# Role of Hibernation Promoting Factor
## 2 Results
aeruginosa_ PAO1 cultured in MOPS-fructose (Figure 6A). Under all four pre-growth conditions, the \(\Delta\)_hpf_ mutant lost recoverable cells over the course of starvation (\(p<0.0001\)) (Figure 6B). Complementation of the _P. aeruginosa_\(\Delta\)_hpf_ mutant with _hpf_ under control of a strong promoter restored resuscitation to levels similar to the wild-type strain (Figure 6C). Figure 4: rRNA analysis of _P. aeruginosa_ and mutant strains prior to starvation (solid bars) and after 6 d of starvation (striped bars). (**A**) Total RNA abundances of pre-starved and 6 d starved cells assayed using the NanoDrop Spectrophotometer. (**B**) 23S/16S rRNA ratios of cells prior to starvation and following 6 d starvation, determined using the Agilent Bioanalyzer. Data show the mean and standard error of the mean for at least three independent replicates. Asterisks (**) for 23S/16S ratio shows significant difference between 0 and 6 d (\(p<0.001\)). The assay for cellular protein over the course of starvation showed reduction in total cellular protein by two-fold over eight days of starvation regardless of the pre-growth condition or the strain (Figure S5). The results of immunoblots show that HPF is produced early in cell growth (day 0, prior to starvation). Surprisingly, even when cells are cultured in MOPS-glucose to early exponential phase (O.D.\({}_{600}\) = 0.3), a band associated with HPF is detected (Figure 7A,B). No HPF band is observed in the \(\Delta\)_lprf_ strain, and an intense band is observed in the \(\Delta\)_lprf_ strain where _hpf_ is expressed from a plasmid and strong promoter. Under all four pre-growth conditions, HPF is maintained in cells through eight days of starvation (\(p\) = 0.57) (Figure 7A-C). _P. aeruginosa_\(\Delta\)_lprf_ with _hpf_ expressed from a strong promoter had high levels of HPF initially, but the abundance of HPF reduced to wild-type levels by two days of starvation and then remained at wild-type levels throughout starvation, suggesting that the excess HPF is degraded during _P. aeruginosa_ starvation. Figure 5: Reporter expression of P_lprf_ when _P. aeruginosa_ is cultured in MOPS minimal medium with glucose or fructose as the sole carbon source. _P. aeruginosa_ was assayed for growth as as O.D.\({}_{600}\) and for _lprf_ expression as fluorescence from the P_lprf_-_lprf_ single-copy reporter. Green symbols show growth and fluorescence in glucose and blue symbols show growth and fluorescence in fructose. Figure 6: Effect of nutrient source and pre-growth conditions on subsequent resuscitation of _P. aeruginosa_ from starvation. Strains were cultured in MOPS minimal medium with glucose (green) or fructose (blue) to O.D.\({}_{600}\) = 0.3 (dashed lines) or O.D.\({}_{600}\) = 1.0 (solid lines). Data are plotted as fold-reduction over 8 d of starvation and compared to strains pre-cultured in TSB to O.D.\({}_{600}\) = 1.0 (black lines). (**A**) Fold-change in recoverable cell numbers for the wild-type strain, _P. aeruginosa_ PAO1. (**B**) Fold-change in recoverable cells for _P. aeruginosa_\(\Delta\)_lprf_. (**C**) Fold-change in recoverable cells for _P. aeruginosa_\(\Delta\)_lprf_ complemented with _hpf_. All data show the mean and standard error of at least three independent biological replicates. We next determined if the pre-cultivation conditions prior to starvation affected rProtein stability of the large subunit or small subunit ribosomal proteins (Figures 8 and 9). In the wild-type strain, the cellular L5 protein abundance reduced slightly over the course of starvation (Figure 8). In contrast, L5 showed significantly reduced abundance over the course of starvation in the \(\Delta\)_hpf_ strain regardless of the pre-cultivation conditions (\(p<0.0001\), MOPS-glucose, MOPS-fructose, O.D.\({}_{600}=0.3\) and 1.0) (Figure 8). Complementation of _hpf_ in the \(\Delta\)_hpf_ strain restored the L5 levels over the course of starvation to wild-type levels. Similarly, S13 was maintained in the wild-type strain and in the complemented strain throughout starvation (Figure 9).