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

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 in bacterial gene expression. Previously, we used laser capture microdissection (LCM) and quantitative reverse transcription (RT)-PCR to target defined biofilm subpopulations for gene expression studies. Here, we combined this approach with quantitative PCR of bacterial DNA to normalize the amount of gene expression per cell. By comparing the ratio of 16S rRNA to 16S rDNA (rRNA gene), we demonstrated that cells at the top of thick Pseudomonas aeruginosa biofilms have 16S rRNA/genome ratios similar to those of cells in a transition from the exponential phase to the stationary phase. Cells in the middle and bottom layers of these biofilms have ratios that are not significantly different from those of stationary-phase planktonic cultures. Since much of each biofilm appeared to be in a stationary-phase-like state, we analyzed the local amounts of the stationary-phase sigma factor rpoS gene and the quorum-sensing regulator rhlR gene per cell. Surprisingly, the amount of rpoS mRNA was largest at the top of the 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 the biofilms, and there was little detectable rhlR expression at the middle or bottom of the biofilms. While the cell density was slightly greater at the bottom of the biofilms, expression of the quorum-sensing regulator occurred primarily at the top of the biofilms, where the cell metabolic activity was greatest, as indicated by local expression of the housekeeping gene acpP and by expression from a constitutive P(trc) promoter. The results indicate that in thick P. aeruginosa biofilms, cells in the 30 microm 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 may be dormant.