1887

Abstract

We have reported previously that the long-term survival of is facilitated by a dual-active enzyme MSDGC-1 (renamed DcpA), which controls the cellular turnover of cyclic diguanosine monophosphate (c-di-GMP). Most mycobacterial species possess at least a single copy of a DcpA orthologue that is highly conserved in terms of sequence similarity and domain architecture. Here, we show that DcpA exists in monomeric and dimeric forms. The dimerization of DcpA is due to non-covalent interactions between two protomers that are arranged in a parallel orientation. The dimer shows both synthesis and hydrolysis activities, whereas the monomer shows only hydrolysis activity. In addition, we have shown that DcpA is associated with the cytoplasmic membrane and exhibits heterogeneous cellular localization with a predominance at the cell poles. Finally, we have also shown that DcpA is involved in the change in cell length and colony morphology of . Taken together, our study provides additional evidence about the role of the bifunctional protein involved in c-di-GMP signalling in .

Funding
This study was supported by the:
  • Department of Biotechnology, India
  • CSIR, India
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2014-10-01
2024-12-13
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References

  1. Aldridge B. B., Fernandez-Suarez M., Heller D., Ambravaneswaran V., Irimia D., Toner M., Fortune S. M. ( 2012). Asymmetry and aging of mycobacterial cells lead to variable growth and antibiotic susceptibility. Science 335:100–104 [View Article][PubMed]
    [Google Scholar]
  2. Banerjee M., Gupta K., Balaram H., Balaram P. ( 2011). Mass spectrometric identification of an intramolecular disulfide bond in thermally inactivated triosephosphate isomerase from a thermophilic organism Methanocaldococcus jannaschii . Rapid Commun Mass Spectrom 25:1915–1923 [View Article][PubMed]
    [Google Scholar]
  3. Barends T. R., Hartmann E., Griese J. J., Beitlich T., Kirienko N. V., Ryjenkov D. A., Reinstein J., Shoeman R. L., Gomelsky M., Schlichting I. ( 2009). Structure and mechanism of a bacterial light-regulated cyclic nucleotide phosphodiesterase. Nature 459:1015–1018 [View Article][PubMed]
    [Google Scholar]
  4. Bharati B. K., Sharma I. M., Kasetty S., Kumar M., Mukherjee R., Chatterji D. ( 2012). A full-length bifunctional protein involved in c-di-GMP turnover is required for long-term survival under nutrient starvation in Mycobacterium smegmatis . Microbiology 158:1415–1427 [View Article][PubMed]
    [Google Scholar]
  5. Bharati B. K., Swetha R. K., Chatterji D. ( 2013). Identification and characterization of starvation induced msdgc-1 promoter involved in the c-di-GMP turnover. Gene 528:99–108 [View Article][PubMed]
    [Google Scholar]
  6. Bhattacharyya M., Gupta K., Gowd K. H., Balaram P. ( 2013). Rapid mass spectrometric determination of disulfide connectivity in peptides and proteins. Mol Biosyst 9:1340–1350 [View Article][PubMed]
    [Google Scholar]
  7. Bitan G., Kirkitadze M. D., Lomakin A., Vollers S. S., Benedek G. B., Teplow D. B. ( 2003). Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways. Proc Natl Acad Sci U S A 100:330–335 [View Article][PubMed]
    [Google Scholar]
  8. Carroll P., Schreuder L. J., Muwanguzi-Karugaba J., Wiles S., Robertson B. D., Ripoll J., Ward T. H., Bancroft G. J., Schaible U. E., Parish T. ( 2010). Sensitive detection of gene expression in mycobacteria under replicating and non-replicating conditions using optimized far-red reporters. PLoS ONE 5:e9823 [View Article][PubMed]
    [Google Scholar]
  9. Chakraborty M., Kuriata A. M., Nathan Henderson J., Salvucci M. E., Wachter R. M., Levitus M. ( 2012). Protein oligomerization monitored by fluorescence fluctuation spectroscopy: self-assembly of rubisco activase. Biophys J 103:949–958 [View Article][PubMed]
    [Google Scholar]
  10. Chan C., Paul R., Samoray D., Amiot N. C., Giese B., Jenal U., Schirmer T. ( 2004). Structural basis of activity and allosteric control of diguanylate cyclase. Proc Natl Acad Sci U S A 101:17084–17089 [View Article][PubMed]
    [Google Scholar]
  11. Chen Z. H., Schaap P. ( 2012). The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium . Nature 488:680–683 [View Article][PubMed]
    [Google Scholar]
  12. Chowdhury R. P., Gupta S., Chatterji D. ( 2007). Identification and characterization of the dps promoter of Mycobacterium smegmatis: promoter recognition by stress-specific extracytoplasmic function sigma factors sigmaH and sigmaF. J Bacteriol 189:8973–8981 [View Article][PubMed]
    [Google Scholar]
  13. Cui T., Zhang L., Wang X., He Z. G. ( 2009). Uncovering new signaling proteins and potential drug targets through the interactome analysis of Mycobacterium tuberculosis . BMC Genomics 10:118 [View Article][PubMed]
    [Google Scholar]
  14. Dahl J. L., Arora K., Boshoff H. I., Whiteford D. C., Pacheco S. A., Walsh O. J., Lau-Bonilla D., Davis W. B., Garza A. G. ( 2005). The relA homolog of Mycobacterium smegmatis affects cell appearance, viability, and gene expression. J Bacteriol 187:2439–2447 [View Article][PubMed]
    [Google Scholar]
  15. De N., Pirruccello M., Krasteva P. V., Bae N., Raghavan R. V., Sondermann H. ( 2008). Phosphorylation-independent regulation of the diguanylate cyclase WspR. PLoS Biol 6:e67 [View Article][PubMed]
    [Google Scholar]
  16. De N., Navarro M. V., Raghavan R. V., Sondermann H. ( 2009). Determinants for the activation and autoinhibition of the diguanylate cyclase response regulator WspR. J Mol Biol 393:619–633 [View Article][PubMed]
    [Google Scholar]
  17. Delogu G., Pusceddu C., Bua A., Fadda G., Brennan M. J., Zanetti S. ( 2004). Rv1818c-encoded PE_PGRS protein of Mycobacterium tuberculosis is surface exposed and influences bacterial cell structure. Mol Microbiol 52:725–733 [View Article][PubMed]
    [Google Scholar]
  18. Engelborghs Y. ( 2012). An elegant way to quantitatively analyze oligomer formation in solution. Biophys J 103:1811–1812 [View Article][PubMed]
    [Google Scholar]
  19. Ferreira R. B., Antunes L. C., Greenberg E. P., McCarter L. L. ( 2008). Vibrio parahaemolyticus ScrC modulates cyclic dimeric GMP regulation of gene expression relevant to growth on surfaces. J Bacteriol 190:851–860 [View Article][PubMed]
    [Google Scholar]
  20. Gibbons H. S., Wolschendorf F., Abshire M., Niederweis M., Braunstein M. ( 2007). Identification of two Mycobacterium smegmatis lipoproteins exported by a SecA2-dependent pathway. J Bacteriol 189:5090–5100 [View Article][PubMed]
    [Google Scholar]
  21. Gupta K., Kumar M., Balaram P. ( 2010a). Disulfide bond assignments by mass spectrometry of native natural peptides: cysteine pairing in disulfide bonded conotoxins. Anal Chem 82:8313–8319 [View Article][PubMed]
    [Google Scholar]
  22. Gupta K., Kumar P., Chatterji D. ( 2010b). Identification, activity and disulfide connectivity of c-di-GMP regulating proteins in Mycobacterium tuberculosis . PLoS ONE 5:e15072 [View Article][PubMed]
    [Google Scholar]
  23. Hengge R. ( 2009). Principles of c-di-GMP signalling in bacteria. Nat Rev Microbiol 7:263–273 [View Article][PubMed]
    [Google Scholar]
  24. Huangyutitham V., Güvener Z. T., Harwood C. S. ( 2013). Subcellular clustering of the phosphorylated WspR response regulator protein stimulates its diguanylate cyclase activity. MBio 4:e00242-13 [View Article][PubMed]
    [Google Scholar]
  25. Indu S., Kumar S. T., Thakurela S., Gupta M., Bhaskara R. M., Ramakrishnan C., Varadarajan R. ( 2010). Disulfide conformation and design at helix N-termini. Proteins 78:1228–1242 [View Article][PubMed]
    [Google Scholar]
  26. Jain V., Saleem-Batcha R., China A., Chatterji D. ( 2006). Molecular dissection of the mycobacterial stringent response protein Rel. Protein Sci 15:1449–1464 [View Article][PubMed]
    [Google Scholar]
  27. Karaolis D. K., Rashid M. H., Chythanya R., Luo W., Hyodo M., Hayakawa Y. ( 2005). c-di-GMP (3′-5′-cyclic diguanylic acid) inhibits Staphylococcus aureus cell–cell interactions and biofilm formation. Antimicrob Agents Chemother 49:1029–1038 [View Article][PubMed]
    [Google Scholar]
  28. Kuchma S. L., Brothers K. M., Merritt J. H., Liberati N. T., Ausubel F. M., O’Toole G. A. ( 2007). BifA, a cyclic-Di-GMP phosphodiesterase, inversely regulates biofilm formation and swarming motility by Pseudomonas aeruginosa PA14. J Bacteriol 189:8165–8178 [View Article][PubMed]
    [Google Scholar]
  29. Lebowitz J., Lewis M. S., Schuck P. ( 2002). Modern analytical ultracentrifugation in protein science: a tutorial review. Protein Sci 11:2067–2079 [View Article][PubMed]
    [Google Scholar]
  30. Levet-Paulo M., Lazzaroni J. C., Gilbert C., Atlan D., Doublet P., Vianney A. ( 2011). The atypical two-component sensor kinase Lpl0330 from Legionella pneumophila controls the bifunctional diguanylate cyclase-phosphodiesterase Lpl0329 to modulate bis-(3′-5′)-cyclic dimeric GMP synthesis. J Biol Chem 286:31136–31144 [View Article][PubMed]
    [Google Scholar]
  31. Li W., He Z. G. ( 2012). LtmA, a novel cyclic di-GMP-responsive activator, broadly regulates the expression of lipid transport and metabolism genes in Mycobacterium smegmatis . Nucleic Acids Res 40:11292–11307 [View Article][PubMed]
    [Google Scholar]
  32. Liu N., Xu Y., Hossain S., Huang N., Coursolle D., Gralnick J. A., Boon E. M. ( 2012). Nitric oxide regulation of cyclic di-GMP synthesis and hydrolysis in Shewanella woodyi . Biochemistry 51:2087–2099 [View Article][PubMed]
    [Google Scholar]
  33. Malone J. G., Jaeger T., Spangler C., Ritz D., Spang A., Arrieumerlou C., Kaever V., Landmann R., Jenal U. ( 2010). YfiBNR mediates cyclic di-GMP dependent small colony variant formation and persistence in Pseudomonas aeruginosa . PLoS Pathog 6:e1000804 [View Article][PubMed]
    [Google Scholar]
  34. Mawuenyega K. G., Forst C. V., Dobos K. M., Belisle J. T., Chen J., Bradbury E. M., Bradbury A. R., Chen X. ( 2005). Mycobacterium tuberculosis functional network analysis by global subcellular protein profiling. Mol Biol Cell 16:396–404 [View Article][PubMed]
    [Google Scholar]
  35. O’Shea T. M., Klein A. H., Geszvain K., Wolfe A. J., Visick K. L. ( 2006). Diguanylate cyclases control magnesium-dependent motility of Vibrio fischeri . J Bacteriol 188:8196–8205 [View Article][PubMed]
    [Google Scholar]
  36. Ojha A. K., Mukherjee T. K., Chatterji D. ( 2000). High intracellular level of guanosine tetraphosphate in Mycobacterium smegmatis changes the morphology of the bacterium. Infect Immun 68:4084–4091 [View Article][PubMed]
    [Google Scholar]
  37. Parrish N. M., Dick J. D., Bishai W. R. ( 1998). Mechanisms of latency in Mycobacterium tuberculosis . Trends Microbiol 6:107–112 [View Article][PubMed]
    [Google Scholar]
  38. Paul R., Weiser S., Amiot N. C., Chan C., Schirmer T., Giese B., Jenal U. ( 2004). Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain. Genes Dev 18:715–727 [View Article][PubMed]
    [Google Scholar]
  39. Paul R., Abel S., Wassmann P., Beck A., Heerklotz H., Jenal U. ( 2007). Activation of the diguanylate cyclase PleD by phosphorylation-mediated dimerization. J Biol Chem 282:29170–29177 [View Article][PubMed]
    [Google Scholar]
  40. Qi Y., Rao F., Luo Z., Liang Z. X. ( 2009). A flavin cofactor-binding PAS domain regulates c-di-GMP synthesis in AxDGC2 from Acetobacter xylinum . Biochemistry 48:10275–10285 [View Article][PubMed]
    [Google Scholar]
  41. Rao F., Qi Y., Chong H. S., Kotaka M., Li B., Li J., Lescar J., Tang K., Liang Z. X. ( 2009). The functional role of a conserved loop in EAL domain-based cyclic di-GMP-specific phosphodiesterase. J Bacteriol 191:4722–4731 [View Article][PubMed]
    [Google Scholar]
  42. Rezwan M., Lanéelle M. A., Sander P., Daffé M. ( 2007). Breaking down the wall: fractionation of mycobacteria. J Microbiol Methods 68:32–39 [View Article][PubMed]
    [Google Scholar]
  43. Römling U., Galperin M. Y., Gomelsky M. ( 2013). Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 77:1–52 [View Article][PubMed]
    [Google Scholar]
  44. Roy A. B., Petrova O. E., Sauer K. ( 2012). The phosphodiesterase DipA (PA5017) is essential for Pseudomonas aeruginosa biofilm dispersion. J Bacteriol 194:2904–2915 [View Article][PubMed]
    [Google Scholar]
  45. Ryjenkov D. A., Tarutina M., Moskvin O. V., Gomelsky M. ( 2005). Cyclic diguanylate is a ubiquitous signaling molecule in bacteria: insights into biochemistry of the GGDEF protein domain. J Bacteriol 187:1792–1798 [View Article][PubMed]
    [Google Scholar]
  46. Schirmer T., Jenal U. ( 2009). Structural and mechanistic determinants of c-di-GMP signalling. Nat Rev Microbiol 7:724–735 [View Article][PubMed]
    [Google Scholar]
  47. Schoonmaker M. K., Bishai W. R., Lamichhane G. ( 2014). Nonclassical transpeptidases of Mycobacterium tuberculosis alter cell size, morphology, the cytosolic matrix, protein localization, virulence, and resistance to β-lactams. J Bacteriol 196:1394–1402 [View Article][PubMed]
    [Google Scholar]
  48. Seshasayee A. S., Fraser G. M., Luscombe N. M. ( 2010). Comparative genomics of cyclic-di-GMP signalling in bacteria: post-translational regulation and catalytic activity. Nucleic Acids Res 38:5970–5981 [View Article][PubMed]
    [Google Scholar]
  49. Sharma I. M., Dhanaraman T., Mathew R., Chatterji D. ( 2012). Synthesis and characterization of a fluorescent analogue of cyclic di-GMP. Biochemistry 51:5443–5453 [View Article][PubMed]
    [Google Scholar]
  50. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R. Jr ( 1990). Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis . Mol Microbiol 4:1911–1919 [View Article][PubMed]
    [Google Scholar]
  51. Stahlberg H., Kutejová E., Muchová K., Gregorini M., Lustig A., Müller S. A., Olivieri V., Engel A., Wilkinson A. J., Barák I. ( 2004). Oligomeric structure of the Bacillus subtilis cell division protein DivIVA determined by transmission electron microscopy. Mol Microbiol 52:1281–1290 [View Article][PubMed]
    [Google Scholar]
  52. Sundriyal A., Massa C., Samoray D., Zehender F., Sharpe T., Jenal U., Schirmer T. ( 2014). Inherent regulation of EAL domain-catalyzed hydrolysis of second messenger cyclic di-GMP. J Biol Chem 289:6978–6990[PubMed] [CrossRef]
    [Google Scholar]
  53. Tarutina M., Ryjenkov D. A., Gomelsky M. ( 2006). An unorthodox bacteriophytochrome from Rhodobacter sphaeroides involved in turnover of the second messenger c-di-GMP. J Biol Chem 281:34751–34758 [View Article][PubMed]
    [Google Scholar]
  54. Thanky N. R., Young D. B., Robertson B. D. ( 2007). Unusual features of the cell cycle in mycobacteria: polar-restricted growth and the snapping-model of cell division. Tuberculosis (Edinb) 87:231–236 [View Article][PubMed]
    [Google Scholar]
  55. Triccas J. A., Parish T., Britton W. J., Gicquel B. ( 1998). An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis . FEMS Microbiol Lett 167:151–156 [View Article][PubMed]
    [Google Scholar]
  56. van den Heuvel R. H. H., Heck A. J. R. ( 2004). Native protein mass spectrometry: from intact oligomers to functional machineries. Curr Opin Chem Biol 8:519–526 [View Article][PubMed]
    [Google Scholar]
  57. van Dieck J., Fernandez-Fernandez M. R., Veprintsev D. B., Fersht A. R. ( 2009). Modulation of the oligomerization state of p53 by differential binding of proteins of the S100 family to p53 monomers and tetramers. J Biol Chem 284:13804–13811 [View Article][PubMed]
    [Google Scholar]
  58. Wan X., Tuckerman J. R., Saito J. A., Freitas T. A., Newhouse J. S., Denery J. R., Galperin M. Y., Gonzalez G., Gilles-Gonzalez M. A., Alam M. ( 2009). Globins synthesize the second messenger bis-(3′-5′)-cyclic diguanosine monophosphate in bacteria. J Mol Biol 388:262–270 [View Article][PubMed]
    [Google Scholar]
  59. Wassmann P., Chan C., Paul R., Beck A., Heerklotz H., Jenal U., Schirmer T. ( 2007). Structure of BeF3 -modified response regulator PleD: implications for diguanylate cyclase activation, catalysis, and feedback inhibition. Structure 15:915–927 [View Article][PubMed]
    [Google Scholar]
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