# Role of Hibernation Promoting Factor
## 1 Introduction  
_Pseudomonas aeruginosa_ is an opportunistic pathogen that causes chronic infections, including pulmonary infections in patients with the genetic disorder, cystic fibrosis (CF) [1; 2]. Chronic infections are often associated with biofilms [3], where bacteria are encased in their extracellular polymers and attached to surfaces [4]. Chronic biofilm infections are difficult to eliminate with antibiotic treatments [5],in part because the biofilms contain cells in a variety of different physiological states, including cells that are dormant [6]. Since most antibiotics only target active metabolic processes, dormant bacteria are tolerant to antibiotics at concentrations that are effective against planktonic cells [7; 8; 9]. Studies of _P. aeruginosa_ isolates from CF patients indicate that even when patients are treated with antibiotics, clones of the original infecting strain reemerge, suggesting that a subpopulation of the founding infections survives the treatment and repopulates the biofilm when treatments are alleviated [10; 11; 12]. In order to target the dormant bacteria of chronic infections, it will be important to gain a better understanding of the physiology of the dormant subpopulation of infectious biofilms [13]. When bacteria undergo a shift from active growth to dormancy, they undergo a variety of metabolic changes [13]. Included in these metabolic processes is a shift away from energy-expensive processes, such as translation, and conservation of energy stores [13; 14]. During starvation, many bacteria also undergo a reduction in cell size and metabolic reprogramming, which includes degradation of certain proteins, to be used as energy, carbon, or nitrogen resources. When bacteria are nutrient-limited, they reduce translation and reduce their number of ribosomes. However, a critical number of ribosomes is essential for resuscitation from dormancy, and therefore a supply of ribosomes is preserved in dormant cells [15]. As shown for _Escherichia coli_ and many other species of bacteria, ribosomes are inactivated during cell starvation by the small ribosome-binding accessory proteins, ribosome modulation factor (RMF) and ribosome hibernation factor (HPF) [16; 17; 18; 19; 20]. In _P. aeruginosa_, HPF is also critical for maintenance of rRNA integrity during starvation and for optimal resuscitation of the cells from starvation [21; 22]. Structural studies have examined the roles of RMF and HPF in ribosome binding and inactivation for a variety of bacteria [23; 24; 25; 26; 27; 28; 29; 30]. In Gram-negative bacteria, RMF binds to ribosomes in the mRNA channel near the small subunit protein, S1, preventing interaction between the anti-Shine Dalgarno site and the Shine Dalgarno sequence of the mRNA [24; 29]. HPF binds near the A-site, inhibiting tRNA binding between the codon and anti-codon [24; 29]. Binding of RMF results in ribosome dimerization and the formation of inactive 90S ribosome dimers [31]. HPF binding converts the ribosomes to an inactive 100S dimer form [31; 32]. In _E. coli_, an HPF homolog, YfiA, competes with HPF, forming inactive 70S ribosome monomers where the C-terminal tail of YfiA inhibits the binding of RMF to the ribosome [31]. In contrast, Gram-positive bacteria do not have the gene for RMF, but have an HPF with a long C-terminal tail [23; 26]. The HPF from Gram-positive bacteria causes the formation of inactive 100S ribosome dimers by protein-protein interactions of its C-terminal domain, caused by formation of HPF homodimers within its binding site of the ribosome [23; 26]. HPF binding to ribosomes causes association of the 30S and 50S ribosomal subunits into inactive 70S ribosome monomers and 100S ribosome dimers. Subunit association hinders cleavage of rRNA by endonucleases, preventing the initial steps in ribosome degradation [33; 34]. In _Bacillus subtilis_, the absence of HPF results in the loss of rProteins proteins S2 and S3 from the ribosomes of stationary phase cells [35].