Current Scholars and Projects

Chromobacterium subtsugae is a non-pathogenic soil-based Proteobacteria that is used as a model to study bacterial interactions. We previously discovered that C. subtsugae uses antibiotic resistance to defend against other bacteria during competition. One mechanism of antibiotic resistance is with the CdeAB-OprM efflux pump, which can pump antibiotics and other substrates from within the cell to the environment. This pump allows the bacteria to defend against competing antibiotic-producing bacteria.
My lab also previously found that the CdeAB-OprM pump is regulated by antibiotics. Regulation is through a transcription regulator, CdeR, that represses the cdeAB-oprM genes. CdeR is a member of the TetR family of gene repressors, which often bind directly to the promoter of their target gene to repress transcription. A potential binding site of CdeR was identified as the cdeA promoter based on conservation with that of other closely related TetR-family proteins. A mutation at this site in the cdeA promoter was found to abolish repression of cdeA transcription by CdeR. This strongly suggests that CdeR directly interacts with the cdeA promoter to regulate the efflux pump genes.
To further this research, I will test whether CdeR directly interacts with the efflux pump gene promoter. I will complete this by first purifying CdeR by expressing a His-tag CdeR in E. coli and filtering through a nickel column; second, testing for binding of purified CdeR to the cdeA promoter using an electrophoretic mobility shift assay, and third, mutating the conserved binding site in the cdeA promoter and testing for reduced binding interactions with CdeR compared with the wild-type cdeA promoter.
Completing these experiments will allow my lab and I to further our understanding of how C. subtsugae uses CdeR to regulate the CdeAB-OprM efflux pump in response to antibiotics. These results will also add to our understanding of how bacteria integrate cues from the environment to optimize strategies to compete with one another.

Meiotic drivers are selfish genetic elements that manipulate gametogenesis to favor their transmission and cheat Mendelian segregation. When a driver is present on a sex chromosome in males, the unequal transmission of sex chromosomes results in biased sex-ratios among progeny, a phenomenon known as sex-ratio meiotic drive. A driving sex chromosome has a significant evolutionary advantage increasing its fitness. Driving X chromosomes skew populations toward female-biased sex ratios, diminishing the average population fitness. Consequently, genomes often evolve resistance on both autosomes and the Y chromosome to counteract drive. Our model organism for sex-ratio meiotic drive is the fruit fly, Drosophila affinis- which harbor 3 different X chromosome haplotypes (two distinct sex-ratio driving: X-SR1 & X-SR2 and one standard/non-driving X: X-ST ). There is considerable evidence that these two X-linked meiotic drivers are distinct: different Y chromosomes are resistant to each driver, the cellular mechanisms of meiotic drive appear different, and preliminary genomic analysis suggests that the two driving X chromosomes are divergent.
I aim to identify resistance alleles segregating for each of the X chromosome driver in lab-reared isofemale lines. We have lab-reared stocks of wild-collected D. affinis from different geographical locations around the United States that were established in the lab as isofemale lines. This research will provide more information on the mechanisms of meiotic drivers and resistance alleles. The unusual co-segregation of drivers and resistances in D. affinis populations has far-reaching implications, from reduced fertility, population extinction, reproductive isolation, and potential for synthetic gene drives. Selfish genetic elements are widespread, comprising nearly half of the human genome. This study bridges a critical gap in understanding how selfish genetic elements shape genomic processes. Meiotic drivers enhance their own transmission at the expense of population fitness, potentially disrupting DNA packaging, meiosis, and reproduction. Without resistance and fitness effects, sex-ratio meiotic drivers can lead to population extinction. Selfish genetic elements are widespread, comprising nearly half of the human genome. Understanding the mechanisms of meiotic drivers is crucial for insights into genome biology.

After the COVID-19 pandemic began, the need for research on coronaviruses increased dramatically due to the global public health crisis the outbreak created. Millions of people died and were disabled from infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19. However, several other coronaviruses, such as Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoV-1) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), have pandemic potential. A critical virulence factor of the coronavirus genome is the highly conserved macrodomain, Mac1, which promotes virus replication, weakens the host immune response, and promotes disease in animal models. When Mac1 is deleted, there is a significant replication defect in the virus, and the activity of the host immune response is rescued, indicating that Mac1 inhibitors could have robust activity against coronaviruses.

Recently, the SARS-CoV-2 macrodomain was incorporated into MHV, using a bacterial artificial chromosome (BAC) based reverse genetic system. This virus allows for the testing of antivirals against SARS-CoV-2 Mac1 in a BSL-2 system, which increases the accessibility of research with SARS-CoV-2, a BSL-3 pathogen. This viral system can also be applied to other coronaviruses, where their macrodomains are inserted into an MHV BAC, allowing for the testing of antivirals for various coronaviruses using a single viral system.

Building on this, the MERS-CoV macrodomain will be incorporated into an MHV BAC using E. coli. I will do this by cloning the MERS-CoV Mac1 gene into MHV BAC DNA in E. coli by Lambda Red Recombination, rescuing the virus by transfection into permissive cells, and characterizing the virus by replication assays, interferon production assays, inhibition assays using macrodomain inhibitors, and evaluating the ability of the virus to cause disease in animal models. After creating the recombinant virus with MERS-CoV, the macrodomains of Porcine Epidemic Diarrhea Virus (PEDV), Infectious Bronchitis Virus (IBV), Hepatitis E Virus (HEV), Human Coronavirus 229E (h-CoV 229E), and Porcine Deltacoronavirus (PD-CoV) will be inserted into this viral system. This could provide insight into the genetic diversity of Mac1 proteins across the coronavirus genera, and pan-coronavirus inhibitors could be identified.