FAST, a method based on split-GFP for the detection in solution of proteins synthesized in cell-free expression systems.

Cell-free protein synthesis (CFPS) systems offer a versatile platform for a wide range of applications. However, the traditional methods for detecting proteins synthesized in CFPS, such as radioactive labeling, fluorescent tagging, or electrophoretic separation, may be impractical, due to environmental hazards, high costs, technical complexity, and time consuming procedures. These limitations underscore the need for new approaches that streamline the detection process, facilitating broader application of CFPS. By harnessing the reassembly capabilities of two GFP fragments-specifically, the GFP1-10 and GFP11 fragments-we have crafted a method that simplifies the detection of in vitro synthesized proteins called FAST (Fluorescent Assembly of Split-GFP for Translation Tests). FAST relies on the fusion of the small tag GFP11 to virtually any gene to be expressed in CFPS. The in vitro synthesized protein:GFP11 can be rapidly detected in solution upon interaction with an enhanced GFP1-10 fused to the Maltose Binding Protein (MBP:GFP1-10). This interaction produces a fluorescent signal detectable with standard fluorescence readers, thereby indicating successful protein synthesis. Furthermore, if required, detection can be coupled with the purification of the fluorescent complex using standardized MBP affinity chromatography. The method's versatility was demonstrated by fusing GFP11 to four distinct E. coli genes and analyzing the resulting protein synthesis in both a homemade and a commercial E. coli CFPS system. Our experiments confirmed that the FAST method offers a direct correlation between the fluorescent signal and the amount of synthesized protein:GFP11 fusion, achieving a sensitivity threshold of 8 ± 2 pmol of polypeptide, with fluorescence plateauing after 4 h. Additionally, FAST enables the investigation of translation inhibition by antibiotics in a dose-dependent manner. In conclusion, FAST is a new method that permits the rapid, efficient, and non-hazardous detection of protein synthesized within CFPS systems and, at the same time, the purification of the target protein.

Antibiotic synergist OM19r reverses aminoglycoside resistance in multidrug-resistant Escherichia coli.

Introduction: The continued emergence and spread of multidrug-resistant (MDR) bacterial pathogens require a new strategy to improve the efficacy of existing antibiotics. Proline-rich antimicrobial peptides (PrAMPs) could also be used as antibacterial synergists due to their unique mechanism of action. Methods: Utilizing a series of experiments on membrane permeability, In vitro protein synthesis, In vitro transcription and mRNA translation, to further elucidate the synergistic mechanism of OM19r combined with gentamicin. Results: A proline-rich antimicrobial peptide OM19r was identified in this study and its efficacy against Escherichia coli B2 (E. coli B2) was evaluated on multiple aspects. OM19r increased antibacterial activity of gentamicin against multidrug-resistance E. coli B2 by 64 folds, when used in combination with aminoglycoside antibiotics. Mechanistically, OM19r induced change of inner membrane permeability and inhibited translational elongation of protein synthesis by entering to E. coli B2 via intimal transporter SbmA. OM19r also facilitated the accumulation of intracellular reactive oxygen species (ROS). In animal models, OM19r significantly improved the efficacy of gentamicin against E. coli B2. Discussion: Our study reveals that OM19r combined with GEN had a strong synergistic inhibitory effect against multi-drug resistant E. coli B2. OM19r and GEN inhibited translation elongation and initiation, respectively, and ultimately affected the normal protein synthesis of bacteria. These findings provide a potential therapeutic option against multidrug-resistant E. coli.

Escherichia coli CspA stimulates translation in the cold of its own mRNA by promoting ribosome progression.

Escherichia coli CspA is an RNA binding protein that accumulates during cold-shock and stimulates translation of several mRNAs-including its own. Translation in the cold of cspA mRNA involves a cis-acting thermosensor element, which enhances ribosome binding, and the trans-acting action of CspA. Using reconstituted translation systems and probing experiments we show that, at low temperature, CspA specifically promotes the translation of the cspA mRNA folded in the conformation less accessible to the ribosome, which is formed at 37°C but is retained upon cold shock. CspA interacts with its mRNA without inducing large structural rearrangements, but allowing the progression of the ribosomes during the transition from translation initiation to translation elongation. A similar structure-dependent mechanism may be responsible for the CspA-dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.

Disparate Phenotypes Resulting from Mutations of a Single Histidine in Switch II of Geobacillus stearothermophilus Translation Initiation Factor IF2.

The conserved Histidine 301 in switch II of Geobacillus stearothermophilus IF2 G2 domain was substituted with Ser, Gln, Arg, Leu and Tyr to generate mutants displaying different phenotypes. Overexpression of IF2H301S, IF2H301L and IF2H301Y in cells expressing wtIF2, unlike IF2H301Q and IF2H301R, caused a dominant lethal phenotype, inhibiting in vivo translation and drastically reducing cell viability. All mutants bound GTP but, except for IF2H301Q, were inactive in ribosome-dependent GTPase for different reasons. All mutants promoted 30S initiation complex (30S IC) formation with wild type (wt) efficiency but upon 30S IC association with the 50S subunit, the fMet-tRNA reacted with puromycin to different extents depending upon the IF2 mutant present in the complex (wtIF2 to IF2H301Q > IF2H301R >>> IF2H301S, IF2H301L and IF2H301Y) whereas only fMet-tRNA 30S-bound with IF2H301Q retained some ability to form initiation dipeptide fMet-Phe. Unlike wtIF2, all mutants, regardless of their ability to hydrolyze GTP, displayed higher affinity for the ribosome and failed to dissociate from the ribosomes upon 50S docking to 30S IC. We conclude that different amino acids substitutions of His301 cause different structural alterations of the factor, resulting in disparate phenotypes with no direct correlation existing between GTPase inactivation and IF2 failure to dissociate from ribosomes.

Characterization of the Self-Resistance Mechanism to Dityromycin in the Streptomyces Producer Strain.

Dityromycin is a peptide antibiotic isolated from the culture broth of the soil microorganism Streptomyces sp. strain AM-2504. Recent structural studies have shown that dityromycin targets the ribosomal protein S12 in the 30S ribosomal subunit, inhibiting translocation. Herein, by using in vitro protein synthesis assays, we identified the resistance mechanism of the producer strain to the secondary metabolite dityromycin. The results show that the self-resistance mechanism of the Streptomyces sp. strain AM-2504 is due to a specific modification of the ribosome. In particular, two amino acid substitutions, located in a highly conserved region of the S12 protein corresponding to the binding site of the antibiotic, were found. These mutations cause a substantial loss of affinity of the dityromycin for the 30S ribosomal subunit, protecting the producer strain from the toxic effect of the antibiotic. In addition to providing a detailed description of the first mechanism of self-resistance based on a mutated ribosomal protein, this work demonstrates that the molecular determinants of the dityromycin resistance identified in Streptomyces can be transferred to Escherichia coli ribosomes, where they can trigger the same antibiotic resistance mechanism found in the producer strain.IMPORTANCE The World Health Organization has identified antimicrobial resistance as a substantial threat to human health. Because of the emergence of pathogenic bacteria resistant to multiple antibiotics worldwide, there is a need to identify the mode of action of antibiotics and to unravel the basic mechanisms responsible for drug resistance. Antibiotic producers' microorganisms can protect themselves from the toxic effect of the drug using different strategies; one of the most common involves the modification of the antibiotic's target site. In this work, we report a detailed analysis of the molecular mechanism, based on protein modification, devised by the soil microorganism Streptomyces sp. strain AM-2504 to protect itself from the activity of the peptide antibiotic dityromycin. Furthermore, we demonstrate that this mechanism can be reproduced in E. coli, thereby eliciting antibiotic resistance in this human commensal bacterium.

Draft Genome Sequence of Streptomyces sp. Strain AM-2504, Identified by 16S rRNA Comparative Analysis as a Streptomyces kasugaensis Strain.

We report here the draft genome sequence of Streptomyces sp. strain AM-2504, a microorganism producing a broad range of biotechnologically relevant molecules. The comparative analysis of its 16S rRNA sequence allowed the assignment of this strain to the Streptomyces kasugaensis species, thus fostering functional characterization of the secondary metabolites produced by this microorganism.

Cold-Responsive Regions of Paradigm Cold-Shock and Non-Cold-Shock mRNAs Responsible for Cold Shock Translational Bias.

In Escherichia coli, the mRNA transcribed from the main cold-shock gene cspA is a thermosensor, which at low temperature adopts a conformation particularly suitable for translation in the cold. Unlike cspA, its paralogue cspD is expressed only at 37 °C, is toxic so cannot be hyper-expressed in E. coli and is poorly translated in vitro, especially at low temperature. In this work, chimeric mRNAs consisting of different segments of cspA and cspD were constructed to determine if parts of cspA could confer cold-responsive properties to cspD to improve its expression. The activities of these chimeric mRNAs in translation and in partial steps of translation initiation such as formation of 30S initiation complexes and 50S subunits docking to 30S complexes to yield 70S initiation complexes were analyzed. We show that the 5' untranslated region (5'UTR) of cspA mRNA is sufficient to improve the translation of cspD mRNA at 37 °C whereas both the 5'UTR and the region immediately downstream the cspA mRNA initiation triplet are essential for translation at low temperature. Furthermore, the translational apparatus of cold-stressed cells contains trans-active elements targeting both 5'UTR and downstream regions of cspA mRNA, thereby improving translation of specific chimeric constructs at both 15 and 37 °C.

Antibiotics Targeting the 30S Ribosomal Subunit: A Lesson from Nature to Find and Develop New Drugs.

The use of antibiotics has revolutionized medicine, greatly improving our capacity to save millions of lives from otherwise deadly bacterial infections. Unfortunately, the health-associated benefits provided by antibiotics have been counteracted by bacteria developing or acquiring resistance mechanisms. The negative impact to public health is now considered of high risk due to the rapid spreading of multi-resistant strains. More than 60 % of clinically relevant antibiotics of natural origin target the ribosome, the supramolecular enzyme which translates the genetic information into proteins. Although many of these antibiotics bind the small ribosomal subunit, only a few are reported to inhibit the initiation of protein synthesis, with none reaching commercial availability. Counterintuitively, translation initiation is the most divergent phase of protein synthesis between prokaryotes and eukaryotes, a fact which is a solid premise for the successful identification of drugs with reduced probability of undesired effects to the host. Such a paradox is one of its kind and deserves special attention. In this review, we explore the inhibitors that bind the 30S ribosomal subunit focusing on both the compounds with proved effects on the translation initiation step and the underreported translation initiation inhibitors. In addition, we explore recent screening tests and approaches to discover new drugs targeting translation.

Development of a graphene oxide-based assay for the sequence-specific detection of double-stranded DNA molecules.

Graphene oxide (GO) is a promising material for the development of cost-effective detection systems. In this work, we have devised a simple and rapid GO-based method for the sequence-specific identification of DNA molecules generated by PCR amplification. The csp genes of Escherichia coli, which share a high degree of sequence identity, were selected as paradigm DNA templates. All tested csp genes were amplified with unlabelled primers, which can be rapidly removed at the end of the PCR taking advantage of the preferential binding to GO of single-stranded versus duplex DNA molecules. The amplified DNAs (targets) were heat-denatured and hybridized to a fluorescently-labelled single strand oligonucleotide (probe), which recognizes a region of the target DNAs displaying sequence variability. This interaction is extremely specific, taking place with high efficiency only when target and probe show perfect or near perfect matching. Upon GO addition, the unbound fraction of the probe was captured and its fluorescence quenched by the GO's molecular properties. On the other hand, the probe-target complexes remained in solution and emitted a fluorescent signal whose intensity was related to their degree of complementarity.

Inhibition of translation initiation complex formation by GE81112 unravels a 16S rRNA structural switch involved in P-site decoding.

In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon-anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state.

Time-resolved assembly of a nucleoprotein complex between Shigella flexneri virF promoter and its transcriptional repressor H-NS.

The virF gene of Shigella, responsible for triggering the virulence cascade in this pathogenic bacterium, is transcriptionally repressed by the nucleoid-associated protein H-NS. The primary binding sites of H-NS within the promoter region of virF have been detected here by footprinting experiments in the presence of H-NS or its monomeric DNA-binding domain (H-NSctd), which displays the same specificity as intact H-NS. Of the 14 short DNA fragments identified, 10 overlap sequences similar to the H-NS binding motif. The 'fast', 'intermediate' and 'slow' H-NS binding events leading to the formation of the nucleoprotein complex responsible for transcription repression have been determined by time-resolved hydroxyl radical footprinting experiments in the presence of full-length H-NS. We demonstrate that this process is completed in ≤1 s and H-NS protections occur simultaneously on site I and site II of the virF promoter. Furthermore, all 'fast' protections have been identified in regions containing predicted H-NS binding motifs, in agreement with the hypothesis that H-NS nucleoprotein complex assembles from a few nucleation sites containing high-affinity binding sequences. Finally, data are presented showing that the 22-bp fragment corresponding to one of the HNS binding sites deviates from canonical B-DNA structure at three TpA steps.

Role of the ribosome-associated protein PY in the cold-shock response of Escherichia coli.

Protein Y (PY) is an Escherichia coli cold-shock protein which has been proposed to be responsible for the repression of bulk protein synthesis during cold adaptation. Here, we present in vivo and in vitro data which clarify the role of PY and its mechanism of action. Deletion of yfiA, the gene encoding protein PY, demonstrates that this protein is dispensable for cold adaptation and is not responsible for the shutdown of bulk protein synthesis at the onset of the stress, although it is able to partially inhibit translation. In vitro assays reveal that the extent of PY inhibition changes with different mRNAs and that this inhibition is related to the capacity of PY of binding 30S subunits with a fairly strong association constant, thus stimulating the formation of 70S monomers. Furthermore, our data provide evidence that PY competes with the other ribosomal ligands for the binding to the 30S subunits. Overall these results suggest an alternative model to explain PY function during cold shock and to reconcile the inhibition caused by PY with the active translation observed for some mRNAs during cold shock.

The cspA mRNA is a thermosensor that modulates translation of the cold-shock protein CspA.

Cold induction of cspA, the paradigm Escherichia coli cold-shock gene, is mainly subject to posttranscriptional control, partly promoted by cis-acting elements of its transcript, whose secondary structure at 37 degrees C and at cold-shock temperature has been elucidated here by enzymatic and chemical probing. The structures, which were also validated by mutagenesis, demonstrate that cspA mRNA undergoes a temperature-dependent structural rearrangement, likely resulting from stabilization in the cold of an otherwise thermodynamically unstable folding intermediate. At low temperature, the "cold-shock" structure is more efficiently translated and somewhat less susceptible to degradation than the 37 degrees C structure. Overall, our data shed light on a molecular mechanism at the basis of the cold-shock response, indicating that cspA mRNA is able to sense temperature downshifts, adopting functionally distinct structures at different temperatures, even without the aid of trans-acting factors. Unlike with other previously studied RNA thermometers, these structural rearrangements do not result from melting of hairpin structures.

Real-time dynamics of ribosome-ligand interaction by time-resolved chemical probing methods.

Three protocols to perform time-resolved in situ probing of rRNA are described. The three methods (chemical modification with DMS and rRNA backbone cleavage by hydroxyl radicals generated by either K-peroxonitrite or Fe(II)-EDTA) make use of a quench-flow apparatus and exploit reactions that are faster than the interactions of ribosomal subunits with their ligands. These methods allow the investigation of the path and dynamics, in a approximately equal 50 to 1500ms time range, of the binding and dissociation of ribosomal ligands.

Cold-stress-induced de novo expression of infC and role of IF3 in cold-shock translational bias.

Expression of Escherichia coli infC, which encodes translation initiation factor IF3 and belongs to a transcriptional unit containing several promoters and terminators, is enhanced after cold shock, causing a transient increase of the IF3/ribosomes ratio. Here we show that after cold shock the two less used promoters (P(T) and P(I1)) remain active and/or are activated, resulting in de novo infC transcription and IF3 synthesis. These two events are partly responsible for the stoichiometric imbalance of the IF3/ribosomes ratio that contributes to establishing the cold-shock translational bias whereby cold-shock mRNAs are preferentially translated by cold-stressed cells while bulk mRNAs are discriminated against. Analysis of the IF3 functions at low temperature sheds light on the molecular mechanism by which IF3 contributes to the cold-shock translational bias. IF3 was found to cause a strong rate increase of fMet-tRNA binding to ribosomes programmed with cold-shock mRNA, an activity essential for the rapid formation of "30S initiation complexes" at low temperature. The increased IF3/ribosome ratio occurring during cold adaptation was also essential to overcome the higher stability of 70S monomers at low temperature so as to provide a sufficient pool of dissociated 30S subunits capable of "70S initiation complex" formation. Finally, at low temperature IF3 was shown to be endowed with the capacity of discriminating against translation of non-cold-shock mRNAs by a cold-shock-specific "fidelity" function operating with a mechanism different from those previously described, insofar as IF3 does not interfere with formation of 30S initiation complex containing these mRNAs, but induces the formation of nonproductive 70S initiation complexes.

Review on bacterial stress topics.

A complex network of regulatory systems ensures a coordinated and effective response to different types of stress that can act on a bacterium. Bacterial stress response generates changes that influence efflux system and virulence factor expression. Thus, partial or total loss of pathogenicity islands in uropathogenic Escherichia coli can be induced by SOS-dependent or SOS-independent pathways related to selection of quinolone-resistant mutants. Likewise, hyperosmolarity and some chemicals, including fluoroquinolones, salicylate, nonantimicrobial medicaments like diazepam and anti-inflammatory drugs are all able to induce an increased active efflux, cyclohexane tolerance, loss of porins, and decreased susceptibility to multiple antimicrobials in enterobacterial strains, suggesting that bacterial response to the stress caused by an increase in osmolarity might be linked to the development of the multidrug-resistant phenotypes. Finally, a sudden downshift of the growth temperature (cold-shock) triggers a drastic reprogramming of bacterial gene expression to allow cell survival under the new unfavorable conditions. The strategy developed by E. coli to reach this goal consists in the induction of a set of (cold-shock) genes whose expression is regulated at both transcriptional and posttranscriptional levels.

Cold-shock-induced de novo transcription and translation of infA and role of IF1 during cold adaptation.

Escherichia coli infA is transcribed from two promoters, P1 and P2, into a longer and a shorter mRNA encoding translation initiation factor IF1. Although P1 is intrinsically stronger than P2, the shorter half-life of its transcripts causes the steady-state level of the P2 transcript to be substantially higher than that of P1 during growth at 37 degrees C. After cold-shock, de novo transcription and translation of infA contribute to the transient increase of the IF1/ribosomes ratio, which is partially responsible for translational bias consisting in the preferential translation of cold-shock mRNAs in the cold. Cold-stress induction of infA expression is mainly due to the high activity of P1 at low temperature, which is further increased by transcriptional stimulation by CspA and by an increased transcript stability. Furthermore, the longer infA mRNA originating from P1 is preferentially translated at low temperature by the translational machinery of cold-shocked cells. The increased level of IF1 during cold adaptation is essential for overcoming the higher stability of the 70S monomers at low temperature and for providing a sufficient pool of dissociated 30S subunits capable of initiating translation.

Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome.

Initiation of protein synthesis is a universally conserved event that requires initiation factors IF1, IF2 and IF3 in prokaryotes. IF2 is a GTPase essential for binding initiator transfer RNA to the 30S ribosomal subunit and recruiting the 50S subunit into the 70S initiation complex. We present two cryo-EM structures of the assembled 70S initiation complex comprising mRNA, fMet-tRNA(fMet) and IF2 with either a non-hydrolyzable GTP analog or GDP. Transition from the GTP-bound to the GDP-bound state involves substantial conformational changes of IF2 and of the entire ribosome. In the GTP analog-bound state, IF2 interacts mostly with the 30S subunit and extends to the initiator tRNA in the peptidyl (P) site, whereas in the GDP-bound state IF2 steps back and adopts a 'ready-to-leave' conformation. Our data also provide insights into the molecular mechanism guiding release of IF1 and IF3.