Quantitative assessment of localized growth rates and gene expression patterns in Pseudomonas aeruginosa biofilms
PÃƒÆ’Ã‚Â©rez-Osorio, Ailyn Cecilia.
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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.