# Role of Hibernation Promoting Factor
## 2 Results
The \(\Delta\)_hpf_ strain pre-cultured to exponential phase had reduced rRNA integrity (\(p\) = 0.0001), with an average 23S/16S rRNA ratio of 0.33 (Figure 4B). The 23S/16S ratio was restored to wild-type levels when the \(\Delta\)_hpf_ strain was complemented with _hpf_ on a plasmid (\(p\) = 0.53). The \(\Delta\)_relAAspoT_ mutation did not have selective loss of the 23S rRNA following starvation (\(p\) = 0.22). The results indicate that when _P. aeruginosa_ is pre-cultured to exponential phase, then starved, HPF is required to maintain rRNA integrity. In addition, the abundances of rProteins is reduced in the \(\Delta\)_hpf_ strain during starvation, but not to the extent of rProtein loss in cells first cultured to stationary phase, then starved. Figure 3: Immunoblot and densitometry analysis of _P. aeruginosa_ L5 and S13 ribosomal proteins over the course of starvation. (**A**) L5 and S13 Immunoblots of _P. aeruginosa_ PAO1, _P. aeruginosa_\(\Delta\)_hpf_, _P. aeruginosa_\(\Delta\)_hpf_ + _lpf_ and _P. aeruginosa_\(\Delta\)_relAAspoT_ over 8 d of starvation. Strains were pre-cultured to O.D.\({}_{600}\) = 6.0 (filled symbols and solid lines) or O.D.\({}_{600}\) = 1.0 (open symbols and dashed lines) prior to starvation. (**B**) Densiometry of L5 immunoblots showing the mean and standard error of the mean of three replicate immunoblots. (**C**) Densiometry of S13 immunoblots showing the mean and standard error of the mean of three replicate immunoblots. **Nutrient sources affect _hpf_ expression and subsequent resuscitation of _P. aeruginosa_ from starvation. ** Previously, we identified the _hpf_ promoter sequence and characterized factors involved in _hpf_ expression [41]. For those studies, we generated a yellow fluorescent protein (YFP) reporter gene to assay for expression from the P_hpf_ promoter (P_hfp_-_hpf_-_vfp_) [41]. _P. aeruginosa_ PAO1 grows in minimal medium with a variety of simple sugars as sole carbon sources, but with varying growth rates depending on the sugar. Here, we used the P_hfp_-_hpf_-_vfp_ reporter to compare the expression of _hpf_ in cells cultured in defined minimal medium (MOPS medium) with either glucose or fructose as the sole carbon source. In MOPS-fructose medium, _P. aeruginosa_ P_hfp_-_hpf_-_vfp_ had an extended lag time compared to growth in MOPS-glucose medium, and in MOPS-fructose, expression from the P_hpf_ promoter closely followed cell growth (Figure 5). In contrast, in MOPS-glucose medium, the lag time was short, and there was an additional lag between growth and expression from the P_hpf_ promoter (Figure 5). Therefore, culturing _P. aeruginosa_ in minimal medium with either glucose or fructose as the sole carbon should result in differing cellular levels of HPF, and possibly differing abilities of the cells to recover from starvation. To test this, we cultured strains in MOPS-fructose and MOPS-glucose to early exponential phase (O.D_e_600 = 0.3 or O.D_e_600 = 1.0). Following growth, the cultures were starved as described above, by washing in PBS, resuspending equivalent cell numbers in PBS (approximately \(2\times 10^{8}\) cells/mL), and incubating in PBS at 37 C for up to eight days. The starved cultures were assayed for resuscitation from starvation, total cellular protein, 23S/16S rRNA ratios, and relative amounts of HPF, L5, and S13. In order to compare strains and growth conditions, the reduction in CFUs was plotted over time of starvation, and the results were compared to those from cells pre-cultured in rich medium (TSB) to O.D_e_600 = 1.0 (Figure 6). Under all pre-growth conditions, _P. aeruginosa_ PAO1 increased in cell numbers after one day of starvation (Figure 6). This phenomenon was observed previously, and is due to reductive cell division early in starvation [21]. For the wild-type strain, cells cultured in MOPS-glucose had an approximately 2.5 fold reduction in cell recovery by day eight of starvation (\(p<0.0001\)), whereas there was no reduction in CFU when cells were first cultured in rich medium (\(p=0.63\)) and little reduction in recovery in _P.