Danielle DeCesaris
Advanced Summer Research Scholar
Major: Microbiology
Mentor: Gail Hardy
Many pathogenic bacteria form multicellular communities called biofilms, which offer support for bacterial proliferation and protection from hazardous environmental conditions. A requirement for biofilm formation is cellular attachment to abiotic or biotic surfaces. Agrobacterium tumefaciens, the causative agent of crown gall disease in plants, initiates biofilm formation by adhering to surfaces as a result of interactions between bacterial cell surface adhesins and the target surface. One of these adhesins is the unipolar polysaccharide (UPP), which is present on a single pole of an A. tumefaciens cell. Previous genetic screens successfully identified UPP-associated genes; however, the previous screens were not ideal for detection of functionally redundant genes or genes necessary for anchoring of the UPP to the cell surface. To improve upon prior screens, we focused on optimizing a genetic screen, TnBarseq, in A. tumefaciens previously used to study the holdfast adhesin of the biofilm forming bacteria Caulobacter crescentus.
TnBarseq utilizes a process called transposon mutagenesis where mobile genetic elements, transposons, insert themselves randomly into the genome, disabling the genes that they land in. The transposons designed for this screen are each linked to short, unique DNA sequences called barcodes. By mutagenizing A. tumefaciens with the barcoded transposons and sequencing the transposons, each unique barcoded transposon will be associated with a gene. The barcoded transposon mutant library will be passaged through a multi-day competitive fitness assay with cheesecloth to enrich for adhesion mutants resulting in UPP mutants remaining in liquid culture while adherent cells with functional UPP are bound to the cheesecloth and removed. Barcode sequencing each day’s culture will identify barcodes enriched during passaging, indicating the associated transposons were inserted into genes necessary for UPP, or decreased, indicating that the associated gene either was unrelated to UPP or potentially decreases attachment.
We have focused on optimizing this powerful genetic screen for usage in A. tumefaciens in order to identify genes of various functions related to the UPP and adherence and to view how they affect adhesion over time.
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