Mutations compensating for the fitness cost of rifampicin resistance in Escherichia coli exert pleiotropic effect on RNA polymerase catalysis.

The spread of drug-resistant bacteria represents one of the most significant medical problems of our time. Bacterial fitness loss associated with drug resistance can be counteracted by acquisition of secondary mutations, thereby enhancing the virulence of such bacteria. Antibiotic rifampicin (Rif) targets cellular RNA polymerase (RNAP). It is potent broad spectrum drug used for treatment of bacterial infections. We have investigated the compensatory mechanism of the secondary mutations alleviating Rif resistance (Rifr) on biochemical, structural and fitness indices. We find that substitutions in RNAP genes compensating for the growth defect caused by ßQ513P and ßT563P Rifr mutations significantly enhanced bacterial relative growth rate. By assaying RNAP purified from these strains, we show that compensatory mutations directly stimulated basal transcriptional machinery (2-9-fold) significantly improving promoter clearance step of the transcription pathway as well as elongation rate. Molecular modeling suggests that compensatory mutations affect transcript retention, substrate loading, and nucleotidyl transfer catalysis. Strikingly, one of the identified compensatory substitutions represents mutation conferring rifampicin resistance on its own. This finding reveals an evolutionary process that creates more virulent species by simultaneously improving the fitness and augmenting bacterial drug resistance.

Higher Concentrations of Bacterial Enveloped Virus Phi6 Can Protect the Virus from Environmental Decay.

Phage Phi6 is an enveloped virus considered a possible nonpathogenic surrogate for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viral pathogens in transmission studies. Larger input amounts of bacteriophage Phi6 are shown to delay and protect the phage from environmental decay, both when the phages are dried in plastic tubes and when they are stored in saline solution at 4°C. In contrast, when bacteriophage Phi6 is placed in LB (Luria-Bertani) growth medium (instead of saline) prior to placement on the plastic surface, the influence of the starting concentration on viral recovery is negligible. Protection is reflected in the phage half-lives at higher concentrations being longer than the half-lives at lower concentrations. Because experiments supporting the possibility of fomite transmission of SARS-CoV-2 and other viruses rely upon the survival of infectious virus following inoculation onto various surfaces, large initial amounts of input virus on a surface may generate artificially inflated survival times compared to realistic lower levels of virus that a subject would normally encounter. This is not only because there are extra half-lives to go through at higher concentrations but also because the half-lives themselves are extended at higher virus concentrations. It is important to design surface drying experiments for pathogens with realistic levels of input virus and to consider the role of the carrier and matrix if the results are to be clinically relevant. IMPORTANCE During the coronavirus disease 2019 (COVID-19) pandemic, much attention has been paid to the environmental decay of SARS-CoV-2 due to the proposed transmission of the virus via fomites. However, published experiments have commenced with inocula with very high virus titers, an experimental design not representative of real-life conditions. The study described here evaluated the impact of the initial virus titer on the environmental decay of an enveloped virus, using a nonpathogenic surrogate for the transmission of SARS-CoV-2, enveloped bacteriophage Phi6. We establish that higher concentrations of virus can protect the virus from environmental decay, depending on conditions. This has important implications for stability studies of SARS-CoV-2 and other viruses. Our results point to a limitation in the fundamental methodology that has been used to attribute fomite transmission for almost all respiratory viruses.

Key features of magnesium that underpin its role as the major ion for electrophilic biocatalysis.

To investigate divalent metal ion (Me2+ ) requirements in electrophilic biocatalysis, we compared Mg2+ , Mn2+ , Co2+ , Zn2+ , Cu2+ , Ni2+ , Cd2+ , Ca2+ , and Fe2+ activities with 13 enzymes executing nucleotidyl and/or phosphoryl transfer. We find that each Me2+ ion was highly catalytically active with one or more of the related enzymes. This result suggests that features of Me2+ coordination at the active center, and/or the enzyme-mediated presentation of the reactants to the chelated Me2+ , rather than the nature of the Me2+ , determine the ability of the Me2+ to support catalysis. At physiological pH, all the tested Me2+ ions, with the exception of Mg2+ , produced insoluble complexes with inorganic phosphate (Pi ) and bicarbonate ( HCO3- ). These data suggest that early in the development of life, bioavailability and biocompatibility with these abundant cellular metabolites may have been decisive factors in the choice of Mg2+ as the major ion for biocatalysis. Taking into account the concentrations of inorganic ions in the ancient environment in which the first cells emerged, as inferred from the 'chemistry conservation principle', the choice of Mg2+ was predetermined prior to the origin of life.

Engineered EF-Tu and tRNA-Based FRET Screening Assay to Find Inhibitors of Protein Synthesis in Bacteria.

Antibiotic-resistant infections that do not respond to available drugs are becoming more common. Methicillin-resistant Staphylococcus aureus, carbapenem-resistant enterobacteria ("superbugs"), and many others pose a continuous threat to public health. To provide tools to combat such deadly infections, we present in this study a homogeneous assay focused on an insufficiently addressed molecular interaction linked to ribosomal translation. We show that a fluorescence resonance energy transfer (FRET) based screening assay can identify antibiotic molecules that inhibit ternary complex (EF-Tu:tRNA:GTP complex) formation, and therefore, protein synthesis in bacteria. Specifically engineered Escherichia coli EF-Tu and tRNAPhe are used to prepare two key components of this assay: (1) Cy5-EF-Tu:GTP and (2) Cy3-Phe-tRNAPhe. When mixed and Cy3 is excited at 532 nm, increased Cy5 fluorescence intensity is observed at 665 nm due to ternary complex formation and FRET. If the same assay is carried out in presence of an inhibitor, such as GE2270A (a known inhibitor of the EF-Tu-tRNA interaction), fluorescence intensity is significantly diminished. To establish proof of principle and to show the adaptability of this assay to high throughput screening (HTS), we analyzed the effect of different classes of antibiotics, including beta-lactams, quinolone compounds, and protein synthesis inhibitors, on fluorescence. The assay was done in a 96-well microplate. We observed inhibition by GE2270A, and no effect of nineteen other tested antibiotics, confirming the ability of this FRET assay to serve as a screen for potential inhibitor molecules of ternary complex formation from libraries of compounds.

New pyrazolobenzothiazine derivatives as hepatitis C virus NS5B polymerase palm site I inhibitors.

We have previously identified the pyrazolobenzothiazine scaffold as a promising chemotype against hepatitis C virus (HCV) NS5B polymerase, a validated and promising anti-HCV target. Herein we describe the design, synthesis, enzymatic, and cellular characterization of new pyrazolobenzothiazines as anti-HCV inhibitors. The binding site for a representative derivative was mapped to NS5B palm site I employing a mutant counterscreen assay, thus validating our previous in silico predictions. Derivative 2b proved to be the best selective anti-HCV derivative within the new series, exhibiting a IC50 of 7.9 µM against NS5B polymerase and antiviral effect (EC50 = 8.1 µM; EC90 = 23.3 µM) coupled with the absence of any antimetabolic effect (CC50 > 224 µM; SI > 28) in a cell based HCV replicon system assay. Significantly, microscopic analysis showed that, unlike the parent compounds, derivative 2b did not show any significant cell morphological alterations. Furthermore, since most of the pyrazolobenzothiazines tested altered cell morphology, this undesired aspect was further investigated by exploring possible perturbation of lipid metabolism during compound treatment.

Design and properties of efficient tRNA:EF-Tu FRET system for studies of ribosomal translation.

Formation of the ternary complex between GTP-bound form of elongation factor Tu (EF-Tu) and aminoacylated transfer RNA (aa-tRNA) is a key event in protein biosynthesis. Here we show that fluorescently modified Escherichia coli EF-Tu carrying three mutations, C137A, C255V and E348C, and fluorescently modified Phe-tRNA(Phe) form functionally active ternary complex that has properties similar to those of the naturally occurring (unmodified) complex. Similarities include the binding and binding rate constants, behavior in gel retardation assay, as well as activities in tRNA protection and in vitro translation assays. Proper labeling of EF-Tu was demonstrated in MALDI mass spectroscopy experiments. To generate the mutant EF-Tu, a series of genetic constructions were performed. Two native cysteine residues in the wild-type EF-Tu at positions 137 and 255 were replaced by Ala and Val, respectively, and an additional cysteine was introduced either in position 324 or 348. The assembly FRET assay showed a 5- to 7-fold increase of Cy5-labeled EF-Tu E348C mutant fluorescence upon formation of ternary complex with charged tRNA(Phe)(Cy3-labeled) when the complex was excited at 532 nm and monitored at 665 nm. In a control experiment, we did not observe FRET using uncharged tRNA(Phe)(Cy3), nor with wild-type EF-Tu preparation that was allowed to react with Cy5 maleimide, nor in the absence of GTP. The results obtained demonstrate that the EF-Tu:tRNA FRET system described can be used for investigations of ribosomal translation in many types of experiments.