Undergraduate Honors Thesis Projects

Date of Award

4-28-2024

Document Type

Honors Paper

Degree Name

Biochemistry and Molecular Biology-BS

Department

Biology & Earth Science

Advisor

Dr. Jennifer Bennett

First Committee Member

Dr. Jennifer Bennett

Second Committee Member

Dr. John Tansey

Third Committee Member

Dr. Anthony DeStefanis

Keywords

Streptomyces bacteria, Microbiology, Agricultural pest, Cyclic-di-GMP

Subject Categories

Agricultural Science | Higher Education | Pathogenic Microbiology

Abstract

Streptomyces scabies is a gram-positive bacterium that causes a scab disease in potatoes. The bacterium itself is filamentous and produces spores which allows it to spread and reproduce. The rmdA, rmdB, and cdgC genes in S. scabies are used in the cyclic-di-GMP signaling pathway. This pathway is responsible for regulating many processes including cell cycle progression and differentiation. RmdA and B are phosphodiesterases that break down the second messenger, cyclic-di-GMP. CdgC is thought to relate to the cyclic di-GMP pathway by making cyclic dimeric guanosine monophosphate. Cyclic dimeric guanosine monophosphate is a second messenger that helps to regulate cellular processes within bacteria. The specific question addressed is what role rmdA, rmdB, and cdgC play in the development of S. scabies and their ability to cause disease in potatoes. One aim of the project will result in the deletion of the rmdA gene. This deletion has been constructed using the lambda Red recombinase system (REDIRECT). REDIRECT works by first designing a forward primer sequence that will provide a region of homology for 39 bases that includes the start codon and flanking DNA directly upstream of the gene. A reverse primer was then designed to provide homology to the stop codon and downstream sequence of rmdA. Both primers also contain 19-20 bases to amplify an apramycin resistance gene cassette that ultimately replaced the rmdA gene. The cosmid containing the wild type rmdA gene has been transformed and the apramycin resistance cassette that has flanking regions of homology to the gene to be deleted into Escherichia coli to create the deletion. The new cosmid containing the deletion of rmdA (pAP1) has been introduced into the mating strain of E. coli and will subsequently be conjugated into the wild-type and rmdB mutant to construct single and double mutant strains, respectively. Meanwhile, the rmdB and cdgC mutants which had been previously designed by the Bennett lab were further characterized. Here, multiple time courses on various media types have been performed to determine phenotypic differences between wild-type and both mutants. Finally, phenotypic differences were analyzed using radish seedling and potato infection assays.

Licensing Permission

Copyright, all rights reserved. Fair Use

Acknowledgement 1

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Acknowledgement 2

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