Sessile bacterial communities which form on the solid surface or solid-liquid interface
are known as biofilms. Both single species and multispecies biofilms are characterized
by an extracellular matrix of polymeric substances which gives them several hundred
times more antibiotic resistances than a planktonic bacterial culture. Though bacteria
are the most common causative agent of various diseases, because of the high
antibiotic resistance, biofilms cause complications of various diseases like cystic
fibrosis, prosthetic valve endocarditis, chronic pulmonary diseases, catheter-associated
urinary tract infections and several other diseases. From past studies,
quorum sensing has been established as a novel target mechanism against biofilms; in
this study, the two-component signal transduction systems (2CSTSs) have been
focused. Once better understood, 2CSTSs can serve as a novel drug target and
prevention mechanism for biofilm associated diseases.
According to prior high-throughput experiments and phenotype microarray
experiments by our lab, several 2CSTSs like OmpR-EnvZ, RcsCDB along with the
global regulator FlhD/FlhC were hypothesized to have an important effect on various
developmental stages of biofilm formation. From that past study, we postulated that
acetate metabolism may be an important aspect for biofilm formation. In this study,
we tested and confirmed this hypothesis. We observed biofilms formed by several
mutants in 2CSTS, as well as mutants in acetate metabolism, using Scanning Electron
Microscopy (SEM). We found quantitative and qualitative differences in the biofilm of
the acetate mutants when compared to their isogenic parental Escherichia coli strain.
An additional mutation in rcsB with acetate mutant strains forms less clumpy biofilms
whereas an additional mutation in dcuR results in the formation of less biofilms. So
the structural and the quantitative differences of acetate mutant biofilms depend on
additional mutations in rcsB and dcuR. Though a number of studies have been done on the temporal gene expression within biofilms, spatial gene expression of the mature biofilm is a big gap of knowledge. The
future aim of this study is to study the temporal as well as the spatial gene expression
of different 2CSTSs in the biofilm. In my MS thesis, I have constructed selected
promoter fused GFP /RFP plasmids and some other fusion plasmids were purchased
from the promoter collections from Open Biosystems, lastly E. coli AJW678 bacterial
strains were transformed with these GFP /RFP fused plasmids. A 96 well microtiter
plate assay was performed to study the temporal expression from the promoters by
quantifying the fluorescence intensity in the planktonic culture. According to this
experiment, the highest expression of flhD was after 20 hours whereas, the expression
of ompR increases up to 7 days, which indicates that the flhD expresses earlier than
ompR. The decreasing phase of flhD expression was paralleled by the sharpest
increase in ompR expression as phosphorylated OmpR is an inhibitor of flhD
expression. / National Institutes of Health (NIH grant 1R15AI089403) / United States. Animal and Plant Health Inspection Service
| Identifer | oai:union.ndltd.org:ndsu.edu/oai:library.ndsu.edu:10365/29330 |
| Date | January 2011 |
| Creators | Samanta, Priyankar |
| Publisher | North Dakota State University |
| Source Sets | North Dakota State University |
| Detected Language | English |
| Type | text/thesis |
| Format | application/pdf |
| Rights | NDSU policy 190.6.2, https://www.ndsu.edu/fileadmin/policy/190.pdf |
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