A possible functional link between RNA degradation and transcription in Bacillus subtilis

Abstract

Cellular levels of RNA depend on the rate of its synthesis and degradation. While synthesis is performed by RNA polymerase conserved in all domains of life, the enzymes responsible for RNA degradation are more unique even among organisms from the same domain. In the best studied bacterium, the gram-negative Escherichia coli, RNA degradation is achieved through a protein complex called RNA degradosome, which is assembled around the essential endoribonuclease RNase E. However, RNase E is not present in the gram-positive model organism Bacillus subtilis. Instead, an enzyme called RNase Y (rny) has been proposed as its functional counterpart responsible for the initiation of RNA degradation. Nevertheless, unlike RNase E of E. coli, it can be deleted from the genome, leaving an open question of its true significance and function. This project was designed to get a deeper understanding of the crucial process of RNA degradation in B. subtilis and of the role RNase Y plays there. Although RNase Y is dispensable for survival, the rny gene deletion leads to detrimental phenotypic effects, including filamentous growth, impaired cellular morphology or defects in the development of genetic competence and sporulation. The rny mutant strain also lyses rapidly and subsequently suppressor colonies appear. Using this natural force of suppressor evolution, we could demonstrate that no other RNase can take over the tasks of RNase Y. Conversely, all identified mutations were aimed to reduce RNA synthesis. This was achieved either by inactivation of transcription factors in conjunction with duplication of core RNA polymerase genes, which results in decreased number of correctly assembled RNA polymerase complexes, or, if the first suppressing mechanism was prevented, by mutations occurring directly in the RNA polymerase core genes, leading to orders of magnitude decrease in transcription. The fact that the mutations always affect RNA synthesis, a process on the opposite side of RNA life to the one RNase Y acts, suggest close collaboration of RNase Y with the RNA polymerase in establishing stable equilibrium between RNA synthesis and degradation. While the suppressor mutant analysis helped to identify the pivotal function of RNase Y, it did not necessarily provide an explanation for all the phenotypes associated with the deletion of the rny gene. In an attempt to better understand such phenotypes, RNA-sequencing analysis revealed global remodeling of gene expression in the rny strain. Furthermore, a screening system to recognize the reasons for the loss of genetic competence was established and helped to decipher the reasons for the loss of competence in the rny mutant as well as in other strains, among them in the ytrA mutant overexpressing putative ABC transporter YtrBCDEF. This was shown to act in remodeling of the cell wall thickness, which hampers development of genetic competence as well as other lifestyles of B. subtilis. The possible influence of a disordered cell wall is also discussed as a potential reason for the loss of competence in the rny mutant