The roles of hibernation promoting factor in resuscitation of Pseudomonas aeruginosa from dormancy

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Montana State University - Bozeman, College of Letters & Science


Microbial biofilms are surface-attached communities of microorganisms. Biofilms are often associated with chronic infections due to antibiotic resistance. Pseudomonas aeruginosa causes chronic pulmonary infections in cystic fibrosis patients and chronic wound infections in diabetic ulcers. One mechanism for biofilm-associated resistance is a formation of non-dividing, metabolically dormant cells resisting antibiotics. The goals of this research were to understand the molecular mechanisms involved in formation, maintenance, and resuscitation of dormant cells, with the ultimate goal of developing enhanced treatment strategies for chronic biofilm-associated infections. While dormant, bacteria must maintain cellular and macromolecular integrity required for resuscitation. Previous study found the high abundance of messenger RNAs for ribosome accessory proteins, hibernation promoting factor (HPF) and ribosome modulation factor (RMF), in the dormant subpopulation of P. aeruginosa biofilms. In this research, we characterized the activity and expression of the ribosome hibernation factor. By exposing the hpf and rmf deletion mutant strains to nutrient starvation, we found that HPF, but not RMF, is essential for cell viability maintenance during starvation-induced dormancy. Viability loss in the hpf mutant strain corresponded to loss of ribosomal RNA, and by inference, loss of cellular ribosome content during dormancy. Single-cell level studies using fluorescence in situ hybridization showed the heterogeneous ribosomal RNA levels for both the hpf and wild-type cells. Single-cell level studies using drop-based microfluidics also showed heterogeneity in resuscitation from dormancy. While the majority lost ability to resuscitate from dormancy, a fraction of hpf mutant cells recovered but with an extended lag time. We also determined the regulation of HPF expression using a transposon-based yellow fluorescent protein (YFP) reporter fused to HPF. The results showed that hpf is expressed from at least two different promoters. HPF expression is also controlled by mRNA folding, and an autofeedback mechanism. The complex regulatory mechanism at transcriptional and post-transcriptional levels may allow the bacteria to respond to nutrient limitation and enter a dormant state. Our results show the importance of HPF on ribosome preservation during starvation, as well as how this hibernation factor is regulated. The results provide new information of this novel target for treatment of dormant infectious bacteria.




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