Abstract
The formation of translationally inactive 70S dimers (called 100S ribosomes) by hibernation-promoting factor is a widespread survival strategy among bacteria. Ribosome dimerization is thought to be reversible, with the dissociation of the 100S complexes enabling ribosome recycling for participation in new rounds of translation. The precise pathway of 100S ribosome recycling has been unclear. We previously found that the heat-shock GTPase HflX in the human pathogen Staphylococcus aureus is a minor disassembly factor. Cells lacking hflX do not accumulate 100S ribosomes unless they are subjected to heat exposure, suggesting the existence of an alternative pathway during nonstressed conditions. Here, we provide biochemical and genetic evidence that two essential translation factors, ribosome-recycling factor (RRF) and GTPase elongation factor G (EF-G), synergistically split 100S ribosomes in a GTP-dependent but tRNA translocation-independent manner. We found that although HflX and the RRF/EF-G pair are functionally interchangeable, HflX is expressed at low levels and is dispensable under normal growth conditions. The bacterial RRF/EF-G pair was previously known to target only the post-termination 70S complexes; our results reveal a new role in the reversal of ribosome hibernation that is intimately linked to bacterial pathogenesis, persister formation, stress responses, and ribosome integrity.
Highlights
The formation of translationally inactive 70S dimers by hibernation-promoting factor is a widespread survival strategy among bacteria
Native 100S ribosomes were isolated to Ն70% homogeneity from methicillin-resistant S. aureus (MRSA) USA300 through a two-step density gradient fractionation and ultracentrifugation protocol
More than 50% of the 100S ribosomes were converted into 70S monomers in the presence of ribosome-recycling factor (RRF), elongation factor G (EF-G), and GTP or HflX and GTP
Summary
The formation of translationally inactive 70S dimers (called 100S ribosomes) by hibernation-promoting factor is a widespread survival strategy among bacteria. Ectopic expression of HflX rescues the growth defect of an RRF-depleted strain, indicating functional redundancy of these factors. Consistent with these findings, the V116D mutant allele fully complemented the growth defect of an RRF-depleted strain (Fig. 3D).
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