The role of elongation factor EF-P in translation and in copy number control of the transcriptional regulator CadC in Escherichia coli

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.