Light Activation of Staphylococcus aureus Toxin YoeB_Sa1 Reveals Guanosine-Specific Endoribonuclease Activity

Abstract

The Staphylococcus aureus chromosome harbors two homologues of the YefM-YoeB toxin-antitoxin (TA) system. The toxins YoeBSa1 and YoeBSa2 possess ribosome-dependent ribonuclease (RNase) activity in Escherichia coli. This activity is similar to that of the E. coli toxin YoeBEc, an enzyme that, in addition to ribosome-dependent RNase activity, possesses ribosome-independent RNase activity in vitro. To investigate whether YoeBSa1 is also a ribosome-independent RNase, YoeBSa1 was expressed using a novel strategy, and its in vitro RNase activity, sequence specificity, and kinetics were characterized. Y88 of YoeBSa1 was critical for in vitro activity and cell culture toxicity. This residue was mutated to ortho-nitrobenzyl tyrosine (ONBY) via unnatural amino acid mutagenesis. YoeBSa1-Y88ONBY could be expressed in the absence of the antitoxin YefMSa1 in E. coli. Photocaged YoeBSa1-Y88ONBY displayed UV light-dependent RNase activity toward free mRNA in vitro. The in vitro ribosome-independent RNase activity of YoeBSa1-Y88ONBY, YoeBSa1-Y88F, and YoeBSa1-Y88TAG was significantly reduced or abolished. In contrast to YoeBEc, which cleaves RNA at both adenosine and guanosine with a preference for adenosine, YoeBSa1 cleaved mRNA specifically at guanosine. Using this information, a fluorometric assay was developed and used to determine the kinetic parameters for ribosome-independent RNA cleavage by YoeBSa1.

Identification and characterization of the chromosomal yefM-yoeB toxin-antitoxin system of Streptococcus suis

“…However, only one TA module, SezAT, has been described in S. suis , yet its function has not been demonstrated 24 . The yefM-yoeB module is one of the best studied TA systems and has been described in various bacteria, including E. coli 27 37 , S. pneumoniae 28 29 , M. tuberculosis 30 , S. aureus 31 32 , Staphylococcus equorum 42 and Streptomyces 33 .…”

“…Overproduction of the YoeB toxin in E. coli Top10 and BL21 (DE3) cells both resulted in toxic effects commonly linked to toxin activity. YoeB homologs were identified as endoribonucleases that inhibit translation by cleaving mRNA, either in a ribosome-dependent or -independent manner 32 35 43 . We therefore reasoned that S. suis YoeB inhibits cell growth via a similar mechanism.…”

“…This TA module was first identified in E. coli , as the homolog of the Enterococcus faecium axe-txe TA system 27 . Since then, the yefM-yoeB TA system has been described in many bacterial species, including major pathogens such as S. pneumoniae 28 29 , M. tuberculosis 30 and Staphylococcus aureus 31 32 . Moreover, the yefM-yoeB locus was identified as the first TA system in Streptomyces 33 .…”

RNA Degradation inStaphylococcus aureus: Diversity of Ribonucleases and Their Impact

“…In E. coli , MazF recognizes the 5′ end of ACA and cleaves just before the cytosine (A∧CA with “∧” represents the cleavage site), whereas in S. aureus , SaMazF cleaves inside a five-base sequence U∧ACAU [ 86 , 87 ]. Up to now, three TA systems exhibiting RNase activity were described in S. aureus , SaMazE/F, SaPemI/K, and YefM-YoeB [ 88 – 91 ].…”

“…The last system, YefM-YoeB, has a ribosome-dependent RNase activity. The toxin binds the A site of the 50S ribosomal subunit and then cleaves the mRNA three base pairs after the start codon [ 91 ]. In addition, SaYoeB exhibits a ribosome-independent RNase activity in vitro by cleaving free mRNA consistent with that previously observed in E. coli [ 91 ].…”