The FAD binding domain of flavin-dependent monooxygenases is required to confer resistance to tetracycline antibiotics 

Antibiotic resistance is a global health crisis that is expected to continue to evolve and cause millions of deaths annually. As increasing amounts of antibiotics are misused in humans, agriculture, and animals, bacteria learn to adapt and become resistant to them, making common infections and diseases  harder to treat and even more deadly. Tetracycline is a common antibiotic used to treat bacterial infections through inhibiting bacterial protein synthesis. Its mechanism of action consists of binding to the 30S ribosomal subunit to block the aminoacyl-tRNA, an essential part of bacterial translation, thus preventing the spread of the bacterial infection. One family of enzymes, named Tetracycline destructases, are the primary group of enzymes that target Tetracycline to render the antibiotic ineffective. These flavoenzymes function as monooxygenases and use reduced flavin adenine dinucleotide (FADH2) and molecular oxygen to covalently modify the Tetracycline scaffold, rendering it inactive. Unlike other mechanisms of antibiotic resistance, the Tetracycline destructase family completely destroys the antibiotic molecule to an irreversible end. The tetX gene codes for the original NADPH-dependent oxidoreductase, TetX, found to cause Tetracycline resistance in Staphylococcus aureus, Escherichia coli, and other gram-positive and gram-negative bacteria. A variant of this resistance enzyme, TetX(1), is a smaller, truncated version missing part of the N-terminal sequence, rendering it incapable of binding the flavin cofactor (FAD). This study aimed to engineer an evolved version of TetX(1), TetX(1)-EVD, which was designed to mimic TetX and contain the full N-terminal sequence for FAD binding. Analysis of TetX(1) versus TetX(1)-EVD protein structures using Alphafold2.0 technology confirmed the missing N-terminus in TetX(1) and added FAD domain in the TetX(1)-EVD construct. Molecular cloning techniques including polymerase chain reaction (PCR), restriction digests, and ligations were employed to insert the new gene construct into the pBAD24 expression vector. Both the tetX(1)-pBAD24 and tetX(1)-EVD-pBAD24 plasmids were transformed into chemically competent E. coli cells to generate working laboratory strains. These strains were used to compare the effectiveness of TetX(1) and TetX(1)-EVD in conferring resistance against Tetracycline antibiotics. Cell-based activity experiments, including minimum inhibitory concentration (MIC) tests, confirmed expected Tetracycline and Doxycycline resistance conferred by TetX, and less noticeable amounts of resistance from  TetX(1) and TetX(1)-EVD. Collectively, these findings confirm the lack of the complete N-terminal sequence in TetX(1) to be a significant hindrance in generating antibiotic resistance, suggesting the possibility that the catalytically active TetX could have evolved from the inactive truncated TetX(1). 

1. Cloning into chemically competent E.coli DH5α for cell-based studies

• Conducted PCR of TetX(1) and TetX(1)-EVD genes followed by DNA extraction for pJET1.2 blunt vector ligation. 

• Perform plasmid preparation and restriction digest using EcoRI and XBaI restriction enzymes into pBAD24 vector. 

• Transformation into DH5α cells. 

2. Expression of TetX, TetX(1), TetX(1)-EVD in E.coli cells 

• Grew 1mL of pET28b(+)-[TetX,TetX(1),TetX(1)-EVD] E.coli BL21(DE3) cells in terrific broth and induced with 1mM IPTG 

• Prepared 12% Polyacrylamide gel and denatured samples at 95 degrees Celsius for three minutes before spinning down at 13,000rpm for three minutes. 

• Loaded the protein gel with 10 microliters of each sample: control BL21 pET vector, TetX, TetX(1), and TetX(1)-EVD 

3. Minimum Inhibitory Concentration Assay Demonstrates Resistance Activity 

• Diluted overnight cultures to an optical density (OD) of 0.004 and prepared a 96 well plate with 1mM IPTG solution.  

• Serial dilution across the plate with 100mM Tetracycline stock and 20mM Doxycycline stock. 

• Added 50 microliters of bacteria to every well and incubated overnight at 37 degrees Celsius, used plate reader for analysis at 600 nm absorbance after 16 hours of incubation. 

Conclusions 

• Lack of a complete N-terminal sequence and FAD-binding domain caused significant hindrance in conferring antibiotic resistance in Tetracycline destructases. 

• Modification of the truncated TetX(1) gene to include the missing FAD domain (TetX(1)-EVD) confers additional but limited resistance when compared to control vectors. 

Future Studies  

• Characterizing TetX variants’ activity in vitro  

  • Truncated TetX(1) and evolved version with added N-terminus will be purified. 

  • Perform binding and activity assays to determine critical function of FAD domain. 

• Cell-based experiments with various antibiotics 

  • Conduct MIC and MBC assays to develop quantitative data for resistance mapping and comparison. 

  • Use time-kill kinetics to test bactericidal activity. 

 Wangrong Yang, et al. 2004. JBC 

Noemi Aniko Bartha, et al. 2011. ScienceDirect 

Mollie C. Shutter, et al. 2023. NIH 

Schematics created with Biorender.com 

•Citi Training courses completed including lab chemical safety, OSHA bloodborne pathogens, initial biosafety training course and basic introduction to biosafety.