Quasi-essentiality of RNase Y inBacillus subtilisis caused by its critical role in the control of mRNA homeostasis

Abstract

RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase -α, β, β′. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the β or β' subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed massive decreases in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.If the continued activity of a protein may become harmful to the bacteria, it is important not only to prevent expression of the corresponding gene but also to take two important measures: (i) switch off the protein's activity and (ii) degrade the mRNA to exclude further production of the protein. The inactivation or even degradation of proteins is well documented in the model bacteria. For example, in both E. coli and B. subtilis the uptake of toxic ammonium is limited by a regulatory interaction of the ammonium transporter with GlnK, a regulatory protein of the PII family (1,2). Similarly, the uptake of potentially toxic potassium can be prevented by inhibition of potassium transporters at high environmental potassium concentrations, either by the second messenger cyclic di-AMP or by interaction with a dedicated modified signal transduction protein, PtsN (3,4,5). To prevent the accumulation of potentially harmful mRNAs, bacteria rely on a very fast mRNA turnover. Indeed, in E. coli and B. subtilis more than 80% of all transcripts have average half-lives of less than 8 minutes, as compared to about 30 minutes and 10 hours in yeast or human cells, respectively (6,7,8,9). Thus, the mRNA turnover is much faster than the generation time. The high mRNA turnover rate in bacteria contributes to the fast adaptation even in rapidly growing cells. The rapid mRNA turnover is therefore a major factor to resolve the apparent growth speed-adaptation trade-off. 4RNases are the key elements to achieve the rapid mRNA turnover in bacteria. Theses enzymes can degrade bulk mRNA in a rather unspecific manner, just depending on the accessibility of the RNA molecules as well as perform highly specific cleavages that serve to process an RNA molecule to its mature form. In all organisms, RNA degradation involves an interplay ...

The Bacillus phage SPβ and its relatives: a temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components

“…Of the SPβ conserved genes, five genes were transcriptionally active, yopR , yopQ , yopP , aimR and yokI (Table S5). The second data set from Popp and colleagues (Popp et al ., 2020) and the third data set from Benda and colleagues (Benda et al ., 2021) were up‐to‐date Illumina RNA‐seq data sets. They were generated as control experiments from cells cultured in LB‐medium at 37°C at vigorous shaking.…”

“…(Popp et al ., 2020) and Benda et al . (Benda et al ., 2021). Raw transcriptome sequence reads were downloaded from the SRA archive using fastq‐dump from sra‐tools (https://github.com/ncbi/sra-tools).…”

TheBacillusphage SPβ and its relatives: A temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components

“…Of the SPβ conserved genes, five genes were transcriptionally active, yopR, yopQ, yopP, aimR and yokI (Table S5). The second data set from Popp and colleagues (Popp et al, 2020) and the third data set from Benda and colleagues (Benda et al, 2021) were up-to-date Illumina RNA-seq data sets. They were generated as control experiments from cells cultured in LB-medium at 37 °C at vigorous shaking.…”

“…Locus tags of genes presented in bold were identified as transcriptionally active on the prophage in microarray experiments (Nicolas et al, 2012). Underlined locus tags were identified as transcriptionally active on the prophage in RNA-seq experiments (Popp et al, 2020;Benda et al, 2021).…”

From annotation to bacterial data models

“…Upon deletion of topA, the bacteria immediately acquire mutations resulting in an increased expression DNA topoisomerase IV which can then take over the role of TopA (Reuß et al, 2019). Similarly, deletion of the rny gene encoding RNase Y results in the acquisition of suppressor mutations that result in a globally reduced level of transcription activity (Benda et al, 2021) and deletion of the unknown gene yqhY triggers mutations that often affect conserved residues of acetyl-CoA synthetase indicating that YqhY prevents a potentially toxic accumulation of malonyl-CoA or of fatty acids (Tödter et al, 2017). Proteins involved in detoxification reactions have recently been recognized as very important for cellular metabolism as they remove harmful by-products or intermediates.…”