Date of Award

4-12-2021

Document Type

Distinction Paper

Degree Name

Biology-BS

Department

Biology & Earth Science

Advisor

Jennifer A. Bennett, PhD

First Committee Member

Jennifer A. Bennett, PhD

Second Committee Member

John T. Tansey, PhD

Third Committee Member

Steffanie Burk, PhD

Keywords

Streptomyces scabies, CRISPR-Cas9, Proteins

Subject Categories

Higher Education

Abstract

Streptomyces scabies is a filamentous bacterial species that is known for infecting tube root organisms such as potatoes. Streptomyces is a pharmacologically important genus that supplies two-thirds of antibiotics in commercial use. Little is known about the effector proteins that bind to cyclic-di GMP, the second messenger that primarily regulates antibiotic production, life cycle progression, and biofilm formation. There are currently only two known effector proteins in Streptomyces that binds to cyclic di-GMP: BldD and RsiG. Three recently discovered genes have been known to code for proteins with potential cyclic nucleotide binding domains. Using bioinformatics analyses and the CRISPR-Cas9 editing system a deletion was initiated for one of the primary genes of interest using the pCRISPomyces-2 protocol designed specifically for this genus, which will generate the pMM3 CRISPR deletion plasmid. This newly created plasmid will be transformed into an E. coli mating strain and then conjugated into S. scabies to create the deletion mutant. The mutant is phenotypically characterized by macroscopic examination of colony phenotype and phase-contrast microscopy and is then compared to the wild-type. Bioinformatics was used to generate protein structures, domain maps, multi-sequence alignments, and BLAST searches. These analyses were used to verify the conservation of the gene, the relatedness and structure driven function. Construction of the final CRISPR deletion plasmid of the pMM3 plasmid into Streptomyces scabies is being finalized and phenotypic analysis from the CRISPR (clustered regularly interspaced short palindromic repeats) deletion will be conducted. In the meantime, bioinformatics confirmed that two of the three putative effector proteins contain a cyclic nucleotide binding domain, and a definitive active site for binding cyclic nucleotides. Their primary sequence appears to also display wide conservation through the phylum taxonomy. We found that the third gene of interest did not contain a cyclic nucleotide binding domain but is a potential reductase. The domain mapping and structure provided little information regarding its definitive function, but the gene appeared to be conserved through its class taxonomy based on BLAST searches. In conclusion, based on bioinformatics two of the three genes of interest indeed possess cyclic nucleotide binding domains and the deletion is anticipated to further elucidate gene function. These genes are highly conserved based upon bioinformatics analyses, and from previous findings in other orthologs we can infer a potential function of binding to cyclic di-GMP for regulation or other cyclic nucleotides. We can also confirm the third gene of interest most likely does not code for a protein containing a cyclic nucleotide binding domain, and therefore does not likely bind to cyclic di-GMP.

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