The tA modification acts as a positive determinant for the anticodon nuclease PrrC, and is distinctively nonessential in Streptococcus mutans.

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 in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (tA) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using tA-deficient yeast derivatives, it is shown that tA is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the tA 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 tA. However, we describe here a novel and a more sensitive hybridization-based tA detection method (compared to HPLC) that showed tA 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 tA synthesis genes (tsaB and tsaC) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of tA modification ratios and of tA synthesis genes mRNA levels in S. mutans suggest they may be regulated by growth phase.