Formation of 100S ribosomes in Staphylococcus aureus by the hibernation promoting factor homolog SaHPF

Abstract

In the stationary growth phase of Escherichia coli, the 70S ribosomes are dimerized by the ribosome modulation factor (RMF) and hibernation promoting factor (HPF) proteins to form 100S ribosomes, which lose translational activity. In this study we found 100S ribosomes in the gram-positive bacterium Staphylococcus aureus, which has an HPF homolog (named SaHPF) but no RMF homolog. Unlike in E. coli, 100S ribosomes exist in all growth phases of S. aureus, with the highest levels at the transition from the exponential phase to the stationary phase. To find the key factors involved in 100S formation, we analyzed proteins associated with crude ribosomes using radical-free and highly reducing 2-D PAGE and MALDI TOF ⁄ MS. Only the SaHPF levels changed in parallel with the changes in 100S levels. SaHPF bound preferentially to 70S components in 100S ribosomes, with a molar ratio of 1 : 1 relative to the 70S, but some SaHPF was also detected in free 70S ribosomes. High-salt washing of the crude ribosomes released SaHPF and dissociated the 100S ribosomes to their 70S components. When these 70S components were incubated with purified SaHPF in vitro, they re-associated to form 100S. These results suggest that SaHPF is a key protein involved in 100S ribosome formation in S. aureus.

The Listeria monocytogenes Hibernation-Promoting Factor Is Required for the Formation of 100S Ribosomes, Optimal Fitness, and Pathogenesis

“…Preparation and fractionation of ribosome samples were performed using a protocol adapted from Ueta et al (9). Cowpea mosaic virus (CPMV) fragments of 68, 95, and 115S were used as sedimentation standards.…”

“…In Escherichia coli, 100S ribosomes are barely detectable in lysates from rapidly growing bacteria but increase dramatically in abundance upon entry to stationary phase, substantially outnumbering 70S ribosomes in stationary-phase lysates (8). In contrast, 100S ribosomes are abundant in log-phase lysates of the Gram-positive bacterium Staphylococcus aureus, increasing to a peak upon entry to stationary phase and decreasing gradually in quantity over the next several hours of post-exponential-phase growth (discussed further below) (9). Given their presumed role in downregulating translation, the function of 100S ribosomes during logarithmic-phase growth in S. aureus remains unclear.…”

“…However, the functions of ribosome hibernation appear to be diverse and have not yet been fully explored. Although ribosome hibernation in S. aureus is dependent on the nutrient content of the growth medium, 100S ribosomes are present in S. aureus extracts at all phases of growth (9). A recent report on hibernation in Bacillus subtilis established the importance of a long HPF homolog in 100S ribosome formation during amino acid starvation but did not discuss the functional importance of 100S ribosomes (16).…”

Resuscitation of Pseudomonas aeruginosa from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation

“…E. coli and most other gamma Proteobacteria have genes for rmf, hpf, and yfiA, whereas bacteria other than the gamma Proteobacteria lack the gene for rmf. Staphylococcus aureus does not encode rmf, but has an hpf with an extended C-terminal tail, termed long HPF (19). Long HPF results in 100S ribosome formation in stationary-phase S. aureus cells, even in the absence of an RMF homolog (19).…”

“…Staphylococcus aureus does not encode rmf, but has an hpf with an extended C-terminal tail, termed long HPF (19). Long HPF results in 100S ribosome formation in stationary-phase S. aureus cells, even in the absence of an RMF homolog (19). P. aeruginosa PAO1 contains genes for rmf (PA3049) and hpf (PA4463), but does not encode the hpf paralog, yfiA (20).…”