phenolic lipids called alkylresorcinols (ARs). The alginate, PHB, and ARs are controlled by the GacS/A two-component system and the small regulatory RNA (sRNA) RsmZ1, belonging to the Rsm (Csr) regulatory system. The Rsm (Csr) systems usually possess two or more sRNAs, in this regard A. vinelandii is the bacterium with the highest number of rsm-sRNAs. Originally, the presence of two sRNAs of the RsmY family (RsmY1 and RsmY2) was reported, but in a subsequent work it was suggested that they conformed to a single sRNA. In this work we provide genetic evidence confirming that rsmY1 and rsmY2 constitute a single gene. Also, it was established that rsmY mutation decreased alginate and ARs production, but did not affect the PHB synthesis. Transcriptional studies showed that rsmY has its higher expression during the stationary growth phase, and in the absence of RsmZ1, rsmY increases its transcription. Interestingly, rsmY expression was influenced by the carbon source, but its expression did not correlate with alginate production.

Quorum-dependent expression of rsmX and rsmY, small non-coding RNAs, in Pseudomonas syringae.

Pseudomonas syringae pathovars are known to produce N-acyl-homoserine lactones (AHL) as quorum-sensing molecules. However, many isolates, including P. syringae pv. tomato DC3000 (PtoDC3000), do not produce them. In P. syringae, psyI, which encodes an AHL synthase, and psyR, which encodes the transcription factor PsyR required for activation of psyI, are convergently transcribed. In P. amygdali pv. tabaci 6605 (Pta6605), there is one nucleotide between the stop codons of both psyI and psyR. However, the canonical stop codon for psyI in PtoDC3000 was converted to the cysteine codon by one nucleotide deletion, and 23 additional amino acids extended it to a C-terminal end. This resulted in overlapping of the open reading frame (ORF) for psyI and psyR. On the other hand, stop codons in the psyR ORF of P. syringae 7 isolates, including pv. phaseolicola and pv. glycinea, were found. These results indicate that many pathovars of P. syringae have genetically lost AHL production ability by the mutation of their responsible genes. To examine whether PtoDC3000 modulates the gene expression profile in a population-dependent manner, we carried out microarray analysis using RNAs prepared from low- and high-density cells. We found the expressions of rsmX and rsmY remarkably activated in high-density cells. The activated expressions of rsmX and rsmY were confirmed by Northern blot hybridization, but these expressions were abolished in a ΔgacA mutant of Pta6605. These results indicate that regardless of the ability to produce AHL, P. syringae regulates expression of the small noncoding RNAs rsmX/Y by currently unknown quorum-sensing molecules.

RsmV, a Small Noncoding Regulatory RNA in Pseudomonas aeruginosa That Sequesters RsmA and RsmF from Target mRNAs.

The Gram-negative opportunistic pathogen Pseudomonas aeruginosa has distinct genetic programs that favor either acute or chronic virulence gene expression. Acute virulence is associated with twitching and swimming motility, expression of a type III secretion system (T3SS), and the absence of alginate, Psl, or Pel polysaccharide production. Traits associated with chronic infection include growth as a biofilm, reduced motility, and expression of a type VI secretion system (T6SS). The Rsm posttranscriptional regulatory system plays important roles in the inverse control of phenotypes associated with acute and chronic virulence. RsmA and RsmF are RNA-binding proteins that interact with target mRNAs to control gene expression at the posttranscriptional level. Previous work found that RsmA activity is controlled by at least three small, noncoding regulatory RNAs (RsmW, RsmY, and RsmZ). In this study, we took an in silico approach to identify additional small RNAs (sRNAs) that might function in the sequestration of RsmA and/or RsmF (RsmA/RsmF) and identified RsmV, a 192-nucleotide (nt) transcript with four predicted RsmA/RsmF consensus binding sites. RsmV is capable of sequestering RsmA and RsmF in vivo to activate translation of tssA1, a component of the T6SS, and to inhibit T3SS gene expression. Each of the predicted RsmA/RsmF consensus binding sites contributes to RsmV activity. Electrophoretic mobility shifts assays show that RsmF binds RsmV with >10-fold higher affinity than RsmY and RsmZ. Gene expression studies revealed that the temporal expression pattern of RsmV differs from those of RsmW, RsmY, and RsmZ. These findings suggest that each sRNA may play a distinct role in controlling RsmA and RsmF activity.IMPORTANCE The members of the CsrA/RsmA family of RNA-binding proteins play important roles in posttranscriptional control of gene expression. The activity of CsrA/RsmA proteins is controlled by small noncoding RNAs that function as decoys to sequester CsrA/RsmA from target mRNAs. Pseudomonas aeruginosa has two CsrA family proteins (RsmA and RsmF) and at least four sequestering sRNAs (RsmV [identified in this study], RsmW, RsmY, and RsmZ) that control RsmA/RsmF activity. RsmY and RsmZ are the primary sRNAs that sequester RsmA/RsmF, and RsmV and RsmW appear to play smaller roles. Differences in the temporal and absolute expression levels of the sRNAs and in their binding affinities for RsmA/RsmF may provide a mechanism of fine-tuning the output of the Rsm system in response to environmental cues.

Functional Analyses of the RsmY and RsmZ Small Noncoding Regulatory RNAs in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA.IMPORTANCE CsrA homologs are present in many bacteria. The opportunistic pathogen Pseudomonas aeruginosa uses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activity in vivo Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activity in vivo and in vitro are more stringent than those for RsmA.

An unusual CsrA family member operates in series with RsmA to amplify posttranscriptional responses in Pseudomonas aeruginosa.

Members of the CsrA family of prokaryotic mRNA-binding proteins alter the translation and/or stability of transcripts needed for numerous global physiological processes. The previously described CsrA family member in Pseudomonas aeruginosa (RsmA) plays a central role in determining infection modality by reciprocally regulating processes associated with acute (type III secretion and motility) and chronic (type VI secretion and biofilm formation) infection. Here we describe a second, structurally distinct RsmA homolog in P. aeruginosa (RsmF) that has an overlapping yet unique regulatory role. RsmF deviates from the canonical 5 ß-strand and carboxyl-terminal α-helix topology of all other CsrA proteins by having the α-helix internally positioned. Despite striking changes in topology, RsmF adopts a tertiary structure similar to other CsrA family members and binds a subset of RsmA mRNA targets, suggesting that RsmF activity is mediated through a conserved mechanism of RNA recognition. Whereas deletion of rsmF alone had little effect on RsmA-regulated processes, strains lacking both rsmA and rsmF exhibited enhanced RsmA phenotypes for markers of both type III and type VI secretion systems. In addition, simultaneous deletion of rsmA and rsmF resulted in superior biofilm formation relative to the wild-type or rsmA strains. We show that RsmF translation is derepressed in an rsmA mutant and demonstrate that RsmA specifically binds to rsmF mRNA in vitro, creating a global hierarchical regulatory cascade that operates at the posttranscriptional level.

Small Regulatory RNA and Legionella pneumophila.

Legionella pneumophila is a gram-negative bacterial species that is ubiquitous in almost any aqueous environment. It is the agent of Legionnaires' disease, an acute and often under-reported form of pneumonia. In mammals, L. pneumophila replicates inside macrophages within a modified vacuole. Many protein regulators have been identified that control virulence-related properties, including RpoS, LetA/LetS, and PmrA/PmrB. In the past few years, the importance of regulation of virulence factors by small regulatory RNA (sRNAs) has been increasingly appreciated. This is also the case in L. pneumophila where three sRNAs (RsmY, RsmZ, and 6S RNA) were recently shown to be important determinants of virulence regulation and 79 actively transcribed sRNAs were identified. In this review we describe current knowledge about sRNAs and their regulatory properties and how this relates to the known regulatory systems of L. pneumophila. We also provide a model for sRNA-mediated control of gene expression that serves as a framework for understanding the regulation of virulence-related properties of L. pneumophila.