1887

Abstract

Branched-chain amino acids (BCAAs) are essential amino acids, but their biosynthetic pathway is absent in mammals. Ketol-acid reductoisomerase (IlvC) is a BCAA biosynthetic enzyme that is coded by Rv3001c in H37Rv (Rv) and MRA_3031 in H37Ra (Ra). IlvCs are essential in Rv as well as in . Compared to wild-type and IlvC-complemented -Ra strains, IlvC knockdown strain showed reduced survival at low pH and under low pH+starvation stress conditions. Further, increased expression of IlvC was observed under low pH and starvation stress conditions. Confirmation of a role for IlvC in pH and starvation stress was achieved by developing BL21(DE3) IlvC knockout, which was defective for growth in M9 minimal medium, but growth could be rescued by isoleucine and valine supplementation. Growth was also restored by complementing with over-expressing constructs of Ra and IlvCs. The knockout also had a survival deficit at pH=5.5 and 4.5 and was more susceptible to killing at pH=3.0. The biochemical characterization of Ra and IlvCs confirmed that both have NADPH-dependent activity. In conclusion, this study demonstrates the functional complementation of IlvC by Ra IlvC and also suggests that IlvC has a role in tolerance to low pH and starvation stress.

Funding
This study was supported by the:
  • Science and Engineering Research Board (Award EMR/20l7/001295)
    • Principle Award Recipient: Kumar SinghSudheer
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001087
2021-09-13
2024-12-09
Loading full text...

Full text loading...

/deliver/fulltext/micro/167/9/mic001087.html?itemId=/content/journal/micro/10.1099/mic.0.001087&mimeType=html&fmt=ahah

References

  1. World Health Organization Global Tuberculosis Report Geneva, Switzerland: WHO; 2020
    [Google Scholar]
  2. Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 2003; 48:77–84 [View Article] [PubMed]
    [Google Scholar]
  3. Kim J, Copley SD. Why metabolic enzymes are essential or nonessential for growth of Escherichia coli K12 on glucose. Biochemistry 2007; 46:12501–12511 [View Article] [PubMed]
    [Google Scholar]
  4. Hondalus MK, Bardarov S, Russell R, Chan J, Jacobs Jr WR et al. Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis. Infect Immun 2000; 68:2888–2898 [View Article] [PubMed]
    [Google Scholar]
  5. Sharma R, Keshari D, Singh KS, Yadav S, Singh SK. MRA_1571 is required for isoleucine biosynthesis and improves Mycobacterium tuberculosis H37Ra survival under stress. Sci Rep 2016; 6:1–2
    [Google Scholar]
  6. Sharma R, Keshari D, Singh KS, Singh SK. Biochemical and functional characterization of MRA_1571 of Mycobacterium tuberculosis H37Ra and effect of its down-regulation on survival in macrophages. Biochem Biophys Res Commun 2017; 487:892–897 [View Article] [PubMed]
    [Google Scholar]
  7. Singh V, Chandra D, Srivastava BS, Srivastava R. Biochemical and transcription analysis of acetohydroxyacid synthase isoforms in Mycobacterium tuberculosis identifies these enzymes as potential targets for drug development. Microbiology (Reading) 2011; 157:29–37 [View Article] [PubMed]
    [Google Scholar]
  8. Amorim Franco TM, Blanchard JS. Bacterial branched-chain amino acid biosynthesis: structures, mechanisms, and drugability. Biochemistry 2017; 56:5849–5865 [View Article] [PubMed]
    [Google Scholar]
  9. Grandoni JA, Marta PT, Schloss JV. Inhibitors of branched-chain amino acid biosynthesis as potential antituberculosis agents. J Antimicrob Chemother 1998; 42:475–482 [View Article] [PubMed]
    [Google Scholar]
  10. Lv Y, Kandale A, Wun SJ, McGeary RP, Williams SJ et al. Crystal structure of Mycobacterium tuberculosis ketol-acid reductoisomerase at 1.0 Å resolution - a potential target for anti-tuberculosis drug discovery. FEBS J 2016; 283:1184–1196 [View Article]
    [Google Scholar]
  11. LaRossa RA, Schloss JV. The sulfonylurea herbicide sulfometuron methyl is an extremely potent and selective inhibitor of acetolactate synthase in Salmonella typhimurium. J Biol Chem 1984; 259:8753–8757 [View Article]
    [Google Scholar]
  12. Chaleff RS, Mauvais CJ. Acetolactate synthase is the site of action of two sulfonylurea herbicides in higher plants. Science 1984; 224:1443–1445 [View Article] [PubMed]
    [Google Scholar]
  13. Jung I-P, Ha N-R, Lee S-C, Ryoo S-W, Yoon M-Y. Development of potent chemical antituberculosis agents targeting Mycobacterium tuberculosis acetohydroxyacid synthase. Int J Antimicrob Agents 2016; 48:247–258 [View Article] [PubMed]
    [Google Scholar]
  14. McCourt JA, Pang SS, King-Scott J, Guddat LW, Duggleby RG. Herbicide-binding sites revealed in the structure of plant acetohydroxyacid synthase. Proc Natl Acad Sci U S A 2006; 103:569–573 [View Article]
    [Google Scholar]
  15. Amorim Franco TM, Favrot L, Vergnolle O, Blanchard JS. Mechanism-based inhibition of the Mycobacterium tuberculosis branched-chain aminotransferase by d-and l-cycloserine. ACS Chem Biol 2017; 12:1235–1244 [View Article] [PubMed]
    [Google Scholar]
  16. Chou PY, Fasman GD. Structural and functional role of leucine residues in proteins. J Mol Biol 1973; 74:263–281 [View Article] [PubMed]
    [Google Scholar]
  17. Betts MJ, Russell RB. Amino acid properties and consequences of substitutions. Bioinformatics for Geneticists 2003; 317:10–02
    [Google Scholar]
  18. Kaiser JC, Heinrichs DE. Branching out: alterations in bacterial physiology and virulence due to branched-chain amino acid deprivation. mBio 2018; 9:e01188-18 [View Article] [PubMed]
    [Google Scholar]
  19. Awasthy D, Gaonkar S, Shandil RK, Yadav R, Bharath S et al. Inactivation of the ilvB1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice. Microbiology (Reading) 2009; 155:2978–2987 [View Article] [PubMed]
    [Google Scholar]
  20. Len AC, Harty DW, Jacques NA. Proteome analysis of Streptococcus mutans metabolic phenotype during acid tolerance. Microbiology (Reading) 2004; 150:1353–1366 [View Article] [PubMed]
    [Google Scholar]
  21. Kim GL, Lee S, Luong TT, Nguyen CT, Park SS et al. Effect of decreased BCAA synthesis through disruption of ilvC gene on the virulence of Streptococcus pneumoniae. Arch Pharm Res 2017; 40:921–932 [View Article] [PubMed]
    [Google Scholar]
  22. Li T, Zhan Z, Lin Y, Lin M, Xie Q et al. Biosynthesis of amino acids in Xanthomonas oryzae pv. oryzae is essential to its pathogenicity. Microorganisms 2019; 7:693 [View Article]
    [Google Scholar]
  23. Li K-H, Yu Y-H, Dong H-J, Zhang W-B, Ma J-C et al. Biological functions of ilvC in branched-chain fatty acid synthesis and diffusible signal factor family production in Xanthomonas campestris. Front Microbiol 2017; 8:2486 [View Article] [PubMed]
    [Google Scholar]
  24. Tyagi R, Lee YT, Guddat LW, Duggleby RG. Probing the mechanism of the bifunctional enzyme ketol‐acid reductoisomerase by site‐directed mutagenesis of the active site. FEBS J 2005; 272:593–602 [View Article] [PubMed]
    [Google Scholar]
  25. Cahn JK, Brinkmann‐Chen S, Buller AR, Arnold FH. Artificial domain duplication replicates evolutionary history of ketol‐acid reductoisomerases. Protein Sci 2016251241–1248 [View Article] [PubMed]
    [Google Scholar]
  26. Goude R, Parish T. Methods in molecular biology. Ch 13. Parish T, Brown A. eds In Mycobacteria Protocols Vol 465 Humana Press; 2008 pp 203–215
    [Google Scholar]
  27. Peña CE, Lee MH, Pedulla ML, Hatfull GF. Characterization of the mycobacteriophage L5 attachment site, attP. J Mol Biol 1997; 266:76–92 [View Article] [PubMed]
    [Google Scholar]
  28. Vandal OH, Nathan CF, Ehrt S. Acid resistance in Mycobacterium tuberculosis. J Bacteriol 2009; 191:4714–4721 [View Article] [PubMed]
    [Google Scholar]
  29. Jordan KN, Oxford L, O’Byrne CP. Survival of low-pH stress by Escherichia coli O157: H7: correlation between alterations in the cell envelope and increased acid tolerance. Appl Environ Microbiol 1999; 65:3048–3055 [View Article] [PubMed]
    [Google Scholar]
  30. Liao CH, Shollenberger LM. Survivability and long‐term preservation of bacteria in water and in phosphate‐buffered saline. Lett Appl Microbiol 2003; 37:45–50 [View Article] [PubMed]
    [Google Scholar]
  31. Hill CM, Duggleby RG. Purified recombinant Escherichia coli ketol-acid reductoisomerase is unsuitable for use in a coupled assay of acetohydroxyacid synthase activity due to an unexpected side reaction. Protein Expr Purif 1999; 15:57–61 [View Article] [PubMed]
    [Google Scholar]
  32. Kim G, Shin D, Lee S, Yun J, Lee S. Crystal structure of IlvC, a ketol-acid reductoisomerase, from Streptococcus pneumoniae. Crystals (Basel) 2019; 9:551 [View Article]
    [Google Scholar]
  33. Chia JS, Lee YY, Huang PT, Chen JY. Identification of stress-responsive genes in Streptococcus mutans by differential display reverse transcription-PCR. Infect Immun 2001; 69:2493–2501 [View Article] [PubMed]
    [Google Scholar]
  34. Sezonov G, Joseleau-Petit D. Escherichia coli physiology in luria-bertani broth. J Bacteriol 2007; 189:8746–8749 [View Article] [PubMed]
    [Google Scholar]
  35. Houser JR, Barnhart C, Boutz DR, Carroll SM, Dasgupta A et al. Controlled measurement and comparative analysis of cellular components in E. coli reveals broad regulatory changes in response to glucose starvation. PLoS Comput Biol 2015; 11:e1004400 [View Article] [PubMed]
    [Google Scholar]
/content/journal/micro/10.1099/mic.0.001087
Loading
/content/journal/micro/10.1099/mic.0.001087
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error