# Role of Hibernation Promoting Factor
## 3 Disscussion  
Heterotrophic bacteria are subject to changing environmental conditions, including periods where nutrients are abundant, as well as periods of starvation. Biofilms contain nutrient gradients, due to diffusion of nutrients into the biofilms and utilization of the nutrients by the bacteria [42]. As a result, biofilms contain bacteria in many different physiological states [6], including cells that are nutrient-limited. The biofilm-forming bacterium, _P. aeruginosa_, has adapted to tolerate nutrient-deficient conditions, and can survive starvation for long periods of time with little loss of recoverable cells [21]. Like most heterotrophic bacteria, _P. aeruginosa_ responds to its changing environmental conditions, including shifts from nutrient abundance to nutrient starvation, through a variety of metabolic processes [13; 14]. Among these changes are conservation of energy resources and degradation of excess cellular proteins and RNA. Ribosomes are an excellent source of resources, including RNA and proteins, that can be used as nitrogen, phosphate, and energy sources under nutrient limiting conditions. As a result, when cells transition from rapid growth to slow growth, they reduce their number of ribosomes [43], presumably to a maintenance level necessary for resuscitation when nutrients become available [15]. In a previous study, we characterized rRNA stability and abundance during _P. aeruginosa_ starvation [21]. In the study here, we further characterized the fate of ribosomes during _P. aeruginosa_ starvation, by assaying protection or loss of two essential ribosomal proteins, L5 and S13, following the transition of _P. aeruginosa_ from active growth to starvation. The results show that these proteins are stably maintained in _P. aeruginosa_ during starvation, and that HPF is required for the maintenance of these proteins. The results also show that, although the stringent response is essential for optimal recovery of _P. aeruginosa_ from starvation, the stringent response is not required for rProtein  
Figure 10: RNA integrity of _P. aeruginosa_ cultured in MOPS-glucose or MOPS-fructose to O.D.\({}_{600}=1.0\), then starved for six days. (**A**) Total cellular RNA levels prior to starvation and after 6 d of starvation. (**B**) 23S/16S rRNA ratios of strains pre-cultured in MOPS-glucose or MOPS-fructose, then starved for 6 d. Data show the mean and standard error of three independent biological replicates. Asterisks (**) for 23S/16S ratio shows significant difference between 0 and 6 d (\(p<0.001\)). maintenance during starvation. Therefore, the mechanisms by which HPF and the stringent response protect _P. aeruginosa_ viability during starvation differ. The pathway for ribosome degradation has been well characterized for _E. coli_[34; 43; 44; 45]. Ribosome degradation is initiated by RNAse E endonuclease cleavage at positions 820 and 919 of the 16S rRNA and position 1942 of the 23S rRNA [34]. The rRNA fragments are then further processed by other ribonucleases, including PNPase, RNAse R, RNAase II, and RNAse R. Cleavage and degradation of rRNA causes the release of ribosomal proteins, which may then either be targeted for degradation by proteases, including Lon protease [46], or recycled for incorporation into new ribosomes [47]. The Lon protease is activated by polyphosphate [46], which accumulates as a starvation response in _P. aeruginosa_[48]. HPF likely evolved to prevent the initial events in ribosome degradation [34; 35; 44], preserving a subset of ribosomes in the inactive 70S or 100S form. Here, we show that protection of rRNA and rProtein degradation is dependent on the presence of HPF. The loss of rRNA is independent of the growth conditions prior to starvation. Regardless of the pre-growth cultivation conditions, in _P. aeruginosa_ _Anfp_ rRNA loses integrity during subsequent starvation, as measured by total RNA and by the reduced 23S/16S rRNA ratio (Figures 4 and 10). The loss of rRNA integrity correlates with the impaired ability of _P. aeruginosa_ to recover from starvation.