Editorial: Interview With the Translational Apparatus: Stories of Intriguing Circuits and Mechanisms to Regulate Translation in Bacteria

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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

Role of the ribosome‐associated protein PY in the cold‐shock response of E scherichia coli

Protein Y ( PY ) is an E scherichia 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 mRNA s 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 mRNA s during cold shock. E scherichia coli responds to cold stress by entering an acclimation phase during which protein synthesis slows down considerably with the exception of a specific set of genes (cold‐shock genes) whose expression is stimulated. In this article, we have investigated in vivo and in vitro the role of PY , a protein that is associated with the ribosome throughout the cold acclimation phase. Our data indicate that protein PY can affect translation initiation but is not responsible for turning off bulk protein synthesis during the cold stress.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97494/1/mbo368-sup-0001-FigureS1-S3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/97494/2/mbo368.pd

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

ackground: Depression and anxiety are two of the most prevalent and disabling mental disorders worldwide, both in the general population and in outpatient clinical settings. Objective: This study aimed to analyze the psychometric properties of the Patient Health Questionnaire-4 (PHQ-4) based on network analysis metrics. Methods: A total of 911 Paraguayans (23.71% women and 76.29% men; mean age 31.25 years, SD = 10.63), selected by non-probabilistic convenience sampling, participated in the study. Network analysis was used to evaluate the internal structure, reliability, and measurement invariance between men and women. Results: The results revealed that the PHQ-4 is a unidimensional measure through Exploratory Graph Analysis (EGA). Reliability, through structural consistency, identified that 100% of the time, only a single dimension was obtained, and all items remained stable, as they were always replicated within the empirical dimension. The unidimensional structure has shown evidence of configural invariance; therefore, the network structure functioned equally among the different sex groups. Conclusion: The PHQ-4 presented optimal preliminary evidence of validity based on its internal structure, reliability, and invariance between sexes. Therefore, it may be useful as an accurate and brief measure of anxiety and depressive symptoms in the Paraguayan context.Horizon 2020 Framework Programm

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

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

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