We hypothesize that the tRNAs encoded by phages are insensitive to tRNA anticodon nuclease activity, preventing depletion of the tRNA pool during phage infection. To investigate this hypothesis, we analyzed the tRNAs encoded by a large and well-characterized dataset of tRNA-rich bacteriophages (33 tRNAs per phage on average) that infect mycobacteria: mycobacteriophage cluster C1 (Russell & Hatfull, 2017) (Fig. 1A,B). The existence of these tRNA-rich phages coincides with a high abundance of tRNA nucleases (tRNAses), including the well-characterized VapCs, MazFs, and RelEs in Mycobacterium (Winther et al., 2016; Chauhan et al., 2022; Cruz et al., 2015; Cintrón et al., 2019; Barth et al., 2021; Pedersen et al., 2003). A subset of these tRNAses target the tRNA anticodon loop and are activated upon a variety of stress responses, including phage infection (Calcuttawala et al., 2022). When activated, these anticodon nucleases cleave specific tRNAs in conserved regions within the anticodon loop to inactivate these tRNAs and thereby regulate protein translation of the host (Winther et al., 2016). The cleavage region within the tRNA anticodon loop is sequence-dependent and highly specific for the type of tRNA. Mutations within the recognition and cleavage site in the anticodon loop have been found to cause insensitivity to these anticodon nucleases (Winther et al., 2016; Cruz et al., 2015). We compared the tRNAs encoded by phages with those of their host and observed all 10 phage-encoded tRNAs that are known to be targeted by anticodon nucleases to contain anticodon loop mutations (Winther et al., 2016; Cruz et al., 2015; Chauhan et al., 2022), reinforcing the idea that phage-encoded tRNAs are likely insensitive to cleavage (Fig. 1C). We hypothesize that these phage tRNAs represent a means to counteract the depletion of tRNAs by anticodon nucleases during phage infection, allowing the phage to translate its proteins and complete its infection cycle (Fig. 1D).
Supporting the selective pressure of the host-encoded tRNA nucleases, we also observed a strong counter-selection for tRNAs that are cleaved in the anticodon itself (Table S1). This is the case for the majority of the serine-coding tRNAs that are cleaved at the GA site within the anticodon: tRNA-Ser(gga), tRNA-Ser(tga), tRNA-Ser(cga), and tRNA-Ser(aga) (Winther et al., 2016). In this instance, the phage encodes an isoacceptor tRNA that is not targeted (tRNA-Ser(gct)) to carry out translation independent from cleaved serine isoacceptor tRNAs. We observed the same counter-selection for the UAN anticodons, which are known targets of RelE in E. coli (Pedersen et al., 2003). Interestingly, we observed that phage genes do not avoid codons of cleaved tRNAs, nor do they have a preference for codons with nuclease insensitivity (Welch Two Sample t-test, t = 0.53848, df = 41.583, p-value > 0.05), suggesting that the selection of phage tRNAs is only determined by their insensitivity to tRNAses and not by codon usage. Altogether, our observations support the hypothesis that phage tRNAs are selected to be insensitive to anticodon nucleases to counteract tRNA-depletion strategies of the host that limit phage propagation. We expect that our hypothesis may be extended outside of Mycobacteria as phage tRNAs and host tRNAses are widespread (Ogawa et al., 2006; Covard & Lazdunski, 1979; Jones et al., 2017).