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Ude, Susanne Caroline Margarethe (2013): The role of elongation factor EF-P in translation and in copy number control of the transcriptional regulator CadC in Escherichia coli. Dissertation, LMU München: Fakultät für Biologie



Enterobacteria have evolved several strategies to survive the acidic environment of the gastrointestinal tract. One of the acid stress resistance systems is the Cad system in Escherichia coli, which is induced by low pH and in the presence of external lysine. It consists of CadA, which catalyzes the decarboxylation of lysine to cadaverine, the lysine/cadaverine antiporter CadB and the pH sensing transcriptional regulator CadC. Moreover, the lysine permease LysP inhibits the induction of cadBA expression when lysine is absent, and the small histon-like molecule H-NS acts as repressor for both cadBA and cadC transcription. Additionally, a random mutagenesis approach revealed that a deletion in yjeK leads to highly reduced cadaverine production. YjeK acts as 2,3-lysine aminomutase (LAM) while catalyzing the isomerization of (S)-α-lysine to (R)-β-lysine. The truncated lysyl-tRNA synthetase YjeA uses (R)-β-lysine as substrate to post-translationally modify and to activate the translation elongation factor EF-P at a conserved lysine residue (K34). EF-P and its ortholog eukaryotic initiation factor 5A (eIF5A) have been investigated for more than thirty years, but their roles in translation remained enigmatic. In this work the role of active EF-P in the Cad system was investigated in more detail. Reduced cadBA expression in ΔyjeA, ΔyjeK642-1029 and Δefp mutants was linked to impaired CadC translation. As the translation of cadA and cadB was EF-P independent, a general role of EF-P in translation could be excluded. The identification of CadC as first direct target for EF-P in E. coli allowed further investigations on the role of EF-P in translation. Determining the β-galactosidase activities of CadC´-LacZ translational fusions of increasing CadC length in efp- and efp+ cells revealed that EF-P is required for translation of the sequence found between codon 108 and 158 in cadC. This region comprises a cluster of three consecutive prolines (Pro120-Pro121-Pro122). Substitution of these prolines by alanines diminished EF-P dependency. Remarkably, cells harboring the CadC-PPPIP/AAAIS variant revealed cadBA expression even under non-inducing conditions. Thus, EF-P tightly controls the CadC copy number, which is crucial for stress dependent regulation of the Cad system. In order to investigate the work mechanism of EF-P in more detail, EF-P independent CadC´-LacZ hybrids were employed to artificially introduce prolines. Three consecutive prolines were sufficient for EF-P dependency, regardless of the codon or the context. The proline-rich proteins AmiB, FlhC, Flk, NlpD, RzoR, TonB and UvrB also showed EF-P dependent expression. Thus, the recognition of three consecutive prolines by EF-P is a general mechanism and not limited to CadC. Dr. Agata Starosta of the group of Dr. Daniel Wilson (Gene Center, LMU Munich) confirmed ribosomal stalling at polyproline-stretches in samples lacking EF-P with in vitro translation assays. Finally it was investigated, if EF-P expression and modification could be stress-dependently regulated. In this work first hints are given that the efp promoter contains a repressor site, and that expression of yjeA and yjeK is dependent on the pH of the medium and the presence of the small RNA binding protein Hfq. This leads to the suggestion that small regulatory RNAs are also involved in regulation of the EF-P modification enzymes. In conclusion, the results obtained in this work reveal a new regulatory mechanism by EF-P dependent translation. 100-1000´s of polyproline rich proteins exist in bacteria, archaea and eukaryotes. Therefore, EF-P and its orthologs aIF5A and eIF5A most likely play an important role in the adjustment of copy numbers of proteins with different functions in all kingdoms of life.