Subject(s)
Antigens, Viral , COVID-19 Testing , COVID-19/diagnosis , Direct-To-Consumer Screening and Testing/methods , Mass Screening/methods , SARS-CoV-2 , Adult , Aged , COVID-19/virology , Female , Humans , Male , Middle Aged , SARS-CoV-2/immunology , Self-Testing , Sensitivity and Specificity , Young AdultABSTRACT
The C-terminal regions (CT) of Pfam PF04740 proteins share significant sequence identity with the toxic CdiA-CT effector domains of contact-dependent growth inhibition (CDI) systems. In accord with this homology, we find that several PF04740 CT domains inhibit cell growth when expressed in Escherichia coli. This growth inhibition is specifically blocked by antitoxin proteins encoded downstream of each PF04740 gene. The YobL-CT, YxiD-CT and YqcG-CT domains from Bacillus subtilis 168 have cytotoxic RNase activities, which are neutralized by the binding of cognate YobK, YxxD and YqcF antitoxin proteins, respectively. Our results show that PF04740 proteins represent a new family of toxin/antitoxin pairs that are widely distributed in Gram-positive bacteria.
Subject(s)
Antitoxins/genetics , Bacillus/genetics , Bacterial Toxins/genetics , Amino Acid Sequence , Antitoxins/chemistry , Antitoxins/metabolism , Bacillus/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Molecular Sequence Data , Multigene Family , Protein Binding , Protein Interaction Domains and Motifs/physiology , Ribonucleases/metabolism , Sequence Homology, Amino AcidABSTRACT
Translational pausing in Escherichia coli can lead to mRNA cleavage within the ribosomal A-site. A-site mRNA cleavage is thought to facilitate transfer-messenger RNA (tmRNA).SmpB- mediated recycling of stalled ribosome complexes. Here, we demonstrate that the aminoglycosides paromomycin and streptomycin inhibit A-site cleavage of stop codons during inefficient translation termination. Aminoglycosides also induced stop codon read-through, suggesting that these antibiotics alleviate ribosome pausing during termination. Streptomycin did not inhibit A-site cleavage in rpsL mutants, which express streptomycin-resistant variants of ribosomal protein S12. However, rpsL strains exhibited reduced A-site mRNA cleavage compared with rpsL(+) cells. Additionally, tmRNA.SmpB-mediated SsrA peptide tagging was significantly reduced in several rpsL strains but could be fully restored in a subset of mutants when treated with streptomycin. The streptomycin-dependent rpsL(P90K) mutant also showed significantly lower levels of A-site cleavage and tmRNA.SmpB activity. Mutations in rpsD (encoding ribosomal protein S4), which suppressed streptomycin dependence, were able to partially restore A-site cleavage to rpsL(P90K) cells but failed to increase tmRNA.SmpB activity. Taken together, these results show that perturbations to A-site structure and function modulate A-site mRNA cleavage and tmRNA.SmpB activity. We propose that tmRNA.SmpB binds to streptomycin-resistant rpsL ribosomes less efficiently, leading to a partial loss of ribosome rescue function in these mutants.
