Heterogeneous rpoS and rhlR mRNA Levels and 16S rRNA/rDNA (rRNA Gene) Ratios within Pseudomonas aeruginosa Biofilms, Sampled by Laser Capture Microdissection

Resuscitation of Pseudomonas aeruginosa from dormancy requires hibernation promoting factor (PA4463) for ribosome preservation

“…Because rRNA is rapidly degraded when not associated with ribosomes (40), quantification of rRNA provides an estimate of the ribosome copy number per cell. From that study, we showed that exponentially growing P. aeruginosa had ∼70,000 ribosomes per cell, whereas cells in the slowly growing subpopulation of biofilms had ∼20,000 ribosomes per cell (39). This relatively high concentration of ribosomes in the slow-growing cells was maintained even for cells with little transcriptional activity, suggesting that ribosomes are preserved in the cells in the interior of the biofilms.…”

“…In a previous study, we measured the ribosome abundance of cells at different vertical strata within P. aeruginosa biofilms by microdissecting the biofilms and then assaying 16S rRNA amounts from the different microzones using RT-qPCR (39). Because rRNA is rapidly degraded when not associated with ribosomes (40), quantification of rRNA provides an estimate of the ribosome copy number per cell.…”

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

“…The results indicated that the abundances of individual mRNA transcripts may vary by several orders of magnitude, even for cells in close proximity to each other. The previous results also suggested that the top of P. aeruginosa biofilms contains cells in transition to stationary phase, while the cells at the bottom of the biofilm may be in a slow-growth or dormant state (51).…”

“…Therefore, these approaches provide transcriptome or proteome information as an average of that for the entire biofilm and may not be adequate for discovering differences in gene expression that occur at localized sites and due to various environmental conditions or stochastic gene expression events. Previously, we used laser capture microdissection (LCM) to isolate clusters of cells from within P. aeruginosa biofilms and quantified the rRNA and mRNA abundances of individual genes from the isolated cell groups (36,51). The results indicated that the abundances of individual mRNA transcripts may vary by several orders of magnitude, even for cells in close proximity to each other.…”

Chronic Wound Biofilm Model

“…These included rpoS, which is implicated in the morphology and antibiotic tolerance of biofilms, 49 and rhlR/ aprA, previously linked to quorum sensing and biofilms. 50,51 The expression of rhlR, rpoS, and arpR was significantly upregulated in our biofilm system. 12 This recent work from our laboratory is the first to provide insight into the progressive development of host-microbe interactions resulting in loss of barrier function of the repaired skin.…”