1887

Abstract

Isoprenoids may be synthesized via one of two pathways, the classical mevalonate pathway or the alternative 2--methyl--erythritol 4-phosphate (MEP) pathway. While the majority of bacteria utilize a single pathway for isoprenoid biosynthesis, is unusual in possessing the complete set of genes for both pathways. Here, we utilized new molecular tools to create precise gene deletions in selected genes encoding enzymes of both pathways, , (encoding proteins in the MEP pathway) and (encoding a protein in the mevalonate pathway). We demonstrate that the gene can only be deleted when the growth medium is supplemented with exogenous mevalonate. Furthermore, full growth of the mutant in the absence of mevalonate was only possible when the intact gene was supplied using an IPTG-inducible expression system. Murine competitive index assays performed via the oral and intraperitoneal routes of infection revealed that the mevalonate mutant could not be recovered from livers and spleens 3 days post-infection. We propose that HmgR in EGDe is involved in essential metabolic functions and that an intact MEP pathway is not capable of sustaining growth.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.056069-0
2012-07-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/7/1684.html?itemId=/content/journal/micro/10.1099/mic.0.056069-0&mimeType=html&fmt=ahah

References

  1. Begley M., Gahan C. G., Kollas A. K., Hintz M., Hill C., Jomaa H., Eberl M. ( 2004). The interplay between classical and alternative isoprenoid biosynthesis controls γδ T cell bioactivity of Listeria monocytogenes . FEBS Lett 561:99–104 [View Article][PubMed]
    [Google Scholar]
  2. Begley M., Bron P. A., Heuston S., Casey P. G., Englert N., Wiesner J., Jomaa H., Gahan C. G. M., Hill C. ( 2008). Analysis of the isoprenoid biosynthesis pathways in Listeria monocytogenes reveals a role for the alternative 2-C-methyl-d-erythritol 4-phosphate pathway in murine infection. Infect Immun 76:5392–5401 [View Article][PubMed]
    [Google Scholar]
  3. Behr C., Poupot R., Peyrat M.-A., Poquet Y., Constant P., Dubois P., Bonneville M., Fournie J.-J. ( 1996). Plasmodium falciparum stimuli for human γδ T cells are related to phosphorylated antigens of mycobacteria. Infect Immun 64:2892–2896[PubMed]
    [Google Scholar]
  4. Boucher Y., Doolittle W. F. ( 2000). The role of lateral gene transfer in the evolution of isoprenoid biosynthesis pathways. Mol Microbiol 37:703–716 [View Article][PubMed]
    [Google Scholar]
  5. Brown A. C., Eberl M., Crick D. C., Jomaa H., Parish T. ( 2010). The nonmevalonate pathway of isoprenoid biosynthesis in Mycobacterium tuberculosis is essential and transcriptionally regulated by Dxs. J Bacteriol 192:2424–2433 [View Article][PubMed]
    [Google Scholar]
  6. Buetow L., Brown A. C., Parish T., Hunter W. N. ( 2007). The structure of Mycobacteria 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase, an essential enzyme, provides a platform for drug discovery. BMC Struct Biol 7:68 [View Article][PubMed]
    [Google Scholar]
  7. Campos N., Rodríguez-Concepción M., Seemann M., Rohmer M., Boronat A. ( 2001). Identification of gcpE as a novel gene of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis in Escherichia coli . FEBS Lett 488:170–173 [View Article][PubMed]
    [Google Scholar]
  8. Considine K. M., Fitzgerald G. F., Kelly A. L., Hill C. ( 2011). EzrA (lmo1594) is essential for growth and survival of Listeria monocytogenes EGDe. Bioeng Bugs 2:150–159 [View Article][PubMed]
    [Google Scholar]
  9. Davey M. S., Lin C. Y., Roberts G. W., Heuston S., Brown A. C., Chess J. A., Toleman M. A., Gahan C. G., Hill C. et al. ( 2011). Human neutrophil clearance of bacterial pathogens triggers anti-microbial γδ T cell responses in early infection. PLoS Pathog 7:e1002040 [View Article][PubMed]
    [Google Scholar]
  10. Dramsi S., Bourdichon F., Cabanes D., Lecuit M., Fsihi H., Cossart P. ( 2004). FbpA, a novel multifunctional Listeria monocytogenes virulence factor. Mol Microbiol 53:639–649 [View Article][PubMed]
    [Google Scholar]
  11. Dubail I., Bigot A., Lazarevic V., Soldo B., Euphrasie D., Dupuis M., Charbit A. ( 2006). Identification of an essential gene of Listeria monocytogenes involved in teichoic acid biogenesis. J Bacteriol 188:6580–6591 [View Article][PubMed]
    [Google Scholar]
  12. Eberl M., Altincicek B., Kollas A. K., Sanderbrand S., Bahr U., Reichenberg A., Beck E., Foster D., Wiesner J. et al. ( 2002). Accumulation of a potent γδT-cell stimulator after deletion of the lytB gene in Escherichia coli . Immunology 106:200–211 [View Article][PubMed]
    [Google Scholar]
  13. Eberl M., Hintz M., Reichenberg A., Kollas A. K., Wiesner J., Jomaa H. ( 2003). Microbial isoprenoid biosynthesis and human γδ T cell activation. FEBS Lett 544:4–10 [View Article][PubMed]
    [Google Scholar]
  14. Glaser P., Frangeul L., Buchrieser C., Rusniok C., Amend A., Baquero F., Berche P., Bloecker H., Brandt P. et al. ( 2001). Comparative genomics of Listeria species. Science 294:849–852[PubMed]
    [Google Scholar]
  15. Goldstein J. L., Brown M. S. ( 1990). Regulation of the mevalonate pathway. Nature 343:425–430 [View Article][PubMed]
    [Google Scholar]
  16. Hara T., Mizuno Y., Takaki K., Takada H., Akeda H., Aoki T., Nagata M., Ueda K., Matsuzaki G., Yoshikai Y. ( 1992). Predominant activation and expansion of V γ 9-bearing γ δ T cells in vivo as well as in vitro in Salmonella infection. J Clin Invest 90:204–210 [View Article][PubMed]
    [Google Scholar]
  17. Hecht S., Eisenreich W., Adam P., Amslinger S., Kis K., Bacher A., Arigoni D., Rohdich F. ( 2001). Studies on the nonmevalonate pathway to terpenes: the role of the GcpE (IspG) protein. Proc Natl Acad Sci U S A 98:14837–14842 [View Article][PubMed]
    [Google Scholar]
  18. Horton R. M., Cai Z. L., Ho S. N., Pease L. R. ( 1990). Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques 8:528–535[PubMed]
    [Google Scholar]
  19. Hunter W. N. ( 2007). The non-mevalonate pathway of isoprenoid precursor biosynthesis. J Biol Chem 282:21573–21577 [View Article][PubMed]
    [Google Scholar]
  20. Kabelitz D., Bender A., Prospero T., Wesselborg S., Janssen O., Pechhold K. ( 1991). The primary response of human γ/δ + T cells to Mycobacterium tuberculosis is restricted to V γ 9-bearing cells. J Exp Med 173:1331–1338 [View Article][PubMed]
    [Google Scholar]
  21. Law J., Buist G., Haandrikman A., Kok J., Venema G., Leenhouts K. ( 1995). A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes. J Bacteriol 177:7011–7018[PubMed]
    [Google Scholar]
  22. Leenhouts K., Buist G., Bolhuis A., ten Berge A., Kiel J., Mierau I., Dabrowska M., Venema G., Kok J. ( 1996). A general system for generating unlabelled gene replacements in bacterial chromosomes. Mol Gen Genet 253:217–224 [View Article][PubMed]
    [Google Scholar]
  23. Leenhouts K., Venema G., Kok J. ( 1998). A lactococcal pWV01-based integration toolbox for bacteria. Methods Cell Sci 20:35–50 [View Article]
    [Google Scholar]
  24. Maguin E., Duwat P., Hege T., Ehrlich D., Gruss A. ( 1992). New thermosensitive plasmid for Gram-positive bacteria. J Bacteriol 174:5633–5638[PubMed]
    [Google Scholar]
  25. McAteer S., Coulson A., McLennan N., Masters M. ( 2001). The lytB gene of Escherichia coli is essential and specifies a product needed for isoprenoid biosynthesis. J Bacteriol 183:7403–7407 [View Article][PubMed]
    [Google Scholar]
  26. Monk I. R., Gahan C. G. M., Hill C. ( 2008a). Tools for functional postgenomic analysis of Listeria monocytogenes . Appl Environ Microbiol 74:3921–3934 [View Article][PubMed]
    [Google Scholar]
  27. Monk I. R., Casey P. G., Cronin M., Gahan C. G., Hill C. ( 2008b). Development of multiple strain competitive index assays for Listeria monocytogenes using pIMC; a new site-specific integrative vector. BMC Microbiol 8:96 [View Article][PubMed]
    [Google Scholar]
  28. Park S. F., Stewart G. S. ( 1990). High-efficiency transformation of Listeria monocytogenes by electroporation of penicillin-treated cells. Gene 94:129–132 [View Article][PubMed]
    [Google Scholar]
  29. Popják G., Boehm G., Parker T. S., Edmond J., Edwards P. A., Fogelman A. M. ( 1979). Determination of mevalonate in blood plasma in man and rat. Mevalonate “tolerance” tests in man. J Lipid Res 20:716–728[PubMed]
    [Google Scholar]
  30. Raya R., Bardowski J., Andersen P. S., Ehrlich S. D., Chopin A. ( 1998). Multiple transcriptional control of the Lactococcus lactis trp operon. J Bacteriol 180:3174–3180[PubMed]
    [Google Scholar]
  31. Rodríguez-Concepción M., Boronat A. ( 2002). Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol 130:1079–1089 [View Article][PubMed]
    [Google Scholar]
  32. Sacchettini J. C., Poulter C. D. ( 1997). Creating isoprenoid diversity. Science 277:1788–1789 [View Article][PubMed]
    [Google Scholar]
  33. Sambrook J., Fritsch E. F., Maniatis T. ( 1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  34. Schauer K., Geginat G., Liang C., Goebel W., Dandekar T., Fuchs T. M. ( 2010). Deciphering the intracellular metabolism of Listeria monocytogenes by mutant screening and modelling. BMC Genomics 11:573 [View Article][PubMed]
    [Google Scholar]
  35. Shin S. J., Wu C. W., Steinberg H., Talaat A. M. ( 2006). Identification of novel virulence determinants in Mycobacterium paratuberculosis by screening a library of insertional mutants. Infect Immun 74:3825–3833 [View Article][PubMed]
    [Google Scholar]
  36. Smith K., Youngman P. ( 1992). Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene. Biochimie 74:705–711 [View Article][PubMed]
    [Google Scholar]
  37. Toledo-Arana A., Dussurget O., Nikitas G., Sesto N., Guet-Revillet H., Balestrino D., Loh E., Gripenland J., Tiensuu T. et al. ( 2009). The Listeria transcriptional landscape from saprophytism to virulence. Nature 459:950–956 [View Article][PubMed]
    [Google Scholar]
  38. Vázquez-Boland J. A., Kuhn M., Berche P., Chakraborty T., Domínguez-Bernal G., Goebel W., González-Zorn B., Wehland J., Kreft J. ( 2001). Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 14:584–640 [View Article][PubMed]
    [Google Scholar]
  39. Wilding E. I., Brown J. R., Bryant A. P., Chalker A. F., Holmes D. J., Ingraham K. A., Iordanescu S., So C. Y., Rosenberg M., Gwynn M. N. ( 2000a). Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in Gram-positive cocci. J Bacteriol 182:4319–4327 [View Article][PubMed]
    [Google Scholar]
  40. Wilding E. I., Kim D.-Y., Bryant A. P., Gwynn M. N., Lunsford R. D., McDevitt D., Myers J. E. Jr, Rosenberg M., Sylvester D. et al. ( 2000b). Essentiality, expression, and characterization of the class II 3-hydroxy-3-methylglutaryl coenzyme A reductase of Staphylococcus aureus . J Bacteriol 182:5147–5152 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.056069-0
Loading
/content/journal/micro/10.1099/mic.0.056069-0
Loading

Data & Media loading...

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