Scholarly Work - Center for Biofilm Engineering
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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 Resuscitation of Pseudomonas aeruginosa from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation(2017-03) Akiyama, Tatsuya; Williamson, Kerry S.; Schaefer, Robert; Pratt, Shawna; Chang, Connie B.; Franklin, Michael J.Pseudomonas aeruginosa biofilm infections are difficult to treat with antibiotic therapy in part because the biofilms contain subpopulations of dormant antibiotic-tolerant cells. The dormant cells can repopulate the biofilms following alleviation of antibiotic treatments. While dormant, the bacteria must maintain cellular integrity, including ribosome abundance, to reinitiate the de novo protein synthesis required for resuscitation. Here, we demonstrate that the P. aeruginosa gene PA4463 [hibernation promoting factor (HPF)], but not the ribosome modulation factor (PA3049), is required for ribosomal NA preservation during prolonged nutrient starvation conditions. Single-cell–level studies using fluorescence in situ hybridization (FISH) and growth in microfluidic drops demonstrate that, in the absence of hpf, the rRNA abundances of starved cells decrease to levels that cause them to lose their ability to resuscitate from starvation, leaving intact nondividing cells. P. aeruginosa defective in the stringent response also had reduced ability to resuscitate from dormancy. However, FISH analysis of the starved stringent response mutant showed a bimodal response where the individual cells contained either abundant or low ribosome content, compared with the wild-type strain. The results indicate that ribosome maintenance is key for maintaining the ability of P. aeruginosa to resuscitate from starvation-induced dormancy and that HPF is the major factor associated with P. aeruginosa ribosome preservation.Item Draft genome sequence and description of Janthinobacterium sp. strain CG3, a psychrotolerant antarctic Supraglacial stream bacterium(2013-11) Smith, Heidi J.; Akiyama, Tatsuya; Foreman, Christine M.; Franklin, Michael J.; Woyke, Tanja; Teshima, H; Davenport, K.; Daligault, H.; Erkkila, T.; Goodwin, L. A.; Gu, W.; Xu, Yan; Chain, P. S.Here we present the draft genome sequence of Janthinobacterium sp. strain CG3, a psychrotolerant non-violacein-producing bacterium that was isolated from the Cotton Glacier supraglacial stream. The genome sequence of this organism will provide insight into the mechanisms necessary for bacteria to survive in UV-stressed icy environments.Item The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP(2016-01) Guragain, Manita; Kinga, Michelle M.; Williamson, Kerry S.; Akiyama, Tatsuya; Khanam, Sharmily; Perez-Osorio, Ailyn C.; Patrauchan, Marianna A.; Franklin, Michael J.Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or with artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca2+ through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+ followed by its removal to the basal sub-micromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+ homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa to elevated [Ca2+] in both planktonic cultures and in biofilms. Among the genes induced by CaCl2 in strain PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators, phoPQ and pmrAB, were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR, and performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 (here called carO) and the inner membrane-anchored five-bladed β-propeller protein, PA0327 (here called carP). Mutations in both carO and carP affected Ca2+ homeostasis, reducing the ability of P. aeruginosa to export excess Ca2+. In addition, a mutation in carP had a pleotropic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+, and responding through its regulatory targets that modulate Ca2+ homeostasis, surface-associated motility, and production of the virulence factor, pyocyanin. IMPORTANCE During infectious disease, Pseudomonas aeruginosa encounters environments with high calcium (Ca2+) concentration, yet the cells maintain intracellular Ca2+ at levels that are orders of magnitude less than the external environment. In addition, Ca2+ signals P. aeruginosa to induce production of several virulence factors. Compared to eukaryotes, little is known about how bacteria maintain Ca2+ homeostasis, or how Ca2+ acts as a signal. In this study, we identified a two-component regulatory system in P. aeruginosa PAO1, termed CarRS, that is induced at elevated Ca2+. CarRS modulates Ca2+ signaling and Ca2+ homeostasis through its regulatory targets, CarO and CarP. The results demonstrate that P. aeruginosa uses a two-component regulatory system to sense external Ca2+, and relays that information for Ca2+-dependent cellular processes.Item New technologies for studying biofilms(2015-08) Franklin, Michael J.; Chang, Connie B.; Akiyama, Tatsuya; Bothner, BrianThe results of recent biofilm characterizations have helped reveal the complexities of these surface-associated communities of microorganisms. The activities of the cells and the structure of the extracellular matrix material demonstrate that biofilm bacteria engage in a variety of physiological behaviors that are distinct from planktonic cells (1 – 3 ). For example, bacteria in biofilms are adapted to growth on surfaces, and most produce adhesins and extracellular polymers that allow the cells to firmly adhere to the surfaces or to neighboring cells ( 4 – 6 ). The extracellular material of biofilms contains polysaccharides, proteins, and DNA that form a glue-like substance for adhesion to the surface and for the three-dimensional (3D) biofilm architecture ( 4 ). The matrix material, although produced by the individual cells, forms structures that provide benefits for the entire community, including protection of the cells from various environmental stresses ( 7 – 9 ). Biofilm cells form a community and engage in intercellular signaling activities ( 10 – 19 ). Diffusible signaling molecules and metabolites provide cues for expression of genes that may benefit the entire community, such as genes for production of extracellular enzymes that allow the biofilm bacteria to utilize complex nutrient sources ( 18 , 20 – 22 ). Biofilm cells are not static. Many microorganisms have adapted to surface-associated motility, such as twitching and swarming motility ( 23 – 28 ). Cellular activities, including matrix production, intercellular signaling, and surface-associated swarming motility suggest that biofilms engage in communal activities. As a result, biofilms have been compared to multicellular organs where cells differentiate with specialized functions ( 2 , 29 ). However, bacteria do not always cooperate with each other. Biofilms are also sites of intense competition. The bacteria within biofilms compete for nutrients and space by producing toxic chemicals to inhibit or kill neighboring cells or inject toxins directly into neighboring cells through type VI secretion ( 30 – 33 ). Therefore, biofilm cells exhibit both communal and competitive activities.Item Genome sequence of Janthinobacterium sp. CG23_2, a violacein-producing isolate from an Antarctic supraglacial stream.(2016-01) Smith, Heidi J.; Foreman, Christine M.; Akiyama, Tatsuya; Franklin, Michael J.; Devitt, N. P.; Ramaraj, ThiruvaranganHere, we present the draft genome sequence for the violacein-producing Janthinobacterium sp. CG23_2 isolated from an Antarctic supraglacial stream. The genome is ~7.85 Mb, with a G+C content of 63.5%. The genome includes 7,247 candidate protein coding genes, which may provide insight into UV tolerance mechanisms.