# Role of Hibernation Promoting Factor
## 2 Results
When the amount of sample loaded onto the gel for the \(\Delta\)_lppf_ mutant was increased four-fold, the intensity of the protein bands increased in the \(\Delta\)_lppf_ mutant, but the banding pattern of the \(\Delta\)_lppf_ mutant still differed from that of the starved wild-type ribosomes (Figure 1C, lane 5). To characterize specific ribosomal proteins, we performed immunoblott analysis of the partially purified ribosomes, using the L5 and S13 antibodies. The immunoblots showed equivalent amounts of L5 (Figure 1C) and S13 (Figure 1D) in the wild-type strain and the \(\Delta\)_lppf_ mutant strain prior to starvation. Following six days of starvation, there was very little detectable L5 or S13 in ribosomes purified from the \(\Delta\)_lppf_ mutant (Figure 1C,D). Faint L5 and S13 bands could be observed in the \(\Delta\)_lppf_ strain when the amount of protein loaded onto the gel was increased two-fold (Figure 1C,D, lanes 5). **Ribosomal protein stability in starved _P. aeruginosa_ is dependent on HPF. ** The experiments using partially purified ribosomes indicated that the amount of both rRNA and rProtein decreased in abundance following _P. aeruginosa_ starvation of the \(\Delta\)_lppf_ strain. However, since the ribosome purification approach requires equivalent efficiency in cell lysis and ribosome extraction, the results are only qualitative. In order to quantify ribosomal protein abundances during _P. aeruginosa_ starvation, we performed experiments on whole cellular extracts over the course of starvation. These time-course studies allowed sample normalization by loading equivalent amounts of total cellular protein per lane. For these experiments, _P. aeruginosa_ PAO1, _P. aeruginosa_\(\Delta\)_lppf_, _P. aeruginosa_\(\Delta\)_lppf_ + _lppf_, and _P. aeruginosa_\(\Delta\)_lpA_\(\Delta\)_spoT_ were cultured to stationary phase (O.D.\({}_{600}\) = 6.0), washed with PBS, then transferred to PBS for starvation at 37 C with shaking for up to eight days. During starvation, cultures were assayed for recoverable cells as colony forming units (CFUs), for total cellular protein, and by immunoblotting for L5 and S13 abundances. All experiments were performed on at least three independent biological replicates, and densitometry was used to compare L5 and S13 band intensities. The results of _P. aeruginosa_ resuscitation over the course of starvation, quantified as CFUs, were similar to our previous studies [21]. _P. aeruginosa_ PAO1 maintained essentially 100% recoverability over eight days of starvation (Figure 2A). In contrast, _P. aeruginosa_\(\Delta\)_lppf_ and _P. aeruginosa_\(\Delta\)_lpA_\(\Delta\)_spoT_ were impaired in their ability to recover from starvation, and had approximately ten-fold fewer recoverable cells by 4 d of starvation. Complementation of the _P. aeruginosa_\(\Delta\)_lppf_ with _lppf_ on a plasmid restored the ability of the mutant strain to resuscitate from starvation. Interestingly, the results of total cellular protein over the course of starvation differed for the \(\Delta\)_lppf_ mutant versus the \(\Delta\)_lpA_\(\Delta\)_spoT_ mutant (Figure 2B). _P. aeruginosa_ PAO1 had consistent amounts of cellular protein throughout starvation (\(p\) = 0.23). Although the \(\Delta\)_lppf_ strain had reduced CFUs over time, the total cellular protein remained similar to that of the wild-type strain (\(p\) = 0.27). In contrast, the \(\Delta\)_lpA_\(\Delta\)_spoT_ mutant lost both recoverable cells (\(p\) < 0.0001) and total cellular protein (\(p\) < 0.0001). The results suggest that the \(\Delta\)_lppf_ mutant cells, although impaired in resuscitation, remained intact during starvation, while a majority of the \(\Delta\)_lpA_\(\Delta\)_spoT_ cells lysed over the course of starvation, resulting in a decrease in the amount of cellular protein. These results are consistent with our previous microfluidics results that showed that the \(\Delta\)_lppf_ mutant cells remained intact, but not dividing, during starvation [21].