# Role of Hibernation Promoting Factor
## 2 Results
The immunoblotting experiments of cells pre-cultured to early stationary phase (O.D.\({}_{600}\) = 6.0) showed that the wild-type strain, the \(\Delta\)_lppf_ mutant, the \(\Delta\)_lppf_ + _lppf_ strain, and the \(\Delta\)_relAASpoT_ mutant strain had similar L5 and S13 levels prior to starvation (Figure 3A). The results of band intensities of triplicate experiments for L5 are shown in Figure 3B and for S13 in Figure 3C. In the wild-type strain, the amounts of L5 and S13 decreased slightly by eight days of starvation (approximately two-fold) (Figure 3A-C). In contrast, the \(\Delta\)_lppf_ strain had approximately ten-fold reduced amounts of L5 and S13 by two days of starvation, and had little detectable L5 and S13 by six days of starvation (Figure 3A-C). These levels were significantly different from the wild type strain (\(p<0.0001\)). The amounts of these proteins were restored to wild-type levels in the \(\Delta\)_lppf_ strain complemented with _lppf_ on a plasmid (\(p=0.91\) for L5; \(p=0.20\) for S13). The stringent response mutant strain (\(\Delta\)_relAASpoT_ strain) had similar levels of L5 (\(p=0.66\)) and S13 (\(p=0.16\)) to the wild-type strain over the course of starvation (Figure 3). We next determined if the growth phase prior to starvation affects the subsequent ability of _P. aeruginosa_ to resuscitate from starvation. For these experiments, _P. aeruginosa_ strains were cultured to exponential phase (O.D.\({}_{600}\) = 1.0) rather than stationary phase, washed in PBS and starved for eight days. Similar to cells precultured to stationary phase, the wild-type strain retained approximately 100% recoverable cells for up to eight days of starvation. The \(\Delta\)_lppf_ mutant and the \(\Delta\)_relA_/\(\Delta\)_spoT_ mutant lost viability faster when precultured to exponential phase than when precultured to stationary phase (Figure S4A). Immunoblot experiments using the L5 and S13 antibodies showed that three of the strains, (wild type, \(\Delta\)_lppf_ + _lppf_, and \(\Delta\)_relA_/\(\Delta\)_spoT_), had reduced abundance of these two proteins after two days of starvation (\(p=0.0005\)), then maintained L5 and S13 proteins throughout the remainder of starvation (\(p=0.33\) for L5, \(p=0.62\) for S13)(Figure 3A-C). In the \(\Delta\)_lppf_ mutant, the L5 protein had reduced abundance compared to the wild type, but the cells did not lose L5 to the extent of the \(\Delta\)_lppf_ cells pre-cultured to stationary phase (\(p=0.0023\)) (Figure 3B,C). Similarly, S13 had reduced abundance at day 4 of starvation (\(p=0.04\)) but was not significantly different from the wild type at the other time points. When cultured to exponential phase, the \(\Delta\)_lppf_ cells did not lose S13 to the extent of cells first cultured to stationary phase (\(p<0.0001\)). Figure 2: **(A)** Resuscitation of _P. aeruginosa_ PAO1, _P. aeruginosa_\(\Delta\)_lppf_, _P. aeruginosa_\(\Delta\)_lppf_ complemented with _lppf_ on a plasmid, and _P. aeruginosa_\(\Delta\)_relAASpoT_ over the course of starvation at 37 C in phosphate buffered saline (PBS). Cultures were diluted to give approximately the same initial concentration of cells. Resuscitation was assayed as colony forming units (CFUs). **(B)** Cellular protein abundances of _P. aeruginosa_ strains over the course of starvation. Data show the mean and standard deviation for three replicates. Ribosome integrity of the cells pre-cultured to exponential phase (OD\({}_{600}\) = 1.0) and then starved was assayed as total cellular RNA and as 23S/16S rRNA ratios, prior to starvation and after six days of starvation. The results showed that all strains had reduced cellular RNA content following starvation (\(p\) < 0.0001) (Figure 4A). Similar to cells cultured to stationary phase [21], the wild-type strain maintained rRNA integrity as assayed by 23S/16S rRNA ratio, maintaining a ratio of approximately 1.4 both before and after six days of starvation (\(p\) = 0.97) (Figure 4B).