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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 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.