The t⁶A modification acts as a positive determinant for the anticodon nuclease PrrC, and is distinctively nonessential in <i>Streptococcus mutans</i>

Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNA^Lys_UUU in the anticodon loop. <i>In vitro</i> studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t⁶A) is required for PrrC activity but this prediction had never been validated <i>in vivo</i>. Here, by using t⁶A-deficient yeast derivatives, it is shown that t⁶A is a positive determinant for PrrC proteins from various bacterial species. <i>Streptococcus mutans</i> is one of the few bacteria where the t⁶A synthesis gene <i>tsaE</i> (<i>brpB</i>) is dispensable and its genome encodes a PrrC toxin. We had previously shown using an HPLC-based assay that the <i>S. mutans tsaE</i> mutant was devoid of t⁶A. However, we describe here a novel and a more sensitive hybridization-based t⁶A detection method (compared to HPLC) that showed t⁶A was still present in the <i>S. mutans</i> δ<i>tsaE</i>, albeit at greatly reduced levels (93% reduced compared to WT). Moreover, mutants in two other <i>S. mutans</i> t⁶A synthesis genes (<i>tsaB</i> and <i>tsaC</i>) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of t⁶A modification ratios and of t⁶A synthesis genes mRNAs levels in <i>S. mutans</i> suggest they may be regulated by growth phase.