Heterogeneous rpoS and rhlR mRNA levels and 16S rRNA/rDNA ratios within Pseudomonas aeruginosa biofilms, sampled by laser capture microdissection

Abstract

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.