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Item The Pseudomonas aeruginosa RpoH (σ32) Regulon and Its Role in Essential Cellular Functions, Starvation Survival, and Antibiotic Tolerance(MDPI AG, 2023-01) Williamson, Kerry S.; Dlakić, Mensur; Akiyama, Tatsuya; Franklin, Michael J.The bacterial heat-shock response is regulated by the alternative sigma factor, σ32 (RpoH), which responds to misfolded protein stress and directs the RNA polymerase to the promoters for genes required for protein refolding or degradation. In P. aeruginosa, RpoH is essential for viability under laboratory growth conditions. Here, we used a transcriptomics approach to identify the genes of the RpoH regulon, including RpoH-regulated genes that are essential for P. aeruginosa. We placed the rpoH gene under control of the arabinose-inducible PBAD promoter, then deleted the chromosomal rpoH allele. This allowed transcriptomic analysis of the RpoH (σ32) regulon following a short up-shift in the cellular concentration of RpoH by arabinose addition, in the absence of a sudden change in temperature. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain grew in the absence of arabinose, indicating that some rpoH expression occurred without arabinose induction. When arabinose was added, the rpoH mRNA abundance of P. aeruginosa ∆rpoH (PBAD-rpoH) measured by RT-qPCR increased five-fold within 15 min of arabinose addition. Transcriptome results showed that P. aeruginosa genes required for protein repair or degradation are induced by increased RpoH levels, and that many genes essential for P. aeruginosa growth are induced by RpoH. Other stress response genes induced by RpoH are involved in damaged nucleic acid repair and in amino acid metabolism. Annotation of the hypothetical proteins under RpoH control included proteins that may play a role in antibiotic resistances and in non-ribosomal peptide synthesis. Phenotypic analysis of P. aeruginosa ∆rpoH (PBAD-rpoH) showed that it is impaired in its ability to survive during starvation compared to the wild-type strain. P. aeruginosa ∆rpoH (PBAD-rpoH) also had increased sensitivity to aminoglycoside antibiotics, but not to other classes of antibiotics, whether cultured planktonically or in biofilms. The enhanced aminoglycoside sensitivity of the mutant strain may be due to indirect effects, such as the build-up of toxic misfolded proteins, or to the direct effect of genes, such as aminoglycoside acetyl transferases, that are regulated by RpoH. Overall, the results demonstrate that RpoH regulates genes that are essential for viability of P. aeruginosa, that it protects P. aeruginosa from damage from aminoglycoside antibiotics, and that it is required for survival during nutrient-limiting conditions.Item Search for a Shared Genetic or Biochemical Basis for Biofilm Tolerance to Antibiotics across Bacterial Species(American Society for Microbiology, 2022-04) Stewart, Philip S.; Williamson, Kerry S.; Boegli, Laura; Hamerly, Timothy; White, Ben; Scott, Liam; Hu, Xiao; Mumey, Brendan M.; Franklin, Michael J.; Bothner, Brian; Vital-Lopez, Francisco G.; Wallqvist, Anders; James, Garth A.Is there a universal genetically programmed defense providing tolerance to antibiotics when bacteria grow as biofilms? A comparison between biofilms of three different bacterial species by transcriptomic and metabolomic approaches uncovered no evidence of one. Single-species biofilms of three bacterial species (Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii) were grown in vitro for 3 days and then challenged with respective antibiotics (ciprofloxacin, daptomycin, and tigecycline) for an additional 24 h. All three microorganisms displayed reduced susceptibility in biofilms compared to planktonic cultures. Global transcriptomic profiling of gene expression comparing biofilm to planktonic and antibiotic-treated biofilm to untreated biofilm was performed. Extracellular metabolites were measured to characterize the utilization of carbon sources between biofilms, treated biofilms, and planktonic cells. While all three bacteria exhibited a species-specific signature of stationary phase, no conserved gene, gene set, or common functional pathway could be identified that changed consistently across the three microorganisms. Across the three species, glucose consumption was increased in biofilms compared to planktonic cells, and alanine and aspartic acid utilization were decreased in biofilms compared to planktonic cells. The reasons for these changes were not readily apparent in the transcriptomes. No common shift in the utilization pattern of carbon sources was discerned when comparing untreated to antibiotic-exposed biofilms. Overall, our measurements do not support the existence of a common genetic or biochemical basis for biofilm tolerance against antibiotics. Rather, there are likely myriad genes, proteins, and metabolic pathways that influence the physiological state of individual microorganisms in biofilms and contribute to antibiotic tolerance.Item Role of hibernation promoting factor in ribosomal protein stability during Pseudomonas aeruginosa dormancy(MDPI, 2020-12) Theng, Sokuntheary; Williamson, Kerry S.; Franklin, Michael J.Pseudomonas aeruginosa is an opportunistic pathogen that causes biofilm-associated infections. P. aeruginosa can survive in a dormant state with reduced metabolic activity in nutrient-limited environments, including the interiors of biofilms. When entering dormancy, the bacteria undergo metabolic remodeling, which includes reduced translation and degradation of cellular proteins. However, a supply of essential macromolecules, such as ribosomes, are protected from degradation during dormancy. The small ribosome-binding proteins, hibernation promoting factor (HPF) and ribosome modulation factor (RMF), inhibit translation by inducing formation of inactive 70S and 100S ribosome monomers and dimers. The inactivated ribosomes are protected from the initial steps in ribosome degradation, including endonuclease cleavage of the ribosomal RNA (rRNA). Here, we characterized the role of HPF in ribosomal protein (rProtein) stability and degradation during P. aeruginosa nutrient limitation. We determined the effect of the physiological status of P. aeruginosa prior to starvation on its ability to recover from starvation, and on its rRNA and rProtein stability during cell starvation. The results show that the wild-type strain and a stringent response mutant (∆relA∆spoT strain) maintain high cellular abundances of the rProteins L5 and S13 over the course of eight days of starvation. In contrast, the abundances of L5 and S13 reduce in the ∆hpf mutant cells. The loss of rProteins in the ∆hpf strain is dependent on the physiology of the cells prior to starvation. The greatest rProtein loss occurs when cells are first cultured to stationary phase prior to starvation, with less rProtein loss in the ∆hpf cells that are first cultured to exponential phase or in balanced minimal medium. Regardless of the pre-growth conditions, P. aeruginosa recovery from starvation and the integrity of its rRNA are impaired in the absence of HPF. The results indicate that protein remodeling during P. aeruginosa starvation includes the degradation of rProteins, and that HPF is essential to prevent rProtein loss in starved P. aeruginosa. The results also indicate that HPF is produced throughout cell growth, and that regardless of the cellular physiological status, HPF is required to protect against ribosome loss when the cells subsequently enter starvation phase.Item Functional Characterization of the Pseudomonas aeruginosa Ribosome Hibernation-Promoting Factor(American Society for Microbiology, 2020-07) Franklin, Michael J.; Sandvik, Elizabeth; Yanardag, Sila; Williamson, Kerry S.Hibernation-promoting factor (HPF) is a ribosomal accessory protein that inactivates ribosomes during bacterial starvation. In Pseudomonas aeruginosa, HPF protects ribosome integrity while the cells are dormant. The sequence of HPF has diverged among bacteria but contains conserved charged amino acids in its two alpha helices that interact with the rRNA. Here, we characterized the function of HPF in P. aeruginosa by performing mutagenesis of the conserved residues and then as-saying mutant HPF alleles for their ability to protect ribosome integrity of starved P. aeruginosa cells. The results show that HPF functionally tolerates point mutations in charged residues and in the conserved Y71 residue as well as a C-terminal trunca-tion. Double and triple mutations of charged residues in helix 1 in combination with a Y71F substitution reduce HPF activity. Screening for single point mutations that caused impaired HPF activity identified additional substitutions in the two HPF alpha helices. However, alanine substitutions in equivalent positions restored HPF activity, indicating that HPF is tolerant to mutations that do not disrupt the protein struc-ture. Surprisingly, heterologous HPFs from Gram-positive bacteria that have long C-terminal domains functionally complement the P. aeruginosa Δhpf mutant, sug-gesting that HPF may play a similar role in ribosome protection in other bacterial species. Collectively, the results show that HPF has diverged among bacteria and is tolerant to most single amino acid substitutions. The Y71 residue in combination with helix 1 is important for the functional role of HPF in ribosome protection dur-ing bacterial starvation and resuscitation of the bacteria from dormancy.Item DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology(2019-09) Pratt, Shawna L.; Zath, Geoffrey K.; Williamson, Kelly S.; Franklin, Michael J.; Chang, Connie B.The physiological heterogeneity of cells within a microbial population imparts resilience to stresses such as antimicrobial treatments and nutrient limitation. This resilience is partially due to a subpopulation of cells that can survive such stresses and regenerate the community. Microfluidic approaches now provide a means to study microbial physiology and bacterial heterogeneity at the single cell level, improving our ability to isolate and examine these subpopulations. Drop-based microfluidics provides a high-throughput approach to study individual cell physiology within bacterial populations. Using this approach, single cells are isolated from the population and encapsulated in growth medium dispersed in oil using a 15 μm diameter drop making microfluidic device. The drops are arranged as a packed monolayer inside a polydimethylsiloxane (PDMS) microfluidic device. Growth of thousands of individual cells in identical microenvironments can then be imaged using confocal laser scanning microscopy (CLSM). A challenge for this approach has been the maintenance of drop stability during extended time-lapse imaging. In particular, the drops do not maintain their volume over time during incubation in PDMS devices, due to fluid transport into the porous PDMS surroundings. Here, we present a strategy for PDMS device preparation that stabilizes drop position and volume within a drop array on a microfluidic chip for over 20 h. The stability of water-in-oil drops is maintained by soaking the device in a reservoir containing both water and oil in thermodynamic equilibrium. This ensures that phase equilibrium of the drop emulsion fluids within the porous PDMS material is maintained during drop incubation and imaging. We demonstrate the utility of this approach, which we label DropSOAC (DropStabilization On AChip), for time-lapse studies of bacterial growth. We characterize growth of Pseudomonas aeruginosa and its Δhpf mutant derivative during resuscitation and growth following starvation. We demonstrate that growth rate and lag time heterogeneity of hundreds of individual bacterial cells can be determined starting from single isolated cells. The results show that the DropSOAC capsule provides a high-throughput approach toward studies of microbial physiology at the single cell level, and can be used to characterize physiological differences of cells from within a larger population.Item Metagenomic Profiling of Microbial Pathogens in the Little Bighorn River, Montana(2019-03) Hamner, Steve; Brown, Bonnie L.; Hasan, Nur A.; Franklin, Michael J.; Doyle, John T.; Eggers, Margaret J.; Colwell, Rita R.; Ford, Tim E.The Little Bighorn River is the primary source of water for water treatment plants serving the local Crow Agency population, and has special significance in the spiritual and ceremonial life of the Crow tribe. Unfortunately, the watershed suffers from impaired water quality, with high counts of fecal coliform bacteria routinely measured during run-off events. A metagenomic analysis was carried out to identify potential pathogens in the river water. The Oxford Nanopore MinION platform was used to sequence DNA in near real time to identify both uncultured and a coliform-enriched culture of microbes collected from a popular summer swimming area of the Little Bighorn River. Sequences were analyzed using CosmosID bioinformatics and, in agreement with previous studies, enterohemorrhagic and enteropathogenic Escherichia coli and other E. coli pathotypes were identified. Noteworthy was detection and identification of enteroaggregative E. coli O104:H4 and Vibrio cholerae serotype O1 El Tor, however, cholera toxin genes were not identified. Other pathogenic microbes, as well as virulence genes and antimicrobial resistance markers, were also identified and characterized by metagenomic analyses. It is concluded that metagenomics provides a useful and potentially routine tool for identifying in an in-depth manner microbial contamination of waterways and, thereby, protecting public health.Item Expression and regulation of the Pseudomonas aeruginosa hibernation promoting factor(2018-10) Akiyama, Tatsuya; Williamson, Kerry S.; Franklin, Michael J.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 5ʹ‐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 (Phpf) within the rpoN gene. Phpf is active when the cells enter stationary phase, and expression from Phpf 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 5ʹ 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.Item Gene expression and protein levels of the stationary phase sigma factors, RpoS, in continuously-fed Pseudomonas aeruginosa biofilms(2001-05) Xu, Karen D.; Franklin, Michael J.; Park, C-H; McFeters, Gordon A.; Stewart, Philip S.Bacteria growing in biofilms experience gradients of environmental conditions, including varying levels of nutrients and oxygen. Therefore, bacteria within biofilms may enter distinct physiological states, depending on the surrounding conditions. In this study, rpoS expression and RpoS levels were measured as indicators of stationary phase growth within thick continuously-fed Pseudomonas aeruginosa biofilms. The level of rpoS expression in a 3-day-old biofilm was found to be three-fold higher than the average expression in stationary phase planktonic cultures. In planktonic cultures, oxygen limitation did not lead to increased levels of RpoS, suggesting that oxygen limitation was not the environmental signal causing increased expression of rpoS. These results suggest that bacteria within P. aeruginosa biofilms may exhibit stationary phase characteristics even when cultured in flow conditions that continually replenish nutrients.Item Compromised host defense on Pseudomonas aeruginosa biofilms: Characterization of neutrophil and biofilm interactions(2003-10) Jesaitis, A. J.; Franklin, Michael J.; Berglund, Deborah L.; Sasaki, Maiko; Lord, Connie I.; Bleazard, Justin Brock; Duffy, James E.; Beyenal, Haluk; Lewandowski, ZbigniewPseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on tissues and other surfaces. We characterized the interaction of purified human neutrophils with P. aeruginosa, growing in biofilms, with regard to morphology, oxygen consumption, phagocytosis, and degranulation. Scanning electron and confocal laser microscopy indicated that the neutrophils retained a round, unpolarized, unstimulated morphology when exposed to P. aeruginosa PAO1 biofilms. However, transmission electron microscopy demonstrated that neutrophils, although rounded on their dorsal side, were phagocytically active with moderate membrane rearrangement on their bacteria-adjacent surfaces. The settled neutrophils lacked pseudopodia, were impaired in motility, and were enveloped by a cloud of planktonic bacteria released from the biofilms. The oxygen consumption of the biofilm/neutrophil system increased 6- and 8-fold over that of the biofilm alone or unstimulated neutrophils in suspension, respectively. H(2)O(2) accumulation was transient, reaching a maximal measured value of 1 micro M. Following contact, stimulated degranulation was 20-40% (myeloperoxidase, beta-glucuronidase) and 40-80% (lactoferrin) of maximal when compared with formylmethionylleucylphenylalanine plus cytochalasin B stimulation. In summary, after neutrophils settle on P. aeruginosa biofilms, they become phagocytically engorged, partially degranulated, immobilized, and rounded. The settling also causes an increase in oxygen consumption of the system, apparently resulting from a combination of a bacterial respiration and escape response and the neutrophil respiratory burst but with little increase in the soluble concentration of H(2)O(2). Thus, host defense becomes compromised as biofilm bacteria escape while neutrophils remain immobilized with a diminished oxidative potential.Item Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin(2003-01) Walters, Marshall C., III; Roe, Frank L.; Bugnicourt, Amandine; Franklin, Michael J.; Stewart, Philip S.The roles of slow antibiotic penetration, oxygen limitation, and low metabolic activity in the tolerance of Pseudomonas aeruginosa in biofilms to killing by antibiotics were investigated in vitro. Tobramycin and ciprofloxacin penetrated biofilms but failed to effectively kill the bacteria. Bacteria in colony biofilms survived prolonged exposure to either 10 micro g of tobramycin ml(-1)or 1.0 micro g of ciprofloxacin ml(-1). After 100 h of antibiotic treatment, during which the colony biofilms were transferred to fresh antibiotic-containing plates every 24 h, the log reduction in viable cell numbers was only 0.49 +/- 0.18 for tobramycin and 1.42 +/- 0.03 for ciprofloxacin. Antibiotic permeation through colony biofilms, indicated by a diffusion cell bioassay, demonstrated that there was no acceleration in bacterial killing once the antibiotics penetrated the biofilms. These results suggested that limited antibiotic diffusion is not the primary protective mechanism for these biofilms. Transmission electron microscopic observations of antibiotic-affected cells showed lysed, vacuolated, and elongated cells exclusively near the air interface in antibiotic-treated biofilms, suggesting a role for oxygen limitation in protecting biofilm bacteria from antibiotics. To test this hypothesis, a microelectrode analysis was performed. The results demonstrated that oxygen penetrated 50 to 90 micro m into the biofilm from the air interface. This oxic zone correlated to the region of the biofilm where an inducible green fluorescent protein was expressed, indicating that this was the active zone of bacterial metabolic activity. These results show that oxygen limitation and low metabolic activity in the interior of the biofilm, not poor antibiotic penetration, are correlated with antibiotic tolerance of this P. aeruginosa biofilm system.