Investigating expression of the AAA+-protease component clpC in the developing Bacillus subtilis spore
Student name: Alex Berg
Project advisor: Amy Hitchcock Camp
Bacillus subtilis is a gram-positive bacterium that is known for its ability to sporulate; create a dormant, spore, version of itself to survive through harsh environmental conditions. Protein degradation is an integral process in sporulation, allowing for the developing spore (the “forespore”) to enter dormancy. Previous literature has shown that proteins are preferentially degraded in the forespore by AAA+ chaperone-protease ClpC-ClpP (ClpCP)1 . The clpC operon is a gene package which transcribes the chaperone component of the ClpCP protease, along with other associated proteins. Data suggests the clpC operon has the potential to be regulated by multiple sporulation-specific sigma factors. In this project, we investigate the activity of two potentially novel σ F promoter regions on the clpC operon. Chemical induction of σ F in growth-phase cells was used to measure the activity of modified clpC operon segments upstream of a lacZ reporter gene. Results indicate both potential σ F promoter regions of interest may show activity. Further experiments will aim to isolate and confirm the activity of these promoter sites during sporulation and, ultimately, assess the importance of σ F -dependent expression of clpC for efficient spore formation.

Characterizing the in vivo interaction of the Bacillus subtilis ClpC chaperone with a novel sporulation adaptor protein
Student name: Ruth Fekade
Project advisor: Dr. Amy Hitchcock Camp
Protein degradation is a complex system that affects biological pathways. Proteolysis in all organisms is carried out by the ATP-dependent AAA+ proteases. The bacterium Bacillus subtilis undergoes sporulation, which results in the formation of a metabolically dormant cell type known as a spore. The molecular mechanisms that drive metabolic dormancy are poorly understood, but research suggests that this is due to the degradation of key metabolic enzymes in the forespore. Recently, we identified an uncharacterized gene MicA expressed in the developing spore; its knockout displays phenotypes consistent with increased metabolic capacity. We are currently testing a working model in which a novel adaptor protein called MicA recruits key metabolic enzymes for the chaperone-protease called ClpCP for degradation. MicA interacts with an interface of the ClpC N-domain at 3 different electrostatic sites confirmed by a co-crystal structure. In this project, we hypothesize that the lysine at position 12 (K12) of ClpC is important for the interaction of MicA with ClpC. We conducted site-directed mutagenesis to confirm the functional importance of the identified residues in vivo. The total loss of ClpC function was then measured using a sporulation assay. Additionally, we investigated the effect of the residue on MicA-ClpC interaction in vegetatively growing B. subtilis. The result showed that the mutants disrupt the specific interaction of ClpC with MicA, but not the overall function of the ClpC protein. Consequently, the data suggests that K12A and K12E are necessary for the toxic phenotype in vegetatively growing cells. Further experiments in vivo in B. subtilis during sporulation confirmed that there is an increase in forespore gene expression similar to ΔmicA. Therefore, the K12 site on the ClpC protein is necessary for MicA and ClpC interaction.

Optimizing CRISPR/Cas9 editing of a bacterial protease adaptor gene, micA, for the study of protein degradation during Bacillus subtilis spore formation
Student name: Siyu Yin
Project advisor: Amy Camp
Sporulation is a process adapted by some bacteria to respond to harsh environmental conditions. During the sporulation, the bacteria form spores, the dormant form of bacteria, to survive in stressful conditions. Bacillus subtilis, the rod-shaped gram-positive bacterium that Camp lab has been working with, has the ability to form spores. The mechanism of a metabolic shutdown during sporulation is the topic that Camp lab has been researching, as this poorly understood molecular mechanism that drives dormancy has important implications for understanding disease states caused by protein aggregation. By using biochemical and genetic strategies, previous members of Camp Lab built up a working model in which a novel adaptor protein, MicA, has been hypothesized to interact with the AAA+-ATPase chaperone-protease ClpCP. The ClpCP degrades the metabolic enzymes recruited by MicA during spore formation, which leads to a metabolic shutdown. Various mutant genes of clpC and micAhave been made by using a pMiniMad plasmid to better understand what specific sites are necessary for the electrostatic interaction between protein MicA and ClpC. However, this editing method is time-consuming and has a low success rate in integrating the mutation into chromosomal DNA, so there is an unmet need to improve the gene engineering process. Therefore, my project aims to leverage CRISPR/Cas9, a natural genome editing system found in bacteria, to make mutants of micA and clpC, investigating its productivity compared to the conventional method. Moreover, a new cloning strategy, FastCloning, was utilized to make plasmids in the project, which is simpler and more economical than the traditional cloning strategy. I have already confirmed the effectiveness of CRISPR/Cas9 in editing genes of interest in Bacillus subtilis. The current project is working on making mutation of micA E161A & E161K, specifically introducing the application of site mutagenesis in FastCloning to make plasmids, and then utilize the CRISPR/Cas9 system to edit gene of interest in B.subtilis.

Tracking and Quantifying Multidrug-Resistant Cancer Cells
Student presenter: Sujin Cha
Project advisor: Shelly Peyton
Lung cancer causes most cancer-related deaths worldwide, and the average survival rate after 5 years of diagnosis for non-small-cell lung cancer (NSCLC) is 28%.1,2 NSCLC is characterized by the expression of epidermal growth factor receptor (EGFR) and several third-generation selective covalent EGFRT790M inhibitors, including AZD9291 (Osimertinib), were developed to avoid acquired drug resistant and low maximum tolerated dose of first and second-generation EGFR inhibitors.3,4 Despite the high response rate of Osimertinib in EGFRT790M-positive NSCLC patients, C797S mutation in the EGFR gene was found to be the leading mechanism of resistance for Osimertinib.4 While additional mechanisms of resistance are to be identified, a way to overcome or avert Osimertinib resistance is required.
Combination of Osimertinib and suicide gene therapy involving cytosine deaminase is proposed as a way to overcome Osimertinib resistance. When administered together, cytosine deaminase converts the non-toxic prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), which is widely used in cancer chemotherapy. 5 In this project, human non-small cell lung carcinoma PC9 cells were used as a model to study the efficacy of combination therapy of Osimertinib with the suicide gene therapy. The efficacy was analyzed by tracking the cell viability overtime using confocal microscopy. The wild type, Osimertinib-resistant, and cytosine deaminase-expressing PC9 cells were either stained with fluorescent dye or expressed different fluorescent proteins, and the dead cells were stained using SYTOX dye. I developed a novel procedure utilizing Fiji software for counting the number of dead and live cells and optimized it to yield the highest accuracy possible. Furthermore, I employed the macro function of Fiji to automate the counting process according to the procedure and analyzed the data using Prism software. Subsequent to the current experiment will be studying efficacy for PC9 cells cultured with Lung Fibroblast, Normal, Human (HLF) cells and 3D cultured PC9 and fibroblast cells, to mimic the real lung environment.

The Role of the Protein-Kinase McsB in Metabolic Shutdown During Bacillus subtilis Sporulation
Fiona Quigley
Senior Symposium Abstract:
The model organism Bacillus subtilis is a Gram-positive bacteria which creates a dormant cell type called a spore when under nutrient stress. Successful dormancy requires selective protein degradation by the ClpCP complex, a member of the universally conserved proteolytic AAA+ complex family. In B. subtilis, ClpCP is used in many protein-regulation applications and employs various protein adaptors to chaperone situation-specific proteins into the complex. The Camp Lab identified the novel protein adaptor MicA as an adaptor candidate to work in degrading proteins during sporulation, however, an in vitro degradation pairing ClpCP and MicA resulted in no protein degradation, possibly indicating another element is needed. We hypothesized the protein-kinase McsB, which marks proteins for degradation and acts as a ClpCP adaptor during heat stress, could be a possible candidate. To test this hypothesis, I created an mcsB knockout to determine whether its phenotype is consistent with a role in MicA/ClpCP-mediated shutdown. In a heat-kill sporulation assay, there was no significant distance between the ∆mcsB and wild type strains. Following that, I used a lacZ reporter assay to determine whether the ∆mcsB strain, like ∆micA, stimulates greater gene expression in the developing spore, a phenotype likely attributable to an inability to enter dormancy. Results from this assay suggest McsB is not crucial in shutdown given that gene expression levels of sporulating ∆mcsB cells were equal to those in wild type cells. To further test our hypothesis, I investigated whether the mcsB knockout, like a clpC knockout, could protect non-sporulating cells from MicA overexpression toxicity. Results from this assay were at first inconclusive, likely due to the importance of McsB’s role in auto-regulating the operon it shares with clpC. Knockouts of the protein seem to confirm this interpretation and showed no phenotypic difference between ∆mcsB and wild type strains. Altogether, results of this study appear to suggest that McsB is not a critical component of the MicA/ClpCP pathway during sporulation and can be eliminated as a possible candidate.