Generation and Characterization of Mycobacterium smegmatis Mutants

1. Generation and Characterization of Mycobacterium smegmatis Mutants Disrupted in Mycothiol Metabolism Kathryn Barretto and Dr. MamtaRawat, PhD Department of Biology, California State University- Fresno 1861 Mst Mst M. smegmatis Genomic DNA Hygromycin Resistance Gene ABSTRACT RESULTS Electroporation into M. smegmatisCell Knockout Construct Hygromycin-Resistant In the cell, low molecular weight thiols are responsible for the reducing environment needed for biochemical reactions to occur, act as cofactors for various metabolic reactions, and protect the cell against oxidants and electrophiles. While glutathione is the major thiol in gram-negative bacteria and higher organisms, most gram-positive bacteria contain other low molecular weight thiols that serve analogous functions to glutathione.  In the high GC Actinomycetes, mycothiol substitutes for glutathione and we have recently demonstrated that bacillithiol is the major thiol in the low GC Firmicutes.  Mycothiol dependent proteins include mycoredoxins (Mrx), which are thioltransferases involved in numerous processes, such as arsenate reduction and putative mycothiolation of cysteine residues. Like S08-24, a Mycobacterium smegmatis disrupted in mshC, which codes for the ligase that catalyzes the fourth step in mycothiol biosynthesis, a mutant disrupted in mrx, Mrxms12, is sensitive to metals, oxidants, and alkylating agents. We have also recently identified a mycothiolS-transferase, Mst, which can catalyze the conjugation of the model substrate, monochlorobimane, and the glutathione-S-transferase substrate, chlorodinitrobenzene, to mycothiol. This Mst is also able to conjugate the DNA damaging mitomycin C. We are currently generating a mutant in the gene coding for mycothiol-S-transferase. Because S-transferases play a major role in detoxification, this mutant may yield clues about the role of MSH in mycobacterial growth and survival. ∆mshC ∆mrx Table 3: Sensitivity of WT and mrxms12 towards metal, cytotoxic, and oxidative stress as determined through Kirby-Bauer disk diffusion assays. All p-values reflect a statistical comparison to the wild type Table 1: Sensitivity of WT, S08-24, and S-0824c towards metal and oxidative stress as determined through Kirby-Bauer disk diffusion assays. All p-values reflect a statistical comparison to the wild type Table 2: Resistance of S08-24 and sensitivity of complemented strain to ethionamide and isoniazid in relation to WT BACKGROUND Figure 10: Minimum inhibitory concentration assay of Potassium Tellurite with WT and mrxms12; standard error of the mean is indicated Figure 11: Minimum inhibitory concentration assay of Copper Chloride with WT and mrxms12; standard error of the mean is indicated • The prevalence of drug resistance in Mycobacteria calls for the legitimization of new and highly specific drug targets, focusing on unique pathways. In Actinomycetes including Mycobacteria, mycothiol replaces glutathione as the primary novel low-molecular-weight thiol, providing redox homeostasis (Figure 1). Mycothiol-associated enzymes display high druggability, and have the potential to show high specificity and essentiality for the organism: three key components of antibiotic drug targets [1]. Several genes encode enzymes of interest, and we study these through the generation of mutants disrupted in the genes, which are then characterized in relation to the wild type. Rather than working directly with Mycobacterium tuberculosis, which requires higher biosafety precautions and is extremely slow growing, we work with Mycobacterium smegmatis, a nonpathogenic model species with a faster doubling time. • We have characterized two existing knockout mutants involved in mycothiol metabolism and biosynthesis. • S08-24: A transposon mutant disrupted in mshC, which codes for a ligase that links GlcN-Ins with cysteine in the fourth step of MSH biosynthesis (Figure 2)[1]. • Mrxms12: A mutant disrupted in one of the genes coding for a putative myxoredoxin, a small redox enzyme and thioltransferase, that utilizes mycothiol as an electron donor (Figure 3) and also mycothiolatescysteine residues [2]. Figure 9: Curve of WT and mrxms12 growth through lag, exponential, stationary, and death phases; standard error of the mean is indicated CONCLUSIONS • Characterization of ∆mshC • Sensitivity towards zinc, selenite, and the strong oxidizing agent chromate have been demonstrated in both disk assays and MICs, indicating that MshC may play a role in the mycobacterial cell environment. We also see increased sensitivity towards the oxidizing agents cumenehydroperoxide and hydrogen peroxide. Reintroduction of the native mshC into the mutant reverts sensitivity that is closer to that of WT. This confirms that sensitivity of S08-24 is due to the mutation, rather than downstream changes or pleiotrophic effects. In comparison to WT, the mutant has slow initial growth, followed by a persisting stationary phase. • Characterization of ∆mrx • The transition metal copper induces stress through cysteineautooxidation. Sensitivity indicates that Mrx may play a role in preventing autooxidation. Sensitivity towards the metalloid oxyaniontellurite was also seen. Sensitivity towards 2,4-Dinitrochloro-benzene (CDNB), a cytotoxin with high thiol affinity often used as a GSH substrate, indicates that M. smegmatis may use Mrx to detoxify some cytotoxins. Sensitivity towards diamide, a thiol oxidizing agent, and hydrogen peroxide, a strong oxidant, emphasizes the role of Mrx in maintaining an intracellular reducing environment. The growth curve shows consistently slow and impaired overall growth. • Mutagenesis of ∆BcpA, ∆BcpB, ∆Mst, and ∆Ohr • Use of the pJV53 and Che9c recombineering system for electrocompetent cells, in tandem with linearization of the knockout construct and removal of the suicide vector, will allow for more efficient electroporation and a decrease in single recombination events. Figure 4: Curve of WT and S08-24 through lag, exponential, and stationary phases; standard error of the mean is indicated Figure 5: MIC assay of Zinc Sulfate with WT and S08-24; standard error of the mean is indicated Mutagenesis Figure 7: Molecular manipulation of knockout construct to reduce frequency of single recombinants using linearization approach Figure 2: Pathway for MSH biosynthesis, with MshC acting as MSH ligase in the fourth step Figure 3: Reduction of mycoredoxin by mycothiol Figure 1: Structure of mycothiol REFERENCES METHODS Figure 6: pJV53 extrachromasomal plasmid with mycobacteriophage Che9c genes 60-61 [3] • 1. Newton, G.L., N. Buchmeier, and R.C. Fahey.(2008). Biosynthesis and Functions of Mycothiol, the Unique Protective Thiol of Actinobacteria. Microbiology and Molecular Biology Reviews. 72(3): 471-494. • 2. Ordonez, E. et. al. (2009). Arsenate Reductase, Mycothiol, and Mycoredoxin Concert Thiol/Disulfide Exchange. Journal of Biological Chemistry. 284(22): 15107-15116. • 3. Van Kessel, J.C. and G. F. Hatfull. (2003.) MycobacterialRecombineering. Methods in Molecular Biology, vol. 435: Chromosomal Mutagenesis. G. Davis and K.J. Kayser, ed. Humana Press Inc, Totowa, NJ. • 4. Jenkins, V., B.D. Robertson, and K.J. Williams. (2012. Aspartate D48 is Essential for the GInR-mediated Transcriptional Response to Nitrogen Limitation in Mycobacterium smegmatis. FEMS Microbiology Letters. 330: 38-45. Figure 8: Methodology for knockout construct formation utilizing disruption of gene of interest using antibiotic resistant marker (Hygromycin resistance gene) ACKNOWLEDGEMENTS Research was supported by a Faculty Sponsored Student Research Award from the College of Science and Mathematics, a Research Grant (rGRANT) from Associated Students Incorporated, an Undergraduate Research Fellowship from the American Society for Microbiology, and an Undergraduate Studies Research Award from the Dean of Undergraduate Studies. We would also like to thank the Department of Biology, College of Science and Mathematics, and members of the Rawat Lab.