Gene transfer agents promote survival and DNA repair during stationary phase for Caulobacter crescentus
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gozzi-kgozzi-phd-biology-2022-thesis .pdf
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Thesis PDF
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5.04 MB
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Author(s)
Gozzi, Kevin Robert
Advisor(s)
Laub, Michael T.
Date Issued
September 2022
Publisher
Massachusetts Institute of Technology
Abstract
Gene transfer agents (GTAs) are prophage-like entities found in many bacterial genomes that cannot propagate themselves and instead package ~5-15 kbp fragments of the host genome that can then be transferred to related recipient cells. Although suggested to facilitate horizontal gene transfer in the wild, no clear physiological role for GTAs has been elucidated. Here, I demonstrate that the α-proteobacterium Caulobacter crescentus produces bona fide GTAs. The production of Caulobacter GTAs is tightly regulated by a newly identified transcription factor, RogA, that represses gafYZ, the direct activators of GTA synthesis. Cells lacking rogA or expressing gafYZ produce GTAs harboring an ~8.3 kbp fragment of the genome that can, after cell lysis, be transferred into recipient cells. Notably, I found that GTAs promote the survival of Caulobacter in stationary phase and following DNA damage by providing recipient cells a template for homologous recombination-based repair. This function may be broadly conserved in other GTA-producing organisms and explain the prevalence of this unusual horizontal gene transfer mechanism. I also found that GTAs act to strongly activate the SOS-independent DNA damage response. Using a combination of biochemical and genetic techniques, I characterize the central regulator DriD and identify that DriD specifically binds ssDNA as a direct ligand. ssDNA acts as an allosteric regulator to induce a conformational change to activate DriD, allowing for DriD to promote transcription at target promoters involved in survival to DNA damage. DriD may serve as a model for other WYLdomain containing proteins, a poorly understood class of proteins commonly found associated with CRISPR loci across bacteria.
MIT Department
Massachusetts Institute of Technology. Department of Biology
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