S1 ribosomal protein and the interplay between translation and mRNA decay

S1 is an ‘atypical’ ribosomal protein weakly associated with the 30S subunit that has been implicated in translation, transcription and control of RNA stability. S1 is thought to participate in translation initiation complex formation by assisting 30S positioning in the translation initiation region, but little is known about its role in other RNA transactions. In this work, we have analysed in vivo the effects of different intracellular S1 concentrations, from depletion to overexpression, on translation, decay and intracellular distribution of leadered and leaderless messenger RNAs (mRNAs). We show that the cspE mRNA, like the rpsO transcript, may be cleaved by RNase E at multiple sites, whereas the leaderless cspE transcript may also be degraded via an alternative pathway by an unknown endonuclease. Upon S1 overexpression, RNase E-dependent decay of both cspE and rpsO mRNAs is suppressed and these transcripts are stabilized, whereas cleavage of leaderless cspE mRNA by the unidentified endonuclease is not affected. Overall, our data suggest that ribosome-unbound S1 may inhibit translation and that part of the Escherichia coli ribosomes may actually lack S1

S1 ribosomal protein over-expression inhibits RNase E-dependent decay in Escherichia coli

It is commonly accepted that in bacteria transcription, translation and degradation of RNA are tightly coordinated processes; however, the molecular bases of this interconnection are poorly understood. S1 is the largest E. coli ribosomal protein; it is very abundant and weakly associated with the 30S ribosomal subunit. Both its over-expression and depletion impair bacterial growth. In a previous work we showed that S1 over-expression leads to a general increase in mRNA stability. To further characterize this phenomenon, we tested the role of 5'-UTR in S1-dependent stabilization. As a model transcript, we chose the cspE mRNA, which is sensitive to S1 stabilization. We showed by EMSA that S1 still binds a cspE RNA lacking the 5'-UTR (cspE-Del), albeit with lower affinity relative to the complete mRNA (cspE-wt), suggesting that for this mRNA different S1 binding sites may exist. In vivo, cspE-Del was less stable than cspE-wt RNA. Upon S1 induction, both the transcripts were stabilized; moreover, different RNA decay intermediates, deriving from cspE-Del degradation at the 5'-end, were detected, whereas no cspE-wt degradation products were present. In an rne thermosensitive mutant expressing cspE-Del, these decay intermediates were produced and stabilized, suggesting that RNase E may be involved in their degradation but not in their production. On the whole, our data suggest that S1 may interact both with the 5'-UTR and/or the 3'-UTR and prevent RNase E-dependent mRNA degradation but not the RNase E-independent endonucleolytic pathway operating on the leaderless cspE mRNA

S1 ribosomal protein and the interplay between translation and mRNA decay

In bacteria, transcription, translation and mRNA decay are tightly interconnected processes; however, little is known about specific factors and molecular mechanisms involved in their co-ordination. The ribosomal protein S1, an \u201catypical\u201d ribosomal protein weakly associated with the 30S subunit of Escherichia coli ribosome, has been implicated in translation, transcription and control of RNA stability. It is thus a good candidate for playing a role in the interplay among these processes. We have addressed S1 function by assaying translation and decay of model full-length and leaderless mRNAs upon modulation of S1 intracellular concentration (from depletion to overexpression). We have shown that S1 over-expression leads to polysome disappearance and translation inhibition. Moreover, in the same condition, RNase E-dependent decay of both the cspE+ and leaderless \u394L-cspE mRNAs is prevented. Conversely, cleavage of \u394L-cspE mRNA by an unidentified endonuclease is not affected. Overall, our data suggest that ribosome-unbound S1 may inhibit translation and stabilize mRNA through the specific inhibition of RNase E-dependent decay

Exploring pyrazinamide derivatives as novel Pseudomonas aeruginosa inhibitors: unexploited antibacterial molecules for a new antibiotics target

Background The ribosomal protein S1 (encoded by rpsA) is a promising target for new antibacterial drugs. In Escherichia coli, S1 has an essential role in translation. S1 is highly conserved among Gram negative bacteria and absent in mammalian cells. Recently, it has been found that pyrazinamide (PZA), a first-line tuberculosis drug, targets S1 protein. PZA derivatives were developed by Bracco SpA in the early \u201860s and roughly characterized for antibacterial activity; interestingly, in preliminary tests, the derivative B2320 seemed to be active against Pseudomonas aeruginosa (Pa). B2320 target in Pa is currently unknown. Hypothesis and objectives 1. Characterizing the anti-Pa activity of B2320. 2. Exploring Pa S1 as a potential target for new antibacterials. 3. Setting-up an E. coli biosensor strain for the screening of S1 inhibitors. Essential methods 1. B2320 will be tested for anti-bacterial activity both against Pseudomonas lab strains and a collection of clinical isolates from cystic fibrosis patients. The mechanism of action of the compound will be investigated. 2. A Pa strain with rpsA conditional expression will be constructed in order to assess its essentiality. 3. A fluorescence-based assay to find inhibitors of S1-dependent translation initiation will be set up in E. coli, transferred in Pa and used to screen a collection of heterogeneous chemical compounds for translation inhibitors. Preliminary results 1. B2320 activity against PAO1 and PA14 lab strains has been tested in different growth conditions. Interestingly, the more virulent PA14 strain seems more sensitive to B2320 than PAO1 strain. 2. The mutant is under construction. We have mapped the 5\u2019-end of rpsA transcript in Pa. This has been instrumental in designing the construct for rpsA mutation. 3. We have developed a simple whole-cell assay in E. coli that allows discriminating antibiotics inhibiting different translation steps. Expected results and their significance P. aeruginosa is the most common pathogen in CF lung infection with a high negative impact on lung functionality and patients\u2019 mortality. The increasing diffusion of multi-resistant strains demands for the development of new anti-Pa agents. These could be identified both among PZA derivatives and by our whole-cell screening of libraries of chemical compounds. Moreover, if Pa S1 will result to be essential as expected, it would be a robust target for the design of new drugs

Evaluation of the infection-relevant role of small RNA-based regulatory systems in the opportunistic pathogen Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important opportunistic pathogen in immune-compromised and cystic fibrosis patients. We have performed a comparative small RNA (sRNA) profiling via deep-sequencing of P. aeruginosa PAO1 and PA14 strains (1) which share the same host range but differ in pathogenicity. We now aim at identifying infection-relevant sRNA-based regulatory systems. To this end, we are focusing on a short list of 8 validated sRNAs which showed, at preliminary screenings, potential virulence hallmarks such as: specific expression in PA14 or PAO1; responsiveness to infection-relevant host stimuli (e.g. oxygen availability, temperature shift) or invasion signals (e.g. quorum sensing). Our approach is to generate sRNA deletion mutants and test them for i) canonical virulence phenotype(s); ii) ability to invade human respiratory epithelial cells and stimulate an immune response; iii) airways infection in both acute and chronic murine models. The above tests are accompanied by in silico, genetic and transcriptomics-based screenings for the genome-wide search of sRNAs target genes. 1. Ferrara S et al. (2012). PLoS One 7: e36553

A genetic approach to thermosensors identification in Pseudomonas aeruginosa

Modulation of mRNA translatability either by trans-acting factors (proteins or sRNAs) or by in cis riboswitches is widespread in Bacteria and controls relevant phenotypic traits. Unfortunately, the identification on a genomic scale of genes post-transcriptionally regulated is not an easy task, as modulation of translation efficiency is not always reflected by changes in the mRNA amount. We devised a reporter genetic system for the identification of post-transcriptionally regulated genes and we applied this system to search for Pseudomonas aeruginosa RNA thermosensors, a class of riboswitches that modulate gene translation in response to temperature changes. As P. aeruginosa is able to thrive in a broad range of ecological niches, genes differentially expressed at 37 \ub0C and at lower temperatures may be implicated in infection and survival in the human host. We prepared in a plasmid vector a translational fusion library of P. aeruginosa DNA fragments (PaDNA) inserted upstream of Tip2, a short peptide able to inactivate the TetR repressor upon expression1. The library was assayed in streptomycin resistant merodiploid rpsL+/rpsL-31 E. coli strain in which the dominant rpsL+ allele (which confers streptomycin sensitivity) was repressed by TetR. PaDNA fragments conferring thermosensitive streptomycin resistance (i.e. putatively expressing PaDNA-Tip2 fusions at 37 \ub0C and not at 28\ub0C) were sequenced. We identified six new putative thermosensors. Interestingly, two of them are located upstream of genes implicated in P.aeruginosa pathogenesis. Experiments are in progress to validate the results of our survey. 1Goeke, D., et al. (2012) J. Mol. Biol. 416:33-4

A genetic approach to thermosensors identification in Pseudomonas aeruginosa

Modulation of mRNA translatability either by trans-acting factors (proteins or sRNAs) or by in cis riboswitches is widespread in Bacteria and controls relevant phenotypic traits. Unfortunately, the identification on a genomic scale of genes post-transcriptionally regulated is not an easy task, as modulation of translation efficiency is not always reflected by changes in the mRNA amount. We devised a reporter genetic system for the identification of post-transcriptionally regulated genes and we applied this system to search for Pseudomonas aeruginosa RNA thermosensors, a class of riboswitches that modulate gene translation in response to temperature changes. As P. aeruginosa is able to thrive in a broad range of ecological niches, genes differentially expressed at 37 \ub0C and at lower temperatures may be implicated in infection and survival in the human host. We prepared in a plasmid vector a translational fusion library of P. aeruginosa DNA fragments (PaDNA) inserted upstream of Tip2, a short peptide able to inactivate the TetR repressor upon expression1. The library was assayed in streptomycin resistant merodiploid rpsL+/rpsL-31 E. coli strain in which the dominant rpsL+ allele (which confers streptomycin sensitivity) was repressed by TetR. PaDNA fragments conferring thermosensitive streptomycin resistance (i.e. putatively expressing PaDNA-Tip2 fusions at 37 \ub0C and not at 28\ub0C) were sequenced. We identified six new putative thermosensors. Interestingly, two of them are located upstream of genes implicated in P.aeruginosa pathogenesis. Experiments are in progress to validate the results of our survey. 1Goeke, D., et al. (2012) J. Mol. Biol. 416:33-4

Comparative small RNA profiling in the PAO1 and PA14 strains of the opportunistic pathogen Pseudomonas aeruginosa

Small RNAs (sRNAs) play fundamental roles in the control of gene expression, both in eukaryotes and prokaryotes, by modulating transcription, translation, mRNA stability, and DNA maintenance or silencing. In bacteria, sRNAs participate in regulatory circuits of cell viability, stress adaptation, metabolism, virulence and pathogenicity. Unlike the most studied bacterial pathogens where hundreds of sRNAs have been identified, only 30 sRNAs have been annotated in the opportunistic pathogen Pseudomonas aeruginosa, in particular in the attenuated strain PAO1. In this work, we aimed at the comparative sRNA profiling of the P. aeruginosa PAO1 and virulent PA14 strains by systematic identification via a massive sequencing approach tailored for low molecular weight RNA. Overall, by this approach we identified about 300 genomic regions significantly expressing short sRNA-like transcripts. Some of these regions are unique to either PAO1 or PA14, whereas the majority is conserved in both strains. In the latter case, we could detect either similar or differential expression levels between the two strains. We also validated by Northern blotting the actual sRNA expression from a selected panel of both unique and orthologous genomic regions. We are currently analyzing their expression in response to pathogenesis-relevant signals such as temperature, O2 availability and quorum sensing. In addition, we aim to build up a genetic system for the identification of target genes in order to dissect sRNA-based regulatory mechanisms involved in cell viability and pathogenesis

Comparative Profiling of <em>Pseudomonas aeruginosa</em> Strains Reveals Differential Expression of Novel Unique and Conserved Small RNAs

<div><p><em>Pseudomonas aeruginosa</em> is a highly adaptable bacterium that thrives in a broad range of ecological niches and can infect multiple hosts as diverse as plants, nematodes and mammals. In humans, it is an important opportunistic pathogen. This wide adaptability correlates with its broad genetic diversity. In this study, we used a deep-sequencing approach to explore the complement of small RNAs (sRNAs) in <em>P. aeruginosa</em> as the number of such regulatory molecules previously identified in this organism is relatively low, considering its genome size, phenotypic diversity and adaptability. We have performed a comparative analysis of PAO1 and PA14 strains which share the same host range but differ in virulence, PA14 being considerably more virulent in several model organisms. Altogether, we have identified more than 150 novel candidate sRNAs and validated a third of them by Northern blotting. Interestingly, a number of these novel sRNAs are strain-specific or showed strain-specific expression, strongly suggesting that they could be involved in determining specific phenotypic traits.</p> </div

Comparative profiling of Pseudomonas aeruginosa strains reveals differential expression of novel unique and conserved small RNAs

In recent years, it has become increasingly clear that regulation of gene expression at both transcriptional and post-transcriptional levels by small RNAs (sRNAs) is widespread in bacteria. Furthermore, several studies suggest that sRNAs can play key roles in regulating cellular processes linked to pathogenesis. Pseudomonas aeruginosa is a widely distributed opportunistic pathogen for which complete genomic sequences of several strains - with varying pathogenic characteristics \u2013 are available. We have performed a comparative sRNA profiling via deep-sequencing of P. aeruginosa PAO1 and PA14 strains which share the same host range but differ in pathogenicity, PA14 being considerably more virulent in several model organisms. Altogether, we have identified more than 150 novel candidate sRNAs and validated a third of them by Northern blotting. Interestingly, a number of these novel sRNAs are strain-specific or showed strain-specific expression, strongly suggesting that they could be involved in determining specific phenotypic traits. About 20 validated sRNAs showed potential virulence hallmarks such as: specific expression in PA14 or PAO1; responsiveness to infection-relevant host stimuli (e.g. oxygen availability, temperature shift) or invasion signals (e.g. quorum sensing). Our future studies will focus on the impact of this panel of sRNAs on P. aeruginosa virulence and infection, and on the dissection of the regulatory role via identification of target mRNAs and characterization of sRNA/target mRNA interactions