S1: A puzzling factor in the stress response of E. coli?

Recently, we have identified a novel post-transcriptional stress response mechanism in Escherichia coli. It is based on the functional specialisation of ribosomes by the stress-induced endoribonucleolytic activity of MazF, the toxin component of the toxin-antitoxin system mazEF. Briefly, the stress-responsive proteolysis of the labile antitoxin MazE results in the activation of MazF. Surprisingly, the endoribonuclease MazF targets the 16S rRNA in the context of the ribosome and removes its 3-terminal region harbouring the anti-Shine-Dalgarno sequence. Consequently, the modified ribosomes exclusively translate leaderless mRNAs that are devoid of a 5-untranslated region and hence lack the Shine-Dalgarno sequence. As the MazF activity concomitantly leads to the generation of distinct leaderless transcripts, the translational program is modulated to adapt protein synthesis to the given conditions. Interestingly, we identified the rpsA mRNA, which encodes ribosomal protein S1, to be a target of the endoribonuclease MazF during our subsequent determination of the leaderless mRNA regulon, i.e. the pool of transcripts that are processed by the MazF toxin and selectively translated. Protein S1 is a peculiar ribosomal protein. In Escherichia coli and most likely all Gram-negative bacteria S1 is pivotal for translation initiation. The multi-domain protein binds to the ribosome via protein-protein interaction and recruits mRNAs to the translational machinery by interacting with their structured 5-untranslated regions. Surprisingly, the processing of the rpsA mRNA by MazF leads to the synthesis of two fragments of protein S1, namely the N-terminal peptide S1222 comprising domains D1, D2 and part of domain D3, and the C-terminal peptide S1MazF consisting of the C- terminal half of domain D3 and domains D4-D6. With respect to the functional characteristics of the different S1 domains, in the frame work of this project proposal we will address the physiological functions of the distinct protein S1 fragments during stress. In addition, this observation raises the intriguing fundamental question whether the MazF- mediated response mechanism has the potential to enhance the transcriptome and proteome diversity. This activity is conceptually related to the mechanism of alternative splicing in eukaryotes, where it significantly enhances the biodiversity of proteins encoded by the genome. Hitherto, such a mechanism is completely unprecedented in bacteria. Hence, in a pilot study we will scrutinize this audacious theory, which has the potential to reveal an entirely novel paradigm for alternative post-transcriptional control in bacteria.

Bacteria frequently encounter changes in environmental conditions, e.g. shifts in temperature or pH, oxidative and osmotic stress or nutrient deprivation. In particular, pathogenic bacteria are subjected to rapidly changing environments when they enter their host. To survive, bacteria have developed several strategies to adapt gene expression and protein activity to the given conditions. Besides, some bacteria are able to form persister cells, which are characterized by a dormant phenotype, enabling the bacteria to sustain antibiotic treatment as well as the host immune response. Recent studies implicated toxin-antitoxin (TA) modules in establishment of persistent infections. The stress-responsive activation of these modules blocks several important processes in the cell, like translation and replication. In our previous studies we described a novel post-transcriptional stress response pathway that is triggered by the TA-system mazEF. Besides degrading the majority of ribonucleases (RNAs) the active endoribonuclease MazF cleaves at specific RNA sequence motives thereby initiating the degradation of the majority of RNAs. However, some RNAs as well as the ribosomal RNA within the translational machinery are processed by MazF. Thereby functionally important regions are removed. Consequently, this subpopulation of ribosomes is formed that selectively translate the mRNAs processed by MazF. Together, this results in a fast and energy-efficient reprogramming of protein synthesis. In the framework of this project we were able to show that this mechanism has the potential to trigger further regulatory pathways in bacteria. The activity of the toxin in concert with an additional factor required for alternative termination of translation can lead to the synthesis of short protein isoforms with regulatory roles. This mechanism represents an entirely novel paradigm for an alternative translational control mechanism in prokaryotes. We were able to obtain evidence that bacteria do have the means to enhance the transcriptome and proteome diversity by stress-responsive RNA processing coupled with an alternative ribosome rescue mechanism. Together, this novel activity that is conceptually related to alternative splicing in eukaryotes significantly enhances the biodiversity of proteins that can be encoded by the bacterial genome and thus opens a novel level of complexity in gene regulation in bacteria.