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.
|Advisor:||Franklin, Michael J.|
|Commitee:||Babbitt, Randall W., Mumey, Brendan M., Starkey, Jean, Teintze, Martin|
|School:||Montana State University|
|School Location:||United States -- Montana|
|Source:||DAI-B 69/12, Dissertation Abstracts International|
|Subjects:||Molecular biology, Microbiology|
|Keywords:||Biofilms, Laser capture microdissection microscopy, PCR, Pseudomonas aeruginosa, QRT-PCR, Ribosomes|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be