Staphylococcus species bacteria are Gram-positive, facultative anaerobic, catalase-
positive cocci, except for Staphylococcus aureus anaerobius and the nonpathogenic S.
saccharolyticus, which are catalase-negative and anaerobic (Zaas 2012; Grüner 2007). While
most Staphylococcus infections are mild, under proper conditions, may cause severe disease or potentially even be fatal. This proposal focuses primarily on S. aureus found in the environment and colonizes human skin and mucous membranes of the nasal region in most individuals.
    Catalase, an iron-containing heme protein, is an important protective enzyme and is
present in most aerobic forms of life. It degrades the toxic reactive oxygen species hydrogen peroxide to water and oxygen. If undegraded, hydrogen peroxide oxidizes proteins, plasma membrane lipids, and DNA, resulting in mutations. It is produced by spontaneous interactions of atmospheric oxygen and water vapor, the rate of which is increased by the presence of ultraviolet light, by aerobic respiration, and by the oxidative burst in neutrophils and monocytes (Gaupp et al. 2012; reviewed by Beavers and Skaar 2016). Catalase is also an important factor in permitting nasal colonization by typical or MSRA strains of S. aureus due to its function in breaking down hydroxyl radicals (Cosgrove et al. 2007).  
    (Move up further) The reactive oxygen species hydrogen peroxide, generated by the reaction of O2 with water, kills many microbes, forcing them to acquire means to survive hydrogen peroxide exposure. Staphylococci use catalase to degrade hydrogen peroxide and survive. 
  Two years ago, I examined the effects of hydrogen peroxide on Staphylococcus epidermis by exposing S. epidermis to hydrogen peroxide-containing discs and this trial revealed unexpected results: production of a double zone of inhibition (ZOI) around disks impregnated with 3% hydrogen peroxide in some types of agar plates. S. epidermis grown on brain-heart infusion (BHI) agar plates produced typical ZOI rings surrounding disks containing 0.3%-3% hydrogen peroxide. On trypticase soy agar (TSA) plates, S. epidermis had less growth and produced highly unusual, double-ZOIs, separated by a ring of hydrogen peroxide - resistant bacteria only at 3% hydrogen peroxide. Typical single-ZOIs were also produced by stationary-phase S. epidermidis grown on TSA plates. The stage of bacterial growth affects double-ZOI production, since S. epidermidis only produced double-ZOIs when in logarithmic - growth, but not stationary cultures. Furthermore, a ZOI was detected on TSA, but not BHI plates. This suggests that growth rate or nutrient availability affects expression of the catalase-regulating gene kat A, with stronger growth rates on BHI plates, but less catalase production. The slower growth of logarithmic-phase S. epidermidis on the TSA versus BHI agar plates may enhance kat A expression, heightening catalase production and growth of hydrogen peroxide - resistant bacteria in the growth ring between the ZOIs. Levels of enzymatically - active catalase differed among S. epidermidis grown on different media or environmental conditions and even among individual cloned colonies from the same plate.
 These double zones of inhibition noted in catalase positive, Staphylococcus species specifically using hydrogen peroxide disks are virtually absent in literature. There have been double zones of inhibition observed in streptomycin, erythromycin, oleandomycin, and spiramycin when grown on Penassay Seed Agar with antibiotic impregnated filter paper disks at a pH of 6.6. These double ZOIs were not observed when the pH was buffered down to a pH of 6.1 (Korkela H, Pekkanen TJ. 1977). Furthermore, a double zone of growth was observed in Staphylococcus haemolyticus around imipenem agar diffusion disks (R. S. Schwalbe, 1990). 
 In our previous work, we did not quantify the number of bacteria in the double ZOI, a prerequisite for publication. For the first phase of my experiment, I wish to repeat the trials I did earlier on S. epidermis on a different bacterium, S. aureus. Furthermore, I want to quantify the number of bacteria in the resistant ring growing between the two ZOIs as well as measure the quantity of catalase produced by the bacteria growing on the outside of the first zone of inhibition versus in the mutant ring of bacteria between the two zones of inhibition using xylenol orange. I will quantify the bacteria utilizing turbidimetric measurement, where the cloudiness of the culture is measured and translated to cell numbers (Salehi, B, 2014). My hypothesis is the catalase production in the mutant ring of bacteria between the two zones of inhibition is significantly higher than in the bacteria growing on the outside of the last zone of inhibition. This would allow the bacteria in the interior growth ring to survive the increased concentration of hydrogen peroxide. 
    These double ZOIs observed in Staphylococcus aureus in the presence of hydrogen peroxide is significant as better understanding the mechanisms of how Staphylococci species utilize catalase to survive hydrogen peroxide exposure is crucial in understanding how these microbes survive in-vitro hydrogen peroxide exposure. S. aureus is the leading cause of nosocomial infections. Both community-acquired and hospital-acquired infections are common, and treatments are difficult to manage due to multi-drug resistant strains like methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA). S. aureus normally does not infect healthy skin. 
The second phase in my trial is to create mutant S. epidermis by mutating the kat A gene and seeing if this affects the double zone of inhibition. This will allow me to see if the mutant ring of bacteria is a result of catalase activity or if there is another factor at play. A precedence for this exists in a catalase-negative S. aureus subsp. aureus clinical isolate UCN61 where the identification of a point mutation in the kat A gene was associated with the loss of catalase activity. To identify the mechanism responsible for the lack of catalase activity, the entire kat A gene of S. aureus UCN61 was amplified via PCR. Two mutations were identified in S. aureus UCN61: a T172C substitution leading to a histidine 58-to-tyrosine change and a G636A substitution leading to an arginine 212-to-histidine substitution (Piau, C, 2018).
   Catalases are heme proteins that detoxify hydrogen peroxide by degrading the molecule to water and oxygen. This rection occurs in two steps. In the first one, the Fe (III) reacts with hydrogen peroxide to form an oxyferryl intermediate. In the second step, this intermediate reacts with a second molecule of hydrogen peroxide and the enzyme returns to the resting state (Rovira, C, 2006). 
  Several catalase structures have been solved, including those of Escherichia coli and Proteus mirabilis. These resolutions have shown the C terminus of the protein of a common highly conserved core. It has been determined that the His 54 of Proteus mirabilis (corresponding to His58 in S. aureus) was crucial for the binding of the peroxide group of the substrate (Gouet, P, 1995). This histidine is part of a distal pocket that is composed of three amino acids, tryptophan, arginine, and histidine that form a major channel (Amara, P., 2001, Regelsberger, G., 2001). Due to its important role, His 54 was fully conserved among catalases. This key amino acid in E. Coli was located at position 128 in the same conserved environment. His128Ala, His128Asn, and His128Glu mutants have been constructed that showed drastically reduced catalase activity (Mate, M., 1999). These observations are confirmation of the important role of this histidine in catalase activity. Furthermore, I am hypothesizing a single point mutation is responsible for the lack of catalase activity in strain UCN61 (Pia, C., 2008). I wish to perform a point mutation of S. aureus at this critical His58 location. These mutants will be grown on NA, TSA, and BHI agar plates with disks containing 0.3%-3% hydrogen peroxide. I am hypothesizing that no double ring will form due to the lack of catalase production. 
 Creating a point mutation has been successfully carried out using single-stranded DNA (ssDNA) oligonucleotide recombineering coupled with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated counterselection (Penewit, K, 2018). This method utilizes equipment outside the scope of Malone University; however, I will be working in a microbiology lab this summer doing research. I will gain a better understanding of this methodology as I will be doing CRISPR genome editing as part of my internship. I am hoping to achieve the desired S. aureus mutation by collaboration with this lab. If creating a mutation does not appear to be feasible, I will consider a second option, which is to degrade the catalase using the catalase inhibitor 3-amino - 1,2,4-triazole. 3-Amino-1,2,4-triazole (pictured at left) irreversibly inhibits catalase and is sold by Santa Cruz Biotechnology.

Materials Needed:
•	Staphylococcus epidermis
•	plates to pour agar
•	BHI agar
•	NA agar
•	TSA agar
•	TSA broth 
•	H2O2
•	Orange xylenol
•	3-amino - 1,2,4-triazole (potentially may use)

Methodology
    Logarithmic-stage (24-hour incubation) or stationary-stage (24-hour incubation, then refrigeration) broth cultures of Staphylococcus aureus will be used to produce bacterial lawns on TSA, BHI, or nutrient agar (NA) plates containing disks with 5μl of 0.03%, 0.3%, or 3% H2O2 or water (control). Following a 24-hour incubation at 35°C, zones of inhibition (ZOI) will be measured.
 I will quantify the bacteria utilizing turbidimetric measurement. Before turbidimetric measurements can be made, the spectrophotometer must be adjusted to one-hundred percent transmittance (0 % absorbance). This is achieved by using a sample of uninoculated medium. The percent transmittance of various dilutions of the bacterial culture is now measures and the values converted to optical density, based on the formula: Absorbance = 2 – log % Transmittance. A wavelength of 420 nm is used when the solution is clear, 540 nm when the solution is light yellow, and 600-625 nm is used for yellow to brown solutions (Not sure how to cite1st two sources https://fire.biol.wwu.edu/cmoyer/zztemp_fire/biol346_W06/labman_week4.pdf
https://fire.biol.wwu.edu/brodham/biol346_S07/labman_week4.pdf,
	Catalase levels in S. aureus colonies from the different or the same plates will be quantified by their ability to remove exogenous H2O2 using the xylenol orange method of H2O2 measurement after incubating in the presence of H2O2 or water for 15 minutes. In this colorimetric assay, the orange xylenol is converted to a purple metabolite in the presence of H2O2. Optical density will be measured by a colorimetric plate reader. 
 My ability to generate a mutant for phase two of my experiment remains unclear at this point. I will gain clarity on this issue over the summer while working in a microbiology research lab at the University of Nebraska Medical Center. If this is not an option, I will consider my second option, which is utilizing the catalase inhibitor 3-amino - 1,2,4-triazole.
 My timeline for this experiment is as follows: 
August 23rd to September 24th will be dedicated to completing phase one, which includes growing the microbes and recording the double ZOIs, quantifying the microbes in the ring of bacteria between the two zones of inhibition, and utilizing the xylenol orange method to determine catalase production. Phase two of my trial will go to November twenty-sixth, where a mutant S. aureus will be exposed hydrogen peroxide saturated disks. The last week will be used to complete writing and defending my thesis.   

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