Center for Biofilm Engineering (CBE)
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/9334
At the Center for Biofilm Engineering (CBE), multidisciplinary research teams develop beneficial uses for microbial biofilms and find solutions to industrially relevant biofilm problems. The CBE was established at Montana State University, Bozeman, in 1990 as a National Science Foundation Engineering Research Center. As part of the MSU College of Engineering, the CBE gives students a chance to get a head start on their careers by working on research teams led by world-recognized leaders in the biofilm field.
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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 Heterogeneous rpoS and rhlR mRNA levels and 16S rRNA/rDNA ratios within Pseudomonas aeruginosa biofilms, sampled by laser capture microdissection(2010-03) Perez-Osorio, Ailyn C.; Williamson, Kerry S.; Franklin, Michael J.The local environmental conditions in biofilms are dependent on the impinging aqueous solution, chemical diffusion, and the metabolic activities of cells within the biofilms. Chemical gradients established in biofilms lead to physiological heterogeneities of bacterial gene expression. Previously, we used laser capture microdissection (LCM) and quantitative RT-PCR to target defined biofilm subpopulations for gene expression studies. Here, we combined that approach with quantitative PCR of bacterial DNA to normalize gene expression per cell. By comparing the ratio of 16S rRNA to 16S rDNA, we demonstrate that cells at the top of thick Pseudomonas aeruginosa biofilms have 16S rRNA/genome ratios similar to cells in a transition between exponential and stationary phase. Cells in the middle and bottom layers of these biofilms have ratios that are not significantly different from stationary phase planktonic cultures. Since much of the biofilm appeared to be in a stationary phase-like state, we analyzed local amounts of the stationary phase sigma factor, rpoS, and a quorum sensing regulator, rhlR, per cell. Surprisingly, the amount of rpoS mRNA was greatest at the top of these biofilms at the air-biofilm interface. Less than one rpoS mRNA transcript per cell was observed in the middle or base of the biofilms. The rhlR mRNA content was also greatest at the top of these biofilms, with little detectable rhlR expression at the middle or bottom of the biofilms. While cell density is slightly greater at the bottom of the biofilms, expression of this quorum sensing regulator occurs primarily at the top of the biofilms, where cell metabolic activity is greatest, as indicated by the local expression of the housekeeping gene, acpP and by expression from a constitutive Ptrc promoter. The results indicate that in thick P. aeruginosa biofilms, cells in the 30 µm adjacent to the air-biofilm interface actively express genes associated with stationary phase, while cells in the interior portions do not express these genes, and therefore are in a late stationary phase-like state and are possibly dormant.Item Heterogeneity in Pseudomonas aeruginosa biofilms includes expression of ribosome hibernation factors in the antibiotic-tolerant subpopulation and hypoxia-induced stress response in the metabolically active population(2012-02) Williamson, Kerry S.; Richards, Lee A.; Perez-Osorio, Ailyn C.; Pitts, Betsey; McInnerney, Kathleen; Stewart, Philip S.; Franklin, Michael J.Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptome responses of Pseudomonas aeruginosa growing in biofilms by using a microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNA levels for these housekeeping genes were low in cells at the bottom of the biofilms. Selective green fluorescent protein (GFP) labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNA levels for genes regulated by the hypoxia-induced regulator Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-term anoxia. Transcripts for ribosomal proteins were associated primarily with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (the rmf and PA4463 genes) at the bottom of the biofilms. The dormant biofilm cells of a P. aeruginosa Δrmf strain had decreased membrane integrity, as shown by propidium iodide staining. Using selective GFP labeling and cell sorting, we show that the dividing cells are more susceptible to killing by tobramycin and ciprofloxacin. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state.