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

Abstract

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.

Using spectral matching to interpret LC‐MS/MS data during RNA modification mapping

“…The synthesis of t 6 A involves the production of l ‐threonylcarbamoyladenylate (TC‐AMP) by the enzyme TsaC or TsaC2. TC‐AMP is added to adenosine 37 by the threonylcarbamoyltransferase complex, which is composed of the three subunits TsaD, TsaB, and TsaE . To elucidate which subunit is essential for the t 6 A37 modification of tRNA Ile GAU in the bacterium S mutans , gene deletions of TsaC, TsaB, and TsaE were prepared, and total tRNA was analyzed by LC‐MS/MS.…”

“…Cryptococcus neoformans was cultured in potato‐dextrose media (pH 5.5). Wild‐type and mutant strains of S mutans were cultured as described . Total tRNA was isolated as previously described …”

Transfer RNA function and evolution

“…These processes include lipid aminoacylation [9] and bacterial conjugation [10]. The contributions to the special issue in this area also focused on the role of tRNA modifications in wobble decoding [11] and bacterial cell death [12].…”

“…Mature tRNAs are rich in post-transcriptional nucleotide base modifications. The modifications have important roles in translation fidelity and reading frame maintenance [12], tRNA stability and transport [14]. Modifications also occur in the anticodon.…”

“…The threonylcarbamoyladenosine (t6A) modification at position 37 is found in most tRNAs that decode ANN codons and it is important for stabilizing the codon anticodon duplex. The t6A modification at position 37, which is adjacent to the anticodon loop, was found to be an essential determinant for the activity of the anticodon nuclease PrrC from Streptococcus mutans [12]. The nuclease promotes bacterial cell death in conditions of stress or upon phage infection when it specifically cleaves the tRNA Lys UUU anticodon loop.…”