Date of Award
1-1-2025
Document Type
Dissertation
Degree Name
Ph.D. in Biological Science
First Advisor
Patrick Curtis
Second Advisor
Colin Jackson
Third Advisor
Erik Hom
School
University of Mississippi
Relational Format
dissertation/thesis
Abstract
The alphaproteobacterium Caulobacter crescentus with a unique dimorphic lifestyle is a model organism for studying prokaryotic development. An important cellular structure is the hair-like appendage called pilus, made up of monomeric subunits called pilins. The pilus helps C. crescentus in surface sensing and attachment, leading to cellular differentiation. Despite these advantages, the C. crescentus cell expresses the pilus filament on its surface for a very short period in its life cycle, during the motile swarmer stage. This study examined the precisely-timed transcriptional regulation of the pilin-encoding gene pilA, and uncovered the impact it has on bacterial survival.
CtrA, a global transcriptional regulator, activates pilA expression by binding to four sites in its promoter. Reporter assays showed that the TSS-proximal Site 1 drives pilA expression and the upstream Sites 2, 3 and 4 repress transcription. Surprisingly, CtrA showed lower binding affinity to Site 1, and higher affinities to Sites 2 and 3, in EMSA experiments. Reporter assays and western blots showed that mutations in Sites 2 and 3 preventing CtrA binding resulted in earlier pilA expression and PilA accumulation, as well as earlier pilus filament expression as observed in streptavidin bead-binding assay. Earlier pilus expression led to reduced survival against ϕCbK bacteriophage infection indicating that C. crescentus delays pilA transcription using additional CtrA-binding sites to increase the probability of its survival against its natural bacteriophage.
Furthermore, pilA transcription was found to be repressed by the methylation-cycle repressor SahR, as well as inhibited by methylation cycle enzymes AhcY and MetK. In addition to AhcY and MetK, SahR showed an interaction with a novel NYN-domain protein CnfP in two-hybrid assays. CnfP also inhibited pilA expression and showed a global effect on transcriptional profiles in RNAseq experiments. EMSAs showed that SahR binds to the pilA promoter specifically in the presence or absence of CtrA, and this binding was reversed in the presence of S-adenosylhomocysteine, resulting in increased CtrA occupancy for the pilA promoter. Together, these results led to a model where a surge in SAH levels in the predivisional stage causes dissociation of SahR from the pilA promoter and allows pilA transcription by CtrA.
Recommended Citation
Rijal, Anurag, "Study of The Mechanism and Physiological Impact of Transcriptional Regulation of Pilin-Encoding Pila Gene in Caulobacter Crescentus" (2025). Electronic Theses and Dissertations. 3370.
https://egrove.olemiss.edu/etd/3370