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 .