Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018;15(4-5):508-517.
doi: 10.1080/15476286.2017.1353861. Epub 2017 Sep 13.

The T 6 A Modification Acts as a Positive Determinant for the Anticodon Nuclease PrrC, and Is Distinctively Nonessential in Streptococcus Mutans

Affiliations
Free PMC article

The T 6 A Modification Acts as a Positive Determinant for the Anticodon Nuclease PrrC, and Is Distinctively Nonessential in Streptococcus Mutans

Jo Marie Bacusmo et al. RNA Biol. .
Free PMC article

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 tRNALysUUU 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.

Keywords: Modified nucleosides; RNA maturation; t6A detection; translation.

Figures

Figure 1.
Figure 1.
Mapping of nucleoside modifications and targets for oligo probes on tRNA. Modifications on positions 34 and 37 are listed in their respective boxes. Target for ASL probe is depicted in green and target for the TΨC probe is depicted in red.
Figure 2.
Figure 2.
Resistance of t6A deficient yeast strains to PrrC toxins. A. The tcs3Δ is resistant to PrrCEc while mitochondrial tcs4Δ is not. B. tcs8Δ is resistant to both PrrCEc and PrrCSm, while the PrrCEc K46A variant has no effect on growth. Cells were grown in synthetic minimal media containing agar and leucine dropout supplement (SD-Leu). Galactose (2% w/v) and raffinose (1% w/v) were added where necessary (SD-Leu Gal/Raf). Strains were incubated at 30°C for ∼72 hours.
Figure 3.
Figure 3.
PHAt6A assay with yeast and E. coli bulk tRNAs. A. PHAt6A with tRNA isolated from wild type yeast and t6A deficient strains. B. Sensitivity of the PHAt6A method using yeast tRNAs. C. PHAt6A with wild type E. coli and in vitro transcribed tRNAs.
Figure 4.
Figure 4.
Detection of t6A in S. mutans wild-type and mutant strains. A. PHAt6A with S. mutans ΔtsaE. B. Mass spec analysis of t6A modification in tRNAIle GAU showing no t6A in ΔtsaC and ΔtsaB but trace amounts are detected in ΔtsaE. C. PHAt6A with t6A deficient strains ΔtsaC, ΔtsaB, ΔtsaE using oligonucleotides specific to tRNAIle GAU. D. PHAt6A with wild type UA159 and ΔtsaE using probes for tRNAiniMet CAU.
Figure 5.
Figure 5.
Detection of t6A in S. mutans UA159 tRNA grown in Biofilm and Rich Media. PHAt6A of tRNA isolated from the following time points: 4h – Early-exponential, 6h – Mid-exponential, 8h – Late-exponential, and 12h – Stationary phase.
Figure 6.
Figure 6.
S. mutans UA159 gene expression profile for ΔtsaC, ΔtsaB, ΔtsaD, ΔtsaE, and prrC genes during growth in Biofilm and Rich media (BHI). A. Fold change difference of each of the genes with respect to 4h time point (calibrator). * indicates statistical significance (P < 0.05, Student-Newman-Keuls Test) relative to calibrator sample. B. Fold change difference in gene expression at each time point in Biofilm media with respect to Rich media (calibrator). * indicates statistical significance (P < 0.05, Two-tailed T-Test) relative to calibrator sample.

Similar articles

See all similar articles

Cited by 3 articles

Publication types

MeSH terms

LinkOut - more resources

Feedback