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Item The role of ribosome hibernation factor and the stringent response in the survival of Pseudomonas aeruginosa during dormancy(Montana State University - Bozeman, College of Letters & Science, 2019) Theng, Sokuntheary; Chairperson, Graduate Committee: Michael FranklinPseudomonas aeruginosa survives in a dormant state in low nutrient environments and is able to resuscitate when favorable conditions are available. In response to stressful environmental conditions including antibiotic stress, osmotic stress, and starvation, P. aeruginosa undergoes the (p)ppGpp-mediated stringent response to induce a variety of genes for entry into the dormant state. One critical mechanism for P. aeruginosa dormancy involves in inactivating translation machinery by converting active ribosomes into inactive 70S and 100S ribosome monomers and dimers. Hibernation promoting factor (HPF~11.6kDa) is a ribosome-associated protein that stabilizes inactive ribosomes. Here, I investigated the relationship between HPF and the stringent response in survival of P. aeruginosa during dormancy. I also investigated role of HPF preserving ribosomes in dormant cells by quantifying the abundance of two ribosomal proteins (L5 of the large ribosomal subunit and S13 of the small ribosomal subunit), during P. aeruginosa starvation. For quantitative analysis during nutrient-limited conditions, I used immunoblotting and image analysis to quantify L5 and S13 abundances in the wild-type strain, PAO1, and in a relA/spoT double mutant strain, which is incapable of producing (p)ppGpp. The results show that the relA/spoT mutant loses HPF proteins after four days of starvation. To explore the role of HPF in preserving ribosomal proteins, I quantified L5 and S13 in wild-type PAO1 and in the Delta hpf deletion mutant and in the relA/spoT mutant. Immunoblots showed that both L5 and S13 rapidly decrease by day 2 of starvation in the Delta hpf mutant strain, but that these proteins are maintained throughout eight days of starvation in the wild-type strain. Notably, L5 and S13 are maintained in the Delta relA/Delta spoT strain throughout starvation. Lastly, I determined if the amount of cellular HPF required for P. aeruginosa ribosome maintenance. Growth in minimal medium (MOPS medium) affects the amount of HPF produced, based on the carbon source. Therefore, I tested ribosome maintenance during starvation of cells first cultured in MOPS-fructose or MOPS-glucose. The results indicate that HPF production during growth in MOPS-fructose is higher than in MOPS-glucose. However, the increased amount of HPF did not affect the amount of L5 and S13 during starvation. Therefore, the amount of HPF is not critical for P. aeruginosa to maintain its ribosomes during starvation. These results demonstrate HPF is essential for maintenance of ribosomal proteins during starvation of P. aeruginosa, and that the ribosomal proteins are likely degraded in the absence of HPF. P. aeruginosa needs a minimum amount of HPF to preserve ribosomes during nutrient-limited condition.Item Characterization of the stability of Pseudomonas aeruginosa ribosomal proteins under stress conditions(Montana State University - Bozeman, College of Letters & Science, 2017) Yanardag, Sila; Chairperson, Graduate Committee: Michael FranklinIn this study, I aimed to standardize western blot methods for probing large and small ribosomal subunits of Pseudomonas aeruginsa grown under different environmetal conditions, and to characterize the stability of ribosomal proteins to bring light to the heterogeneous composition of the population, which is hypothesized as one mechanism for antibiotic tolerance. Long-term studies done with P.aeruginosa PAO1 showed that mRNA transcripts of two proteins, RMF and HPF, are highly abundant at the biofilm-nutrient interface of the thick P.aeruginosa biofilms. Also, it was previously shown by Perez et al. and Williamson et al. (Pérez-Osorio et al., 2010; Williamson et al., 2012) that the cells located at the oxygen limited interphase of the biofilm were metabolically inactive or slow-growing. Akiyama et al. (Akiyama et al., 2017) and Williamson et al. (Williamson et al., 2012) found that HPF is a critical protein for the maintenance of 23S rRNA and overall ribosomal RNA stability after prolonged stress exposure (Akiyama et al., 2017; Williamson et al., 2012). In light of this information, Akiyama et al. (Akiyama et al., 2017) showed that in the absence of the HPF protein, P.aeruginosa cannot protect its ribosome integrity and cannot resuscitate from dormancy after the environmental stressors are gone. Perez et al. (Pérez-Osorio et al., 2010) showed that P.aeruginosa biofilms are heterogeneous in physiology, and it is posited that persister cells of the biofilm are located at the bottom of the biofilm, unaffected by the antibiotic exposure and therefore can repopulate the biofilm (Williamson et al., 2012). Localization of ribosomal subunits and determination of the abundance of ribosomes within the heterogeneous biofilms will provide valuable insights on the mechanisms of persister cell formation, dormancy, and resusication from dormancy. In order to do so, I have isolated two ribosomal proteins, L5 and S13, and HPF. In this study, I generated polyclonal antibodies against those three proteins. I used the antibodies to determine the abundance of these proteins during the normal course of growth of the wild type and Deltahpf mutant strains. Growth analysis in nutrient rich media gave us an understanding of the stability of 70S ribosomes when the bacterium was growing without any stress. Later, the wild type and Deltahpf strain were grown in a carbon and nitrogen-limited environment for seven days to examine the response of the cells to the starvation stress regarding ribosomal stability. Finally, I tested the hypothesis that cells located at the bottom of the biofilm are abundant in the HPF protein, and therefore contain more inactive ribosomes compared to the cells located at the top of biofilm.Item The roles of hibernation promoting factor in resuscitation of Pseudomonas aeruginosa from dormancy(Montana State University - Bozeman, College of Letters & Science, 2018) Akiyama, Tatsuya; Chairperson, Graduate Committee: Michael Franklin; Kerry S. Williamson, Robert Schaefer, Shawna Pratt, Connie B. Chang and Michael J. Franklin were co-authors of the article, 'Resuscitation of Pseudomonas aeruginosa from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation' in the journal 'Proceedings of the National Academy of Sciences of the United States of America' which is contained within this thesis.; Kerry S. Williamson and Michael J. Franklin were co-authors of the article, 'Expression and regulation of the Pseudomonas aeruginosa hibernation promoting factor' submitted to the journal 'Molecular microbiology' which is contained within this thesis.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.