The t6A 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(Lys) (UUU) in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t(6)A) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using t(6)A-deficient yeast derivatives, it is shown that t(6)A is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the t(6)A 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 t(6)A. However, we describe here a novel and a more sensitive hybridization-based t(6)A detection method (compared to HPLC) that showed t(6)A was still present in the S. mutans tsaE, albeit at greatly reduced levels (93% reduced compared with WT). Moreover, mutants in 2 other S. mutans t(6)A synthesis genes (tsaB and tsaC) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of t(6)A modification ratios and of t(6)A synthesis genes mRNA levels in S. mutans suggest they may be regulated by growth phase.