Center for Biofilm Engineering

Thesis Abstract:  

"Quantitative assessment of localized growth rates and gene expression patterns in Pseudomonas aeruginosa biofilms" 

      This dissertation work provides evidence of heterogeneity in the distribution of
gene expression and growth rates among surface associated cells of Pseudomonas
aeruginosa. Currently, methodologies used for characterizing biofilm heterogeneity are
constrained by the need of in vitro biofilm growth and by the need to genetically
manipulate bacteria.
      This dissertation describes findings obtained by using LCMM, qRT-PCR, qPCR
and microarrays. Through combining LCMM with qRT-PCR a housekeeping gene and
two quorum sensing induced genes were found to be differentially expressed at the
periphery of P. aeruginosa biofilms. qPCR also enabled the growth rate of cells in
discrete locations of biofilms to be determined. Cells localized to the deep layers of
biofilms were found in a growth state analogous to stationary phase in planktonic
cultures, while cells localized to the biofilm periphery were slightly more active with
growth rates that approached cells growing exponentially in planktonic cultures. By
elucidating the growth rates of subpopulations within the biofilm it was subsequently
possible to determine that the most active cells had approximately 7 copies of the mRNA
of housekeeping and stationary phase associated genes. Each of the least active cells,
those found in the deeper sections of the biofilms, had less than one copy of any of the
mRNAs measured. No significant differences in the distribution of 16S rRNA were found
along the sections analyzed. The microarray studies revealed several genes, known to be
involved in the pathogenesis of P. aeruginosa, to be undergoing active transcription in
young biofilms under conditions of low calcium concentrations. This is significant
because calcium homeostasis is known to be out of balance in the lungs of cystic fibrosis
patients, where P. aeruginosa biofilms grow causing life threatening infections.
      These results suggest that spatial and temporal heterogeneity within biofilms
underscores their ability to not only survive in diverse and sometimes harsh
environmental conditions, but to exploit those environments. The methods described in
this work are suitable for characterizing heterogeneity of gene expression and growth rate
in biofilms collected from their natural environment. These also represent an alternative
method for assessing the distribution of populations in multispecies biofilms.
 

Quantitative assessment of localized growth rates and gene expression patterns in Pseudomonas aeruginosa biofilms, Thesis Defense by Ailyn Cecilia Pérez-Osorio, PhD  Candidate in Microbiology, Montana State University, January 2009.