S19: Laser microdissection of microbial biofilms

Biofilms are communities of microorganisms and their secreted exopolymers associated with surfaces. As nutrients and oxygen are utilized by the cells and microbial waste products are generated, concentration gradients of chemicals are established, creating heterogeneous environmental conditions throughout the biofilms. The cells respond to their local environment through gene induction/repression, resulting in cells in a wide range of physiological states.  Cellular physiological heterogeneity affects microbial processes, including the tolerance of cells to antimicrobials.  We characterized heterogeneous gene expression within biofilms by combining laser capture microdissection (LCM) with transcriptomic analyses of cell subpopulations. Using quantitative RT-PCR, we analyzed the local abundance of RNA for a gene involved in membrane fatty acid production and for ribosomal RNA in P. aeurginosa biofilms.  The results indicated that membrane biosynthetic activity (cell growth) was associated with the top 30 µm of the biofilm. The abundance of ribosomal RNA was relatively uniform throughout the biofilms.  Using DNA as a normalizing factor, we quantified the amounts of mRNA on a per cell basis.  Cells at the air-biofilm interface had the highest abundance of mRNA for rpoS and rhlR, genes associated with stationary phase growth.  These results, combined with the cellular ratios of rRNA/genome suggested that cells at the top of the biofilms were in a transition phase between exponential and stationary phase.  Cells in the middle and bottom layers were likely dormant. Finally, we combined LCM with microarray analyses to provide information on expression processes that may be unique to localized regions within the biofilms.