The (p)ppGpp-binding GTPase Era promotes rRNA processing and cold shock survival in Staphylococcus aureus

Abstract

Ribosome assembly cofactors are widely conserved across all domains of life. One such group, the ribosome-associated GTPases (RA-GTPase), act as molecular switches to coordinate ribosome assembly. We previously identified the Staphylococcus aureus RA-GTPase Era as a target for the stringent response alarmone (p)ppGpp, although the function of Era in ribosome assembly is unclear. Era is highly conserved throughout the bacterial kingdom and is essential in many species. Here we show that Era is not essential in S. aureus but is important for 30S ribosomal subunit assembly. Protein interaction studies reveal that Era interacts with the endonuclease YbeY and the DEAD-box RNA helicase CshA. We determine that both Era and CshA are required for growth at suboptimal temperatures, virulence and 16S rRNA processing. Era and CshA also form direct interactions with the (p)ppGpp synthetase RelSA, with RelSA positively impacting the GTPase activity of Era. We propose that Era acts to direct enzymes involved in rRNA maturation and ribosome subunit assembly to their site of action, an activity that is regulated by components of the stringent response. Author summary The bacterial ribosome is an essential cellular component and as such is the target for a number of currently used antimicrobials. Correct assembly of this complex macromolecule requires a number of accessory enzymes, the functions of which are poorly characterised. Here we examine the function of Era, a GTPase enzyme involved in 30S ribosomal subunit biogenesis in the important human pathogen S. aureus. We uncover that Era is not an essential enzyme in S. aureus, as it is in many other species, but is important for correct ribosome assembly. In a bid to uncover the cellular function of this enzyme, we identify a number of protein interaction partners with roles in ribosomal RNA maturation and degradation, supporting the idea that Era acts as an intermediary protein facilitating ribosomal biogenesis. We also uncover a link between Era and the (p)ppGpp synthetase RelSA, revealing an additional level of control of rRNA levels by the stringent response. With this study we elaborate on the functions of GTPases in ribosomal assembly, processes that are controlled at multiple points by the stringent response.