1887

Abstract

The Emb proteins (EmbA, EmbB, EmbC) are mycobacterial arabinosyltransferases involved in the biogenesis of the mycobacterial cell wall. EmbA and EmbB are predicted to work in unison as a heterodimer. EmbA and EmbB are involved in the formation of the crucial terminal hexaarabinoside motif [Ara(1→2)Ara(1→5)] [Ara(1→2)Ara(1→3)]Ara(1→5)Ara1→(Ara) in the cell wall polysaccharide arabinogalactan. Studies conducted in revealed that mutants with disruptions in or are viable, although the growth rate was affected. In contrast, we demonstrate here that is an essential gene in , since a deletion of the chromosomal gene could only be achieved when a second functional copy was provided on an integrated vector. Complementation of an mutant of by confirmed that it encodes a functional arabinosyltransferase. We identified a promoter for located immediately upstream of the gene, indicating that it is expressed independently from the upstream gene, . Promoter activity from P was sevenfold lower when assayed in compared to , indicating that the latter is not a good host for genetic analysis of expression. P activity remained constant throughout growth phases and after stress treatment, although it was reduced during hypoxia-induced non-replicating persistence. Ethambutol exposure had no effect on P activity. These data demonstrate that encodes a functional arabinosyltransferase which is constitutively expressed and plays a critical role in .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/012153-0
2008-01-01
2019-11-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/1/240.html?itemId=/content/journal/micro/10.1099/mic.0.2007/012153-0&mimeType=html&fmt=ahah

References

  1. Belanger, A. E., Besra, G. S., Ford, M. E., Mikusova, K., Belisle, J. T., Brennan, P. J. & Inamine, J. M. ( 1996; ). The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol. Proc Natl Acad Sci U S A 93, 11919–11924.[CrossRef]
    [Google Scholar]
  2. Berg, S., Torrelles, J. B., Chatterjee, D., Crick, D. C., Escuyer, V. E. & Brennan, P. J. ( 2003; ). Point mutations in embC affect synthesis of lipoarabinomannan in Mycobacterium smegmatis. Abstract submitted for the 8th Annual Conference of the Society for Glycobiology. Glycobiology 13, 853
    [Google Scholar]
  3. Blokpoel, M. C. J., Murphy, H. N., O'Toole, R., Wiles, S., Runn, E. S. C., Stewart, G. R., Young, D. B. & Robertson, B. D. ( 2005; ). Tetracycline-inducible gene regulation in mycobacteria. Nucleic Acids Res 33, e22 [CrossRef]
    [Google Scholar]
  4. Brennan, P. J. ( 2003; ). Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis. Tuberculosis (Edinb) 83, 91–97.[CrossRef]
    [Google Scholar]
  5. Carroll, P., Muttucumaru, D. G. N. & Parish, T. ( 2005; ). Use of a tetracycline-inducible system for conditional expression in Mycobacterium tuberculosis and Mycobacterium smegmatis. Appl Environ Microbiol 71, 3077–3084.[CrossRef]
    [Google Scholar]
  6. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S. & other authors ( 1998; ). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[CrossRef]
    [Google Scholar]
  7. Daffe, M., Brennan, P. J. & McNeil, M. R. ( 1990; ). Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass spectrometry and by 1H and 13C NMR analysis. J Biol Chem 265, 6734–6743.
    [Google Scholar]
  8. Dussurget, O., Timm, J., Gomez, M., Gold, B., Yu, S. W., Sabol, S. Z., Holmes, R. K., Jacobs, W. R. & Smith, I. ( 1999; ). Transcriptional control of the iron-responsive fxbA gene by the mycobacterial regulator IdeR. J Bacteriol 181, 3402–3408.
    [Google Scholar]
  9. Ehrt, S., Guo, X. Z. V., Hickey, C. M., Ryou, M., Monteleone, M., Riley, L. W. & Schnappinger, D. ( 2005; ). Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor. Nucleic Acids Res 33, e21 [CrossRef]
    [Google Scholar]
  10. Escuyer, V. E., Lety, M. A., Torrelles, J. B., Khoo, K. H., Tang, J. B., Rithner, C. D., Frehel, C., McNeil, M. R., Brennan, P. J. & Chatterjee, D. ( 2001; ). The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan. J Biol Chem 276, 48854–48862.[CrossRef]
    [Google Scholar]
  11. Hinds, J., Mahenthiralingam, E., Kempsell, K. E., Duncan, K., Stokes, R. W., Parish, T. & Stoker, N. G. ( 1999; ). Enhanced gene replacement in mycobacteria. Microbiology 145, 519–527.[CrossRef]
    [Google Scholar]
  12. Khasnobis, S., Zhang, J., Angala, S. K., Amin, A. G., McNeil, M. R., Crick, D. C. & Chatterjee, D. ( 2006; ). Characterization of a specific arabinosyltransferase activity involved in mycobacterial arabinan biosynthesis. Chem Biol 13, 787–795.[CrossRef]
    [Google Scholar]
  13. Khoo, K. H., Douglas, E., Azadi, P., Inamine, J. M., Besra, G. S., Mikusova, K., Brennan, P. J. & Chatterjee, D. ( 1996; ). Truncated structural variants of lipoarabinomannan in ethambutol drug- resistant strains of Mycobacterium smegmatis: inhibition of arabinan biosynthesis by ethambutol. J Biol Chem 271, 28682–28690.[CrossRef]
    [Google Scholar]
  14. Mahenthiralingam, E., Marklund, B. I., Brooks, L. A., Smith, D. A., Bancroft, G. J. & Stokes, R. W. ( 1998; ). Site-directed mutagenesis of the 19-kilodalton lipoprotein antigen reveals no essential role for the protein in the growth and virulence of Mycobacterium intracellulare. Infect Immun 66, 3626–3634.
    [Google Scholar]
  15. McNeil, M. R., Chatterjee, D., Hunter, S. W. & Brennan, P. J. ( 1989; ). Mycobacterial glycolipids: isolation, structures, antigenicity, and synthesis of neoantigens. Methods Enzymol 179, 215–242.
    [Google Scholar]
  16. McNeil, M. R., Robuck, K. G., Harter, M. & Brennan, P. J. ( 1994; ). Enzymatic evidence for the presence of a critical hexa-arabinoside in the cell walls of Mycobacterium tuberculosis. Glycobiology 4, 165–173.[CrossRef]
    [Google Scholar]
  17. Mikusova, K., Slayden, R. A., Besra, G. S. & Brennan, P. J. ( 1995; ). Biogenesis of the mycobacterial cell wall and the site of action of ethambutol. Antimicrob Agents Chemother 39, 2484–2489.[CrossRef]
    [Google Scholar]
  18. Miller, J. H. ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  19. Papavinasasundaram, K. G., Chan, B., Chung, J. H., Colston, M. J., Davis, E. O. & Av-Gay, Y. ( 2005; ). Deletion of the Mycobacterium tuberculosis pknH gene confers a higher bacillary load during the chronic phase of infection in BALB/c mice. J Bacteriol 187, 5751–5760.[CrossRef]
    [Google Scholar]
  20. Parish, T. & Stoker, N. G. ( 2000; ). Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology 146, 1969–1975.
    [Google Scholar]
  21. Parish, T. & Wheeler, P. R. ( 1998; ). Preparation of cell-free extracts from mycobacteria. In Mycobacteria Protocols, pp. 77–90. Edited by T. Parish & N. G. Stoker. Totowa: Humana Press.
  22. Pashley, C. A. & Parish, T. ( 2003; ). Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis. FEMS Microbiol Lett 229, 211–215.[CrossRef]
    [Google Scholar]
  23. Scherman, M. S., Kalbe-Bournonville, L., Bush, D., Xin, Y., Deng, L. & McNeil, M. ( 1996; ). Polyprenylphosphate-pentoses in mycobacteria are synthesized from 5-phosphoribose pyrophosphate. J Biol Chem 271, 29652–29658.[CrossRef]
    [Google Scholar]
  24. Seidel, M., Alderwick, L. J., Sahm, H., Besra, G. S. & Eggeling, L. ( 2007; ). Topology and mutational analysis of the single Emb arabinofuranosyltransferase of Corynebacterium glutamicum as a model of Emb proteins of Mycobacterium tuberculosis. Glycobiology 17, 210–219.
    [Google Scholar]
  25. Sharma, K., Gupta, M., Pathak, M., Gupta, N., Koul, A., Sarangi, S., Baweja, R. & Singh, Y. ( 2006; ). Transcriptional control of the mycobacterial embCAB operon by PknH through a regulatory protein, EmbR, in vivo. J Bacteriol 188, 2936–2944.[CrossRef]
    [Google Scholar]
  26. Sreevatsan, S., Pan, X., Zhang, Y., Kreiswirth, B. N. & Musser, J. M. ( 1997; ). Mutations associated with pyrazinamide resistance in pncA of Mycobacterium tuberculosis complex organisms. Antimicrob Agents Chemother 41, 636–640.
    [Google Scholar]
  27. Telenti, A., Philipp, W. J., Sreevatsan, S., Bernasconi, C., Stockbauer, K. E., Wieles, B., Musser, J. M. & Jacobs, W. R., Jr ( 1997; ). The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol. Nat Med 3, 567–570.[CrossRef]
    [Google Scholar]
  28. Wayne, L. G. & Sohaskey, C. D. ( 2001; ). Nonreplicating persistence of Mycobacterium tuberculosis. Annu Rev Microbiol 55, 139–163.[CrossRef]
    [Google Scholar]
  29. Xin, Y., Lee, R. E., Scherman, M. S., Khoo, K. H., Besra, G. S., Brennan, P. J. & McNeil, M. ( 1997; ). Characterization of the in vitro synthesized arabinan of mycobacterial cell walls. Biochim Biophys Acta 1335, 231–234.[CrossRef]
    [Google Scholar]
  30. Zhang, N., Torrelles, J., McNeil, M., Escuyer, V. E., Khoo, K. H., Brennan, P. J. & Chatterjee, D. ( 2003; ). The Emb proteins of mycobacteria direct arabinosylation of lipoarabinomannan and arabinogalactan via an N-terminal recognition region and a C-terminal synthetic region. Mol Microbiol 50, 69–76.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/012153-0
Loading
/content/journal/micro/10.1099/mic.0.2007/012153-0
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