# Role of Hibernation Promoting Factor
## 2 Results
aeruginosa_ growth and cellular protein abundance over growth was not affected by the \(\Delta\)_relAASpoT_ mutations (Figure S2A,B). In early exponential phase, HPF production was delayed in the \(\Delta\)_relAASpoT_ strain compared to the wild-type strain (Figures S2C and S3A), similar to the modulatory effect we observed for _lppf_ expression in the \(\Delta\)_relAASpoT_ mutant [41]. HPF protein amounts in the \(\Delta\)_relAASpoT_ mutant ultimately reached similar levels to that of the wild-type strain (_p_ = 0.50) (Figures S2C and S3A). The ribosomal protein L5 showed consistent levels of protein abundance over the course of exponential growth in the wild-type strain, the \(\Delta\)_tpf_ mutant, and the \(\Delta\)_relAASpoT_ mutant, although the L5 protein had reduced abundances at 8 hours and into stationary phase in PAO1 and the \(\Delta\)_tpf_ mutant (Figures S2C and S3B). The ribosomal protein S13 abundance was not affected by the \(\Delta\)_tpf_ or the \(\Delta\)_relA_/_AspoT_ mutations, but the abundance of S13 reduced approximately two-fold from exponential phase to stationary phase (Figures S2C and S3C). **Ribosomal proteins are degraded in _P. aeruginosa_\(\Delta\)_tpf_ when cells are starved. ** We previously observed a loss of ribosome integrity during starvation of the _P. aeruginosa_\(\Delta\)_tpf_ mutant, by measuring rRNA using fluorescence in situ hybridization (FISH) and by comparing 23S/16S rRNA ratios of starved cells [21]. In those studies, we showed loss of rRNA and a reduction in the 23S/16S rRNA ratio in the \(\Delta\)_tpf_ mutant following starvation, whereas the wild-type strain retained a 23S/16S rRNA ratio of approximately 1.5 throughout starvation. Here, we further characterized ribosome integrity of starved cells by assaying rProtein stability of _P. aeruginosa_ and the \(\Delta\)_tpf_ mutant. Ribosomes from _P. aeruginosa_ PAO1 and from _P. aeruginosa_\(\Delta\)_tpf_ were partially purified using sucrose cushions at two time-points: (i) stationary-phase cultures (O.D.\({}_{600}\) = 6.0) that were washed and resuspended in PBS and (ii) stationary-phase cultures that were washed and resuspended in PBS, then starved by incubation in PBS for six days. Purified ribosomes were assayed for the rRNA component by agarose gels (Figure 1A), and for the rProtein component by SDS-PAGE (Figure 1B), and by immunoblotting (Figure 1C,D). The rRNA results showed that both the wild-type strain and the \(\Delta\)_tpf_ mutant strain had abundant 16S and 23S rRNAs prior to starvation. Following six days of starvation, both strains had reduced amounts of rRNA, and the 23S and 16S rRNAs were reduced further in the starved \(\Delta\)_tpf_ mutant, compared to the wild-type cells. Almost no rRNA was detected until the sample amount was increased five-fold (Figure 1A, lane 5). Figure 1: Analysis of _P. aeruginosa_ ribosomes, partially purified using sucrose cushions. Ribosomes were purified from pre-starved cells and from 6 d starved cultures. (**A**) rRNA extracts of purified ribosomes from _P. aeruginosa_ and from the \(\Delta\)_tpf_ strain prior to starvation and after 6 d of starvation. (**B**) SDS-PAGE of purified ribosomes of pre-starved and 6 d starved cultures. (**C**) Immunoblot analysis of protein L5 from pre-starved cultures and 6 d starved cultures. (**D**) Immunoblot analysis of protein S13 from pre-starved cultures and from 6 d starved cultures. Shown are representative images from three independent replicate gels. The SDS-PAGE results of purified ribosomes showed similar banding patterns and intensities for the wild-type strain and for the \(\Delta\)_lppf_ mutant prior to starvation (Figure 1B). The protein banding pattern for the wild-type strain was similar in the pre-starved cells and six days-starved cells (Figure 1C, lane 3). In contrast, the banding pattern for the \(\Delta\)_lppf_ mutant strain following starvation differed from the pre-starved cells (Figure 1C, lane 4).