RESUMO
Adenosine-to-inosine (A-to-I) RNA editing plays an important role in the post-transcriptional regulation of eukaryotic cell physiology. However, our understanding of the occurrence, function and regulation of A-to-I editing in bacteria remains limited. Bacterial mRNA editing is catalysed by the deaminase TadA, which was originally described to modify a single tRNA in Escherichia coli. Intriguingly, several bacterial species appear to perform A-to-I editing on more than one tRNA. Here, we provide evidence that in the human pathogen Streptococcus pyogenes, tRNA editing has expanded to an additional tRNA substrate. Using RNA sequencing, we identified more than 27 editing sites in the transcriptome of S. pyogenes SF370 and demonstrate that the adaptation of S. pyogenes TadA to a second tRNA substrate has also diversified the sequence context and recoding scope of mRNA editing. Based on the observation that editing is dynamically regulated in response to several infection-relevant stimuli, such as oxidative stress, we further investigated the underlying determinants of editing dynamics and identified mRNA stability as a key modulator of A-to-I editing. Overall, our findings reveal the presence and diversification of A-to-I editing in S. pyogenes and provide novel insights into the plasticity of the editome and its regulation in bacteria.
RESUMO
Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. To overcome this limitation and enable the straightforward investigation of gene functions in S. pyogenes, we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for S. pyogenes. The new replicative plasmids feature low- and high-copy replicons combined with different resistance cassettes and a standardized multiple cloning site for rapid cloning procedures. We designed site-specific integrative plasmids and verified their integration by nanopore sequencing. To minimize the effect of plasmid integration on bacterial physiology, we screened publicly available RNA-sequencing datasets for transcriptionally silent sites. We validated this approach by designing the integrative plasmid pSpy0K6 targeting the transcriptionally silent gene SPy_1078. Analysis of the activity of different constitutive promoters indicated a wide variety of strengths, with the lactococcal promoter P 23 showing the strongest activity and the synthetic promoter P xylS2 showing the weakest activity. Further, we assessed the functionality of three inducible regulatory elements including a zinc- and an IPTG-inducible promoter as well as an erythromycin-inducible riboswitch that showed low-to-no background expression and high inducibility. Additionally, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. We therefore adapted the chemically defined medium called RPMI4Spy that showed reduced autofluorescence and enabled efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS*, which enabled the scarless deletion of the sagB gene. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.