Expression and regulation of the Pseudomonas aeruginosa hibernation promoting factor

Bacterial biofilms contain subpopulations of cells that are dormant and highly tolerant to antibiotics. While dormant, the bacteria must maintain the integrity of macromolecules required for resuscitation. Previously, we showed that hibernation promoting factor (HPF) is essential for protecting Pseudomonas aeruginosa from ribosomal loss during dormancy. In this study, we mapped the genetic components required for hpf expression. Using 5'-RACE and fluorescent protein reporter fusions, we show that hpf is expressed as part of the rpoN operon, but that hpf also has a second promoter (P_hpf ) within the rpoN gene. P_hpf is active when the cells enter stationary phase, and expression from P_hpf is modulated, but not eliminated, in mutant strains impaired in stationary phase transition (ΔdksA2, ΔrpoS and ΔrelA/ΔspoT mutants). The results of reporter gene studies and mRNA folding predictions indicated that the 5' end of the hpf mRNA may also influence hpf expression. Mutations that opened or that stabilized the mRNA hairpin loop structures strongly influenced the amount of HPF produced. The results demonstrate that hpf is expressed independently of rpoN, and that hpf regulation includes both transcriptional and post-transcriptional processes, allowing the cells to produce sufficient HPF during stationary phase to maintain viability while dormant.