# Role of Hibernation Promoting Factor
## 2 Results
The \(\Delta\)_hpf_ strain had reduced abundance of S13 over the course of starvation (\(p=0.0002\)), regardless of the pre-cultivation conditions (Figure 9A-C). However, both L5 and S13 were detected even after eight days of starvation, when the cells were first cultured in minimal medium prior to starvation. Figure 7: Effect of nutrient source and pre-growth conditions on cellular HPF abundances. (**A**) Immunoblots of HPF over 8 d of starvation for strains _P. aeruginosa_ PAO1, \(\Delta\)_hpf_, \(\Delta\)_hpf_ + _hpf_, pre-cultured in minimal medium with glucose or fructose to early exponential phase O.D.\({}_{600}=0.3\) or O.D.\({}_{600}=1.0\). (**B**) Mean and standard error of the mean for immunoblot HPF band intensities for PAO1 and \(\Delta\)_hpf_ + _hpf_ strains pre-cultured in minimal medium to O.D.\({}_{600}=0.3\). (Results from \(\Delta\)_hpf_ strain were below detection). (**C**) Mean and standard error of the mean for immunoblot HPF band intensities for PAO1 and \(\Delta\)_hpf_ + _hpf_ strains pre-cultured in minimal medium to O.D.\({}_{600}=1.0\). Figure 8: Quantification of _P. aeruginosa_ L5 band intensity over the course of 8 d of starvation, from whole cell extracts. Strains were pre-cultured in MOPS minimal media to early exponential phase (O.D.\({}_{600}\) = 0.3 or 1.0) with either fructose or glucose as the sole carbon source. (**A**) Representative immunoblot of L5 from whole cell extracts. (**B**) Quantification of L5 signal intensity obtained by densitometry of three immunoblots, when strains were cultured in MOPS-fructose. Solid lines and symbols indicate cells were pre-cultured to O.D.\({}_{600}\) = 1.0 and dashed lines and open symbols indicate cells were pre-cultured to O.D.\({}_{600}\) = 0.3. (**C**) Quantification of L5 signal intensity when strains were cultured in MOPS- glucose. Data show the mean and standard error of three independent biological replicates. Figure 9: Quantification of _P. aeruginosa_ S13 band intensity over the course of 8 d of starvation, from whole cell extracts. Strains were pre-cultured in MOPS minimal media with either fructose or glucose as the sole carbon source. (**A**) Representative immunoblot of S13 from whole cell extracts. (**B**) Quantification of S13 signal intensity obtained by densitometry of three immunoblots, when strains were cultured in MOPS-fructose. Solid lines and symbols indicate cells were pre-cultured to O.D.\({}_{600}\) = 1.0 and dashed lines and open symbols indicate cells were pre-cultured to O.D.\({}_{600}\) = 0.3. (**C**) Quantification of S13 signal intensity when strains were cultured in MOPS- glucose.. Data show the mean and standard error of three independent biological replicates. We determined if pre-growth of strains to early exponential phase in MOPS-glucose or MOPS-fructose influenced rRNA integrity following starvation, by measuring total cellular RNA (Figure 10A) and the 23S/16S rRNA ratios before and after six days of starvation (Figure 10B). Prior to starvation, all strains had similar 23S/16S rRNA ratios regardless of the pre-growth conditions (\(p=0.90\)) (Figure S6). Following six days of starvation, the wild-type strain had preserved 23S/16S rRNA ratios of approximately 1.5 (Figure 10B). However, the \(\Delta\)_lnpf_ strain had selective loss of the 23S rRNA component in the \(\Delta\)_lnpf_ strain compared to the wild-type strain after six days of starvation (\(p<0.0001\)). The results show that, even when _P. aeruginosa_ is cultured to early exponential phase in minimal medium where _lnpf_ expression is low, there is sufficient HPF to protect against subsequent loss of rRNA and rProteins during starvation.