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Advances in characterizing the gene-encoded subunits that compose the nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase complexes within the Bacillus niacini nicotinic acid degradation pathway

Emilee R. Haines

Name: Emilee R. Haines
Major: Biochemistry and Molecular Biology
Minor: Mathematics
Advisors: Dr. Mark Snider and Dr. Marie Southerland

N-Heterocyclic aromatic compounds (NHACs) are constituents of many manufactured products where their widespread use has led to contamination of groundwater and soils, posing environmental concerns. Nicotinic acid (NA) is an example of an NHAC where its degradation has been studied in a variety of bacteria such as Pseudomonas, Eubacterium, and Bacillus. While NA degradation has been elucidated in bacteria such as P. putida and E. barkeri, the majority of B. niacini’s NA degradation pathway is uncharacterized. Previous work identified a nic gene cluster that had been found to be upregulated in the presence of NA and 6-hydroxynicotinic acid (6-HNA), leading to the hypothesis that the set of protein subunits NicA1, NicA2, NicB1, NicB2and CoxG are involved in the first two hydroxylation steps of B. niacini’s NA degradation pathway. These proteins are thought to form distinct multi-subunit protein complexes with nicotinate dehydrogenase (NDH) and 6-hydroxynicotinate dehydrogenase (6-HNDH) activities. A vector (pBBrMCS_START2) specific for expressing the genes in Pseudomonas was engineered to contain the set of five B. niacini genes hypothesized to code for these hydroxylating enzymes and was transformed into Pseudomonas fluorescens 1f2, a strain shown to be unable to degrade nicotinic acid. Analysis of the media upon incubation of the P1f2 transformants with NA or6-HNA in minimal media by C-18chromatography (HPLC) showed rapid and complete hydroxylation of NA to 6-HNA, as well as the hydroxylation of 6-HNA to 2,6-dihydroxynicotinic acid (2,6-DHNA). However, the combination of gene-encoded subunits that compose the NDH and 6-HNDH complexes is unknown. Using deletion mutagenesis, specific combinations of genes were removed from the vector, allowing for expression of recombinant variant complexes that lack a specific subunit to provide insight on both substrate specificity and oligomeric structure of the NDH and 6-HNDHcomplexes.

Posted in Comments Enabled, Independent Study, Symposium 2022.


7 responses to “Advances in characterizing the gene-encoded subunits that compose the nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase complexes within the Bacillus niacini nicotinic acid degradation pathway”

  1. Dante King says:

    Hi Emilee! Amazing job!! I will admit that this is way over my head, but I can tell that you put an incredible amount of time and effort into your research, so thank you so much for sharing! Best wishes for what’s ahead! 😀

  2. Emilee Haines says:

    Thank you so much, Dante!! I appreciate you reading my page! I.S. was such a great experience and made me realize I wanted to go into a research-based career!

  3. Dante King says:

    You’re very welcome! I’m glad it was such a rewarding experience for you!!

  4. Christina Mathews says:

    Way to go, Emilee!! Way over my head, too. You amaze me.

  5. Emilee Haines says:

    Thank you so much!! Also, thank you for being my accompanist for several bassoon juries. Music was another highlight of my experience here. 😊

  6. Ed Haines says:

    Your research is very interesting and I hope we can go over, step by step, when I take you out for celebration. Let’s make it next time I see you! Great work!!

  7. Emilee Haines says:

    Thank you so much!! I’d love that! 😊

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