The t⁶A modification acts as a positive determinant for the anticodon nuclease PrrC, and is distinctively...

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. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t⁶A) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using t⁶A-deficient yeast derivatives, it is shown that t⁶A is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the t⁶A synthesis gene tsaE (brpB) is dispensable and its genome encodes a PrrC toxin. We had previously shown using an HPLC-based assay that the S. mutans tsaE 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 S. mutans ΔtsaE, albeit at greatly reduced levels (93% reduced compared with WT). Moreover, mutants in 2 other S. mutans t⁶A synthesis genes (tsaB and tsaC) 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 mRNA levels in S. mutans suggest they may be regulated by growth phase.