Short-term stability of wastewater samples for storage and shipment in the context of the EU Sewage Sentinel System for SARS-CoV-2.

In the context of an EU-wide surveillance system for SARS-CoV-2 in wastewater, recommended by the European Commission, this study aims to provide scientific support to the adequacy of transport and storage conditions of samples both in terms of duration and samples temperature. Three laboratories in Slovenia, Cyprus and Estonia investigated the short-term, one-week, isochronous stability of wastewater samples by RT-qPCR based detection of SARS-CoV-2 genes. The results were tested for statistical significance to determine uncertainty of quantification and shelf-life, at testing temperatures of + 20 °C and - 20 °C, relative to reference at + 4 °C. Samples were collected from three urban wastewater treatment plant influents and analysed respectively for SARS-CoV-2 genes N1, N2 (Laboratory 1), N2, E (Laboratory 2) and N3 (Laboratory 3), with various analytical methods. For a period of 7/8 days at + 20 °C, decreasing trends of measured concentrations were observed for all genes resulting in instability according to the statistical analysis, while at - 20 °C the trend of variation was stable only for N1, N2 (Laboratory 1) and N3 (Laboratory 3). Trends for gene E concentrations at - 20 °C (Laboratory 2) could not be tested statistically for stability because of lack of data. Over a period of just 3 days at + 20 °C, the variation was statistically non-significant indicating stability for genes N1, E and N3 for laboratories 1, 2 and 3, respectively. Nonetheless, the outcome of the study presents evidence to support the choice of the selected temperature at which samples shall be preserved during storage before analysis or transport to the laboratory. The conditions (+4 °C, ∼ few days) chosen for EU wastewater surveillance are in accordance with these results, highlighting the importance of stability testing of environmental samples to determine the short-term analytical uncertainty.

Short peptides conferring resistance to macrolide antibiotics.

Translation of specific short peptides can render the ribosome resistant to macrolide antibiotics such as erythromycin. Peptides act in cis upon the ribosome on which they have been translated. Amino acid sequence and size are critical for peptide activity. Pentapeptides with different consensus sequences confer resistance to structurally different macrolide antibiotics, suggesting direct interaction between the peptide and the drug on the ribosome. Translation of resistance peptides may result in expulsion of the macrolide antibiotics from the ribosome. The consensus sequence of peptides conferring erythromycin resistance is similar to the sequence of the leader peptide involved in translational attenuation of erythromycin resistance genes, indicating that a similar type of interaction between the nascent peptide and antibiotics can occur in both cases.

A novel site of antibiotic action in the ribosome: interaction of evernimicin with the large ribosomal subunit.

Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants of Halobacterium halobium contained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2 in vitro, suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.

Inhibition of translation and cell growth by minigene expression.

A random five-codon gene library was used to isolate minigenes whose expression causes cell growth arrest. Eight different deleterious minigenes were isolated, five of which had in-frame stop codons; the predicted expressed peptides ranged in size from two to five amino acids. Mutational analysis demonstrated that translation of the inhibitory minigenes is essential for growth arrest. Pulse-labeling experiments showed that expression of at least some of the selected minigenes results in inhibition of cellular protein synthesis. Expression of the deleterious minigenes in cells deficient in peptidyl-tRNA hydrolase causes accumulation of families of peptidyl-tRNAs corresponding to the last minigene codon; the inhibitory action of minigene expression could be suppressed by overexpression of the tRNA corresponding to the last sense codon in the minigene. Experimental data are compatible with the model that the deleterious effect of minigene expression is mediated by depletion of corresponding pools of free tRNAs.

Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA.

Functionally active large ribosomal subunits of thermophilic bacterium Thermus aquaticus have been assembled in vitro from ribosomal proteins and either natural or in vitro-transcribed 23S rRNA and 5S rRNA. Sedimentation properties of reconstituted subunits were similar to those of native ribosomal 50S subunits. Subunits reconstituted with in vitro-transcribed rRNAs exhibited high activity in the peptidyl transferase assay and in a poly(U)-dependent cell-free translation system (22 and 30%, respectively, compared to that of native 50S subunits). Catalytic activity of reconstituted subunits critically depended on the presence of 5S rRNA. rRNA mutations known to affect functions of the native ribosome produced similar effects in reconstituted T. aquaticus 50S subunits. Subunits assembled with in vitro-transcribed T. aquaticus 23S rRNA containing the G2267A mutation (G2252A in Escherichia coli), which interferes with binding of peptidyl-tRNA in the ribosomal P-site, showed drastically reduced peptidyl transferase activity, whereas clindamycin resistance mutation A2084G (A2058G in E. coli) rendered assembled subunits tolerant to clindamycin inhibition. Thus, reconstitution of functional subunits with in vitro-transcribed rRNA makes possible the use of in vitro genetics for mutational analysis of 23S rRNA functions in translation. In addition, the ability to assemble catalytically active 50S subunits from the rRNA transcript lacking any posttranscriptional modifications clearly demonstrates that modified nucleotides in 23S rRNA are dispensable for the principal activities of the ribosome.

Multiple functions of the transcribed spacers in ribosomal RNA operons.

rRNA operons contain about 25% transcribed spacer sequences in addition to the 16S, 23S, 5S and tRNA genes. The spacer sequences are removed from the primary rRNA transcript by a series of co-ordinated nucleolytic events. Besides the role in rRNA processing, the spacer sequences are also involved in transcription and the ribosome assembly. In this study we analyze the spacer between tRNA and 23S rRNA genes. Based on computer modeling and chemical probing data, a model for the transient secondary structure of the intergenic spacer is proposed. Mutational analysis has shown that the transient secondary structure around the 5' end of 23S rRNA is involved in ribosome assembly. We propose that the transient structure at the 5' end of 23S rRNA directs 23S rRNA folding into the mature structure and facilitates ribosomal large subunit assembly.

Erythromycin resistance peptides selected from random peptide libraries.

Translation of a 5-codon mini-gene encoded in Escherichia coli 23 S rRNA was previously shown to render cells resistant to erythromycin (Tenson, T., DeBlasio, A., and Mankin, A. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5641-5646). Erythromycin resistance was mediated by a specific interaction of the 23 S rRNA-encoded pentapeptide with the ribosome. In the present study, peptides conferring erythromycin resistance were selected from in vivo expressed random peptide libraries to study structural features important for peptide activity. Screening of a 21-codon mini-gene library (the general structure ATG (NNN)20 TAA) demonstrated that only short peptides (3-6 amino acids long) conferred erythromycin resistance. Sequence comparison of erythromycin resistance peptides isolated from the 5-codon library (ATG (NNN)4 TAA) revealed a strong preference for leucine or isoleucine as a third amino acid and a hydrophobic amino acid at the C terminus of the peptide. When tested against other antibiotics, erythromycin resistance peptides rendered cells resistant to other macrolides, oleandomycin and spiramycin, but not to chloramphenicol or clindamycin. Defining the consensus amino acid sequence of erythromycin resistance peptides provided insights into a possible mode of peptide action and the nature of the peptide binding site on the ribosome.

Analysis of the ribosome large subunit assembly and 23 S rRNA stability in vivo.

The ability of mutant 23 S ribosomal RNA to form particles with proteins of the large ribosomal subunit in vivo was studied. A series of overlapping deletions covering the entire 23 S rRNA, were constructed in the plasmid copy of an E. coli 23 S rRNA gene. The mutant genes were expressed in vivo using an inducible tac promoter. Mutant species of 23 S rRNA, containing deletions between positions 40 and 2773, were incorporated into stable ribonucleoprotein particles. In contrast, if one end of the 23 S rRNA was deleted, the mutant rRNA was unstable and did not form ribosomal particles. Protein composition of the mutant particles was specific; the presence of the primary rRNA-binding proteins corresponded to their known binding sites. Furthermore, several previously unknown ribosomal protein binding sites in 23 S rRNA were identified. Implications of the results on ribosome assembly are discussed.

A functional peptide encoded in the Escherichia coli 23S rRNA.

A pentapeptide open reading frame equipped with a canonical ribosome-binding site is present in the Escherichia coli 23S rRNA. Overexpression of 23S rRNA fragments containing the mini-gene renders cells resistant to the ribosome-inhibiting antibiotic erythromycin. Mutations that change either the initiator or stop codons of the peptide mini-gene result in the loss of erythromycin resistance. Nonsense mutations in the mini-gene also abolish erythromycin resistance, which can be restored in the presence of the suppressor tRNA, thus proving that expression of the rRNA-encoded peptide is essential for the resistance phenotype. The ribosome appears to be the likely target of action of the rRNA-encoded pentapeptide, because in vitro translation of the peptide mini-gene decreases the inhibitory action of erythromycin on cell-free protein synthesis. Thus, the new mechanism of drug resistance reveals that in addition to the structural and functional role of rRNA in the ribosome, it may also have a peptide-coding function.

Mutations in domain II of 23 S rRNA facilitate translation of a 23 S rRNA-encoded pentapeptide conferring erythromycin resistance.

Mutations in domain II of Escherichia coli 23 S rRNA that cause resistance to erythromycin do so in a manner fundamentally different from mutations at the drug binding site in domain V of the 23 S rRNA. The domain II mutations are located in a hairpin structure between nucleotides 1198 and 1247. This is close to a short open reading frame in the 23 S rRNA that encodes a pentapeptide (E-peptide) whose expression in vivo renders cells resistant to erythromycin. Therefore, a possible mechanism of resistance caused by domain II mutations may be related to an increased expression of the E-peptide. To test this hypothesis, a range of point mutations was generated in domain II of 23 S rRNA in the vicinity of the E-peptide open reading frame. We find a correlation between erythromycin resistance of the mutant clones and increased accessibility of the ribosome binding site of the E-peptide gene. Furthermore, the erythromycin resistance determinant in the mutants was shown to be confined to a small 23 S rRNA segment containing the coding region and the ribosome binding site of the E-peptide open reading frame. It thus appears that the domain II mutations mediate erythromycin resistance by increasing expression of the 23 S rRNA-encoded E-peptide.

Comparison of functional peptide encoded in the Escherichia coli 23S rRNA with other peptides involved in cis-regulation of translation.

A new approach for studying functional rRNA fragments has been developed based on using a plasmid library expressing random fragments of rRNA. A 34 nucleotide long fragment of Escherichia coli 23S rRNA has been identified that renders cells resistant to erythromycin, when expressed in vivo. The rRNA fragment contains a five codon long open reading frame, initiating at GUG and terminating at UAA, with a Shine-Dalgarno sequence located at an appropriate distance from the initiator codon. Translation of this mini-gene is required for the observed erythromycin resistance. Experiments with in vitro translated, or synthetic, peptide indicate the ribosome as a likely target for the action of the identified rRNA-encoded peptide, which apparently remains associated with the ribosome after completion of its translation. The known properties of the rRNA-encoded peptide are compared with information about other functionally active short peptides that can be involved in regulation of translation.