INTRODUCTION
Ribosomal biogenesis is a highly regulated process encompassing
concomitant transcription, processing, degradation, modification and
folding of ribosomal RNAs, equimolar synthesis, and incorporation into
ribosomes of more than 50 different ribosomal proteins (Davis and
Williamson, 2017). In bacteria, this is catalyzed, chaperoned and
generally facilitated by dozens of dedicated proteins working in tandem
in several partially overlapping and redundant pathways (Shajaniet al. , 2011). However, due to its sheer complexity, our
understanding of this process is bounded to isolated fragments of
processing/folding pathways, with minimal knowledge of many individual
factors’ precise mechanisms of action.
It has long been known that Mg2+ is necessary for
ribosomal assembly and translation (McCarthy et al. , 1962). More
recently, it was discovered that intracellular free
Mg2+ and rRNA transcription are actively co-regulated
for achieving optimal ribosomal assembly and translation (Ponteset al. , 2016). Also, it has been shown that
Mg2+ influx can provide an active mechanism to
alleviate ribosomal stress phenotypes, probably by stabilizing ribosomal
structure (Lee et al. , 2019).
ybeX encodes a putative
Co2+/Mg2+ efflux protein, which is
highly conserved in bacteria but poorly characterized (Kazakov et
al. , 2003; Anantharaman and Aravind, 2003). In the genome of E.
coli , it is located in the ybeZYX-Int operon (Fig. 1A ),
transcripts of which have not been fully mapped. The lnt gene,
which encodes an essential inner membrane
protein, is predicted to be under
the control of the minor heat shock sigma factor σ24(RpoE) (Keseler et al. , 2013), while transcription ofybeY , ybeZ and ybeX is regulated by the primary
heat shock sigma factor σ32 (RpoH) (Nonaka et
al. , 2006). In low-magnesium conditions, the levels of YbeX (but not of
YbeY and YbeZ) mRNA and protein are about two-fold reduced, consistently
with its proposed role in Mg+2 efflux (Caglar et
al. , 2017).
The most-studied member of the ybeZYX-lnt operon is theybeY , whose importance in ribosomal metabolism is beyond dispute,
while the precise mode of action of YbeY remains unclear (Davieset al. , 2010). The YbeY is, by sequence homology and structural
studies, a zinc-dependent RNA endonuclease. YbeY is universally
conserved over the three domains of life, has very strong, albeit
heterogeneous, phenotypes in every organism that has been looked into,
and it has been shown by genetical methods to be required for the
correct processing of the 3’ end of 16S rRNA (Liao et al. , 2021).
Moreover, YbeY mutants have been shown to be defective in translation
and accumulate defective ribosomes in several bacterial species,
mitochondria and chloroplasts (Liu et al. , 2015; Liao et
al. , 2021; D’Souza et al. , 2021). And yet, in the purified form,
its RNase activity seems to be limited to short RNA oligonucleotides
(Jacob et al. , 2013; Babu et al. , 2020), while in
vitro processing of the 16S rRNA 3’-end can be achieved without it
(Smith et al. , 2018).
The ybeZ gene is located upstream of ybeY , having four
nucleotides overlap. ybeZ encodes a phosphate
starvation-regulated PhoH subfamily protein with the NTP hydrolase
domain (Kim et al. , 1993). YbeZ has phosphatase activity and is a
putative RNA helicase through sequence homology (Kazakov et al. ,
2003; Andrews and Patrick, 2022). A physical interaction between YbeY
and YbeZ was suggested based on bacterial two-hybrid system experiments
in E. coli (Vercruysse et al. , 2016). Their interaction
has been biochemically verified in Pseudomonas aeruginosa (Xiaet al. , 2020).
Here we characterise the growth and ribosomal homeostasis phenotypes of
the deletion of ybeX in Escherichia coli .