# Role of Hibernation Promoting Factor
## 2 Results  
**Effect of HPF and the stringent response on ribosomal proteins during _P. aeruginosa_ growth**. In order to monitor ribosomal protein abundance during _P. aeruginosa_ cell growth and starvation, we induced polyclonal antibody production to the _P. aeruginosa_ HPF protein and to the large and small subunit ribosomal proteins, L5 and S13. L5 and S13 were chosen because these proteins interact during active translation, forming the B1b bridge, and are essential for ribosome ratcheting during translation [36, 37]. Antibodies to these proteins were generated by fusing the _P. aeruginosa hpf_, _rplE_, and _rpsM_ genes to 6X-poly-histidine tags, purifying the proteins, and using the purified proteins to induce rabbit polyclonal antibodies. The IgG fraction was purified from the antisera and tested for efficacy in immunoblot analyses against the _P. aeruginosa_ proteins. For HPF, a band near the molecular mass of HPF (11.7 kDa) was observed in the _P. aeruginosa_ PAO1 cell extracts, but not in extracts from the _P. aeruginosa_\(\Delta\)_lppf_ mutant strain (Figure S1A). Complementation of _P. aeruginosa_\(\Delta\)_lppf_ with _lppf_ on a plasmid resulted in high expression of HPF (Figure S1A). For the large subunit protein L5, antibodies reacted with a band at approximately 20 kDa, the calculated molecular mass of L5 (Figure S1B). Since L5 is essential [38], no deletion mutant was available to serve as a negative control in immunoblots. Therefore, we tested the antibodies against L5 fusion proteins. Antibodies reacted with the L5-6X-His fusion protein, giving a band of 21 kDa, and the L5-YFP (yellow fluorescent protein) fusion protein, giving a band of 48 kDa (Figure S1B). Similarly, for the small ribosomal subunit protein S13, a band was observed at approximately the molecular mass of S13 (13.3 kDa) (Figure S1C), and the antibodies reacted with an S13-YFP fusion protein, producing a band at 41 kDa. The results indicated that these antibodies are effective for use in immunoblot analysis to detect these ribosomal proteins during _P. aeruginosa_ growth and starvation. We first used the antibodies in immunoblots to determine the relative amounts of HPF, L5, and S13 over the course of _P. aeruginosa_ growth. Growth curves were performed over 24 h and assayed for colony forming units (CFUs) (Figure S2A), total protein content (Figure S2B), and abundances of HPF, L5 and S13 (Figure S2C). Growth curves and total cellular protein amounts showed that there was no effect of the \(\Delta\)_lppf_ deletion during _P. aeruginosa_ growth in TSB medium. Overexpression of _lppf_ also did not influence cell growth. For immunoblot analysis, total cellular protein concentrations were determined and used to load 15 ug cellular protein per lane (Figure S2B,C). For all rProtein abundance studies, band intensities from at least three independent biological replicates were quantified by densitometry. Immunohistochemistry over the course of cell growth showed that HPF amounts were relatively constant throughout exponential phase and into early stationary phase in wild-type _P. aeruginosa_ PAO1 (_p_ = 0.46) (Figures S2C and S3A). No HPF bands were observed in the \(\Delta\)_lppf_ strain. _P. aeruginosa_\(\Delta\)_lppf_ containing _lppf_ on a plasmid under control of a strong promoter (P\({}_{trc}\)-_lppf_) resulted in increased amounts of HPF compared to the wild-type strain (_p_ < 0.0001) (Figures S2C and S3A). The stringent response is a nutrient starvation response caused by ribosome stalling due to insufficient amino acid-charged tRNAs. The stringent response results from the production of the starvation signaling molecule, guanosine tetraphosphate (or pentaphosphate), (p)ppGpp, through the activities of ReIA and SpoT (reviewed in [39]). Since the stringent response is involved in ribosome dimerization in _E. coli_[40], and has a modulatory effect on _lppf_ expression in _P. aeruginosa_[41], here, we determined if the stringent response affected HPF levels in _P. aeruginosa_ over the course of growth and starvation. _P.