Browsing by Author "Perez-Osorio, Ailyn C."

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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 Î”rmf 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.
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 Âµm 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.
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 Î²-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.