Impact of Inhibition of the TCA cycle on ATP and NADH levels in Escherichia coli during Bacterial Replication Raina De La Cruz, Briana Mercado, Aiswarya Suriaprakash, and Jessica O?Hara EXPANSION OF RESEARCH INTRODUCTION TO FUMA AND ICD GENES PLAQUE ASSAYS SHOW BACTERIAL LYSIS ? Quantify ATP, NADH, and glucose levels before and after cell lysis to further The tricarboxylic acid (TCA) is the metabolic pathway understand the impacts of each knockout on cellular metabolism and the resulting that both genes of interest are part of. The TCA cycle impact on T4 replication. ? Expand range of genes in the TCA cycle to manipulate (gltA, maeA, acnB, sucC) consists of a series of reactions that occur within - This will be quantified by utilizing a plate reader (@600nm) in both growth and aerobic respiration. This is not the most ATP lysis curves over eight hours. generative part of aerobic respiration; rather, it creates - The growth curve will measure the growth of the new genes compared to wildtype high energy carriers, NADH and FADH . (WT) E. coli 2 - The double agar overlay method will be utilized to grow and analyze T4 and T4r The fumA gene is one of the three fumarase genes bacteriophage growth with the wild type and gene knockout of choice. that creates the enzyme need for the conversion of fumarate to S-malate, in the last few processes of the Gene TCA Cycle Enzyme TCA cycle. Figure 1. ?fumA in the TCA cycle Illustration of the TCA cycle and gene gltA Citrate Synthase icd knockout implications. maeA Pyruvate Dehydrogenase Isocitrate ? ?-ketoglutarate ? Normal ATP acnB Aconitase Isocitrate ? ?-ketoglutarate ? Decreased ATP The icd gene connects the TCA cycle to the glyoxylate bypass Figure 3. ?fumA plaque assays sucC Succinyl-CoA Synthetase icd A double agar overlay method was used with T4 pathway. It is the first enzyme in a bacteriophage in the wild type (A and C) and Figure 4. ?icd plaque assays ?fumA E.coli strains (B and D) at dilutions of 10-6 Objective: Understand ways to treat bacterial diseases in the face of antibiotic resistance, Figure 2. ?icd Reaction in Krebs Cycle. Diagram of the bacterial pathway to be regulated The plaque assays feature plaques of similar size and by phosphorylation. (A and B) and 10 -7 (C and D). There are less PFU specifically phage therapy. TCA cycle and gene knockout implications. shape, indicating similar lysis of bacterial cells by phage.in the ?fumA strain than the wild type. ?FUMA AND ?ICD LED TO DELAYED BACTERIOPHAGE REPLICATION ?ICD FOUND TO DECREASE ATP LEVELS IN E. COLI A) B) ? Deletion of the icd gene led to reduced specific growth rate and reduced specific glucose consumption Figure 5. Growth Curves rate because of the lower intracellular ATP/ADP and NADPH/NADP+ ratios compared to the parent A) Growth of ?icd and ?fumA in LB strain5. Media Growth of both knockout strains were ? Since ?fumA followed the same trends in growth and lysis curves, it is assumed that the quantification of not impacted in LB media. ATP/ADP and NADPH/NADP+ ratios in this strain and other TCA cycle enzyme knockout strains will B) Growth of ?icd and ?fumA in M9 provide similar results. Media Growth of the ?icd strain was significantly less in M9 media. QUANTIFICATION METHODS These assays will be performed to quantify NAD-NADH ratios, glucose consumption rate, and ATP levels during growth and viral replication. Figure 6. Lysis Curves A) ?fumA A) B) Lysis of the parent and ?fumA strain NAD-NADH Pool Isolation and Sigma-Aldrich Glucose (HK) NAD Cycling Assay6 Assay Kit4 ATP Assay Kit (Colorimetric) 1 with T4 phage was observed over time by measuring absorbance at 1. Samples will be centrifuged and 1. Glucose is phosphorylated by 1. ATP is made through metabolic 600nm. The ?fumA strain experienced slower lysis by phage the subsequent supernatant will ATP with the help of hexokinase reactions in the cells ? ATP is than the parent strain. be removed and will immediately ? Glucose-6-Phosphate (G6P) then used to phosphorylate B) ?icd be frozen in a dry ice-ethanol and ADP are products. glycerol. Lysis of the parent and ?icd strain bath. 2. G6P is oxidized to 2. The phosphorylation of glycerol with T4r phage was observed over 2. HCl (for NAD extraction) or 6-phosphogluconate in the reaction produces byproduct time by measuring absorbance at NaOH (for NADH extraction) is presence of NAD in a reaction (Resorufin) ? byproduct is then r 600nm. The ?ics strain experienced added to the pellets. catalyzed by stained red via assay and able to slower lysis by phage than the 3. Cycling assays will be performed glucose-6-phosphate be quantified by optical density parent strain for both, the reduction of dehydrogenase (G6PDH) (OD) readings @ 570 nm. REFERENCES & ACKNOWLEDGEMENTS 3-(4,5-dimethylthiazol-2-yl)-2,5-di 3. During the oxidation, an phenyl tetrazolium bromide equimolar amount of NAD is (MTT) is directly proportional to reduced to NADH. 1. ATP assay Kit (Colorimetric/Fluorometric) (ab83355). (2021, April 12). Retrieved April 16, 2021, from https://www.abcam.com/atp-assay-kit-colorimetricfluorometric-ab83355.html levels of NAD and NADH @ 570 4. The resulting increase in 2. ?Escherichia Coli K-12 Substr. MG1655 Icd.? https://biocyc.org/gene?orgid=ECOLI&id=EG10489 (April 14, 2021). 3. Gerdes SY, et. al. Experimental determination and system level analysis of essential genes in Escherichia coli MG1655. J Bacteriol. 2003 Oct;185(19):5673-84. nm. absorbance @ 340 nm is directly 4. Glucose (HK) Assay Kit GAHK20. Sigma-Aldrich. Retrieved April 16, 2021, from https://www.sigmaaldrich.com/catalog/product/sigma/gahk20 proportional to the concentration 5. Kabir, M.M., and Shimizu, K. (2004). Metabolic regulation analysis of icd-gene knockout Escherichia coli based on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements. Appl Microbiol Biotechnol 65, 84?96. of glucose. 6. Leonardo, M R, Y Dailly, and D P Clark. 1996. ?Role of NAD in Regulating the AdhE Gene of Escherichia Coli.? Journal of bacteriology 178(20): 6013?18. 5. Glucose consumption rate is 7. Li, J., Li, Y., Cui, Z. et al. Enhancement of succinate yield by manipulating NADH/NAD+ ratio and ATP generation. Appl Microbiol Biotechnol 101, 3153?3161 (2017). https://doi.org/10.1007/s00253-017-8127-6 measured by measuring glucose 8. M R Leonardo, Y Dailly, D P Clark Journal of Bacteriology Oct 1996, 178 (20) 6013-6018; DOI: 10.1128/jb.178.20.6013-6018.1996 concentration (intra or Financial support for this project was provided by the First-Year Innovation and Research Experience at the University of Maryland, College Park. extracellular) over time.