INTRODUCTION
Ribosome 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 several
partially overlapping and redundant pathways (Shajani et al. ,
2011). However, due to its sheer complexity, our understanding of this
process relies on 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+ concentrations and rRNA transcription rates are
actively co-regulated for achieving optimal ribosomal assembly and
translation (Pontes et al. , 2016). Also, Mg2+influx can alleviate ribosomal stress phenotypes, probably by
stabilizing the ribosomal structure (Lee et al. , 2019).ybeX encodes a putative
Co2+/Mg2+ efflux protein, which,
while highly conserved in bacteria, is poorly characterized (Kazakovet al. , 2003; Anantharaman and Aravind, 2003). TheybeX /corC gene was initially discovered inSalmonella enterica serovar Typhimurium in a screen for cobalt
resistance and was proposed to contribute, possibly as a co-effector of
the metal transport protein CorA, to the efflux of divalent cations
(Gibson et al. , 1991). Notwithstanding over 97% sequence
similarity between E. coli and SalmonellaybeX /corC , a corresponding study in E. coli is
absent. The levels of YbeX (but not YbeY and YbeZ) mRNA and protein are
about two-fold reduced under low-magnesium conditions (Caglar et
al. , 2017). Intriguingly, as a serendipitous finding, E. colicells that rely for growth on an artificial ribosome variant, where
fused rRNAs covalently tether the subunits, have increased growth rate
caused by a nonsense mutation in the ybeX gene together with a
missense mutation in the ribosomal protein gene rpsA (Orelleet al. , 2015).
In the E. coli genome, ybeX is located in theybeZYX-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 of ybeZ , ybeY , ybeX and lnt is
regulated by the primary heat shock sigma factor σ32(RpoH) (Nonaka et al. , 2006).
The most-studied member of the ybeZYX-lnt operon is theybeY , whose importance in ribosomal metabolism is beyond dispute,
while its precise mode of action remains unclear (Davies et al. ,
2010). The YbeY is, by sequence homology and structural studies, a
zinc-dependent RNA endonuclease. It is extremely highly conserved, has
strong, albeit heterogeneous phenotypes in every organism that has been
looked into, and is required to correctly process the 3‘ end of 16S rRNA
(Liao et al. , 2021). Moreover, ybeY mutants have been
shown to be defective in translation and to 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). In an E. coli interactome study, it has
been observed that YbeZ not only interacts with YbeY but also exhibits
interactions with numerous ribosomal proteins (Butland et al. ,
2005).
In this work, we investigate the effects of ybeX deletion onE. coli growth and ribosomal metabolism.