Date of Completion
Molecular and Cell Biology
In this study, we performed transposon mutagenesis to create a library of Enterobacter hormaechei mutants and developed a melanization assay to identify mutants that could not induce the typical melanization phenotype in Cryptococcus neoformans. Relevant phenotype labels included: “hypomelanizer”, a total lack of melanization; “hypermelanizer”, an increased melanization; or “abnormal”, an increased melanization with complete color change of the assay plate to a reddish-brown. Genomic sequencing of 47 mutants and bioinformatic analysis allowed us to pinpoint the transposon insertion site in each E. hormaechei mutant to identify the genes that were affected. A single mutant that induced the abnormal phenotype in C. neoformans was further analyzed and observed to harbor a disruption of the gene that encodes the enzyme 3,4-dihydroxyphenylacetate 2,3-dioxygenase. This phenotype may be attributable to the excess production of homogentisic acid, a byproduct of tyrosine catabolism. However, additional biochemical tests to demonstrate this possibility are necessary, and complementation of the mutant should be performed. Additionally, the detection of dopamine in all mutant cultures should be performed to determine if any genes with a transposon insertion contribute to dopamine production in E. hormaechei.
Nelson, Amy, "3,4-Dihydroxyphenylacetate 2,3-dioxygenase modulates the ability of Enterobacter hormaechei to induce Cryptococcus neoformans melanization" (2020). Honors Scholar Theses. 734.