| dc.description.abstract | Biological processes are inherently noisy, and such stochasticity can result in significant
cell to cell variability in isogenic bacterial populations. Functionally, cellular
heterogeneity can have significant impact on the survival of the bacterial population in
nature, as well as in our ability to understand, predict and control them. Since most of our
knowledge in biology comes from ensemble experiments which measure average
behaviour, there is a need to design studies that can identify and characterize variability
present in the population. In this thesis, cellular heterogeneity and its consequences in
bacterial inducible gene expression systems and in bacterial response to antimicrobial
treatment was studied, using a combination of complementary bulk and single cell assays.
In the first part of the thesis, antimicrobial action on bacterial populations was examined.
Bacterial resistance to antibiotics is a major threat to public health today. Significant
efforts in research and health policy have been directed towards understanding and
combatting this issue. While genetic mechanisms of resistance development are well
understood, there are several phenotypic responses such as persistence, tolerance and
heteroresistance resulting due to transient variations within sub populations of cells that
allow them to survive an antimicrobial stress. Clinically, these phenomena have been
linked to recurrence of various infections such as tuberculosis, cystic fibrosis and urinary
tract infections. To overcome these issues, several alternatives to antibiotics are being
tested, including antimicrobial peptides (AMPs). These molecules are part of the innate
immune system, known for their broad-spectrum activity and immunomodulatory effects.
While they have shown promising results in combatting resistant bacteria, the efficacy of
these peptides on persistent and heteroresistant cells haven’t been thoroughly
investigated.
With this background, the action of antibiotics and AMPs on bacterial populations was
investigated for their ability to kill such phenotypically different cells. Significant
difference was observed in the efficacy of these antimicrobials, when tested on actively
growing and growth arrested E. coli. While, both are equally efficient on exponentially
growing cells, diverse responses were observed with stationary phase cells. Variability
existed even amongst the peptides. Particularly, colistin was found to induce
heteroresistant behaviour. This observation was further studied and found to have link
with hypermutations in the bacterial genome. In addition, cross resistance to other
antimicrobials was also observed. Finally, single cell imaging of this heteroresistant
population confirmed the variability in response of individual cells to colistin treatment.
Overall, deeper understanding of the interaction between bacterial cells and these
antimicrobials was obtained, which can form a basis for designing efficient treatment
strategies in the future.
In the second part of the thesis, heterogeneity of gene expression from the inducible
arabinose operon in E. coli was studied. The arabinose operon is known for its graded
response to arabinose concentrations at the population level. At the individual cell level,
however, it exhibits an all-or-none response due to stochasticity in the numbers of the
arabinose import proteins, AraE and AraFGH. By using different mutant strains of E. coli
with varying levels of these transporters, the gene expression response from this operon
was characterized for its uniformity, tunability and sensitivity to a range of arabinose
concentrations. Varying the levels of each transporter was found to influence the system
in a different way, and maximising for one property usually led to a trade-off with another
property. In addition, analysis of the temporal behaviour of the gene expression process
using a fast-degrading green fluorescent protein (GFP) reporter revealed a dependency on
the physiological state of the cells. Overall, information obtained from this study will aid
in engineering of inducible gene expression systems for better control | en_US |
