Almost all bacteria contain genes that may lead to their growth stasis and death.Normally, these toxins are believed to be neutralized with their cognate antitoxinsfrom a toxin-antitoxin (TA) operon. These modules are also abundant in pathogenic bacteria suggesting a role for them both in normal bacterial physiology and pathogenicity. Their functions have been subject to intense debates. Due to the cell killing capability of the toxin and the gene silencing capability of the antitoxin, they have been utilized for basic research, biotechnology and medical applications. However, further advancements of these applications have been impeded by our limited knowledge of the biology of TAs. Among these TA systems is the Ibs/Sib (A-E) family. Here, we discuss our efforts in characterizing these systems, with a focus on the IbsC/SibC member. Studying them has shown to not be straightforward due to the complexity of their underlying mechanisms and the current approaches being laborious and lacking sensitivity to be applied to these low abundant molecules. We have developed fluorescence-based platforms to take advantage of sensitive and high throughput and resolution techniques such as Fluorescence Assisted Cell Sorting (FACS) to study these molecules instead of
relying on traditional culturing methods. While developing these platforms, we gained insights about the biology and regulation of these molecules. To expand this knowledge, we actively pursued investigating the regulation of these molecules at the transcriptional and post-transcriptional levels, both in their native context and in artificial systems. The rest of this thesis summarizes our efforts in solving one of the biggest pieces of the Ibs/Sib puzzle, namely their physiological expressions. With the strategies we have optimized for specific detection of these low abundance molecules, and the knowledge of their biology and regulation presented, we are now at an exciting phase to interrupt the long pause in the study of functions by these molecules and advancement of TA-based applications. / Thesis / Doctor of Philosophy (PhD) / Almost all bacteria contain genes that may lead to their growth stasis or death. Normally, these toxins are believed to be neutralized with their cognate antitoxins. In spite of the efforts to understand these toxin-antitoxin (TA) systems, their physiological roles are subject to intense debate. These systems are hard to study mainly because 1) they are only activated under specific conditions and 2) they are low in abundance. Current approaches are not high throughput and sensitive enough. In this thesis, we developed DNA constructs, bacterial strains and methodologies to facilitate the study of these molecules, particularly the Ibs-Sib family. We next employed these tools to gain a fundamental knowledge of their
expression under different conditions, which revealed surprising information about the function of these molecules. We believe that future studies can greatly benefit from the tools offered here to tremendously enhance our understanding of these systems and lead to useful applications.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/24983 |
| Date | January 2019 |
| Creators | Jahanshahi, Shahrzad |
| Contributors | Li, Yingfu, Biomedical Engineering |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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