1887

Abstract

The opportunistic pathogen causes a wide range of infections, including chronic biofilm infections in the lungs of individuals with cystic fibrosis. We previously found that the inner-membrane protein MgtE can function both as a magnesium transporter and a virulence modulator, although the exact mechanism governing these activities is unclear. To address this issue, we carried out an experimental characterization of MgtE and generated a computer-rendered model. Our analysis demonstrated the structural similarity of MgtE to that of the crystal structure of MgtE in . Experimentally, we verified that MgtE is not essential for growth and found that it may not be involved directly in biofilm formation, even under low-magnesium conditions. We demonstrated both magnesium transport and cytotoxicity-regulating functions, and showed that magnesium-binding sites in the connecting helix region of MgtE are vital in coupling these two functions. Furthermore, limiting magnesium environments stimulated transcriptional responses. Our results suggested that MgtE might play an important role in linking magnesium availability to pathogenesis.

Funding
This study was supported by the:
  • Research Support Funds Grant
  • Purdue Research Foundation
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.075275-0
2014-06-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/160/6/1200.html?itemId=/content/journal/micro/10.1099/mic.0.075275-0&mimeType=html&fmt=ahah

References

  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. ( 1997). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [View Article][PubMed]
    [Google Scholar]
  2. Anderson G. G., Moreau-Marquis S., Stanton B. A., O’Toole G. A. ( 2008). In vitro analysis of tobramycin-treated Pseudomonas aeruginosa biofilms on cystic fibrosis-derived airway epithelial cells. Infect Immun 76:1423–1433 [View Article][PubMed]
    [Google Scholar]
  3. Anderson G. G., Yahr T. L., Lovewell R. R., O’Toole G. A. ( 2010). The Pseudomonas aeruginosa magnesium transporter MgtE inhibits transcription of the type III secretion system. Infect Immun 78:1239–1249 [View Article][PubMed]
    [Google Scholar]
  4. Balasubramanian D., Schneper L., Kumari H., Mathee K. ( 2013). A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence. Nucleic Acids Res 41:1–20 [View Article][PubMed]
    [Google Scholar]
  5. Bateman A. ( 1997). The structure of a domain common to archaebacteria and the homocystinuria disease protein. Trends Biochem Sci 22:12–13 [View Article][PubMed]
    [Google Scholar]
  6. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F. Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. ( 1977). The Protein Data Bank. A computer-based archival file for macromolecular structures. Eur J Biochem 80:319–324 [View Article][PubMed]
    [Google Scholar]
  7. Bruce M. C., Poncz L., Klinger J. D., Stern R. C., Tomashefski J. F. Jr, Dearborn D. G. ( 1985). Biochemical and pathologic evidence for proteolytic destruction of lung connective tissue in cystic fibrosis. Am Rev Respir Dis 132:529–535[PubMed]
    [Google Scholar]
  8. Carugo O., Pongor S. ( 2001). A normalized root-mean-square distance for comparing protein three-dimensional structures. Protein Sci 10:1470–1473 [View Article][PubMed]
    [Google Scholar]
  9. Colovos C., Yeates T. O. ( 1993). Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci 2:1511–1519 [View Article][PubMed]
    [Google Scholar]
  10. Cozens A. L., Yezzi M. J., Kunzelmann K., Ohrui T., Chin L., Eng K., Finkbeiner W. E., Widdicombe J. H., Gruenert D. C. ( 1994). CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 10:38–47 [View Article][PubMed]
    [Google Scholar]
  11. Dann C. E. III, Wakeman C. A., Sieling C. L., Baker S. C., Irnov I., Winkler W. C. ( 2007). Structure and mechanism of a metal-sensing regulatory RNA. Cell 130:878–892 [View Article][PubMed]
    [Google Scholar]
  12. Diaz M. R., King J. M., Yahr T. L. ( 2011). Intrinsic and extrinsic regulation of type III secretion gene expression in Pseudomonas aeruginosa. Front Microbiol 2:89[PubMed]
    [Google Scholar]
  13. Dötsch A., Eckweiler D., Schniederjans M., Zimmermann A., Jensen V., Scharfe M., Geffers R., Häussler S. ( 2012). The Pseudomonas aeruginosa transcriptome in planktonic cultures and static biofilms using RNA sequencing. PLoS ONE 7:e31092 [View Article][PubMed]
    [Google Scholar]
  14. Estévez R., Pusch M., Ferrer-Costa C., Orozco M., Jentsch T. J. ( 2004). Functional and structural conservation of CBS domains from CLC chloride channels. J Physiol 557:363–378 [View Article][PubMed]
    [Google Scholar]
  15. Eswar N., Webb B., Marti-Renom M. A., Madhusudhan M. S., Eramian D., Shen M. Y., Pieper U., Sali A. ( 2006). Comparative protein structure modeling using Modeller.. Curr Protoc Bioinformatics 5:6.1–6.30 [View Article][PubMed]
    [Google Scholar]
  16. Folkesson A., Jelsbak L., Yang L., Johansen H. K., Ciofu O., Høiby N., Molin S. ( 2012). Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective. Nat Rev Microbiol 10:841–851 [View Article][PubMed]
    [Google Scholar]
  17. Guerrero-Romero F., Bermudez-Peña C., Rodríguez-Morán M. ( 2011). Severe hypomagnesemia and low-grade inflammation in metabolic syndrome. Magnes Res 24:45–53[PubMed]
    [Google Scholar]
  18. Guina T., Wu M., Miller S. I., Purvine S. O., Yi E. C., Eng J., Goodlett D. R., Aebersold R., Ernst R. K., Lee K. A. ( 2003). Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation. J Am Soc Mass Spectrom 14:742–751 [View Article][PubMed]
    [Google Scholar]
  19. Gupta A., Eastham K. M., Wrightson N., Spencer D. A. ( 2007). Hypomagnesaemia in cystic fibrosis patients referred for lung transplant assessment. J Cyst Fibros 6:360–362 [View Article][PubMed]
    [Google Scholar]
  20. Hattori M., Tanaka Y., Fukai S., Ishitani R., Nureki O. ( 2007). Crystal structure of the MgtE Mg2+ transporter. Nature 448:1072–1075 [View Article][PubMed]
    [Google Scholar]
  21. Hattori M., Iwase N., Furuya N., Tanaka Y., Tsukazaki T., Ishitani R., Maguire M. E., Ito K., Maturana A., Nureki O. ( 2009). Mg2+-dependent gating of bacterial MgtE channel underlies Mg2+ homeostasis. EMBO J 28:3602–3612 [View Article][PubMed]
    [Google Scholar]
  22. Hicks D. B., Wang Z., Wei Y., Kent R., Guffanti A. A., Banciu H., Bechhofer D. H., Krulwich T. A. ( 2003). A tenth atp gene and the conserved atpI gene of a Bacillus atp operon have a role in Mg2+ uptake. Proc Natl Acad Sci U S A 100:10213–10218 [View Article][PubMed]
    [Google Scholar]
  23. Hmiel S. P., Snavely M. D., Florer J. B., Maguire M. E., Miller C. G. ( 1989). Magnesium transport in Salmonella typhimurium: genetic characterization and cloning of three magnesium transport loci. J Bacteriol 171:4742–4751 [View Article][PubMed]
    [Google Scholar]
  24. Holloway B. W. ( 1955). Genetic recombination in Pseudomonas aeruginosa. J Gen Microbiol 13:572–581 [View Article][PubMed]
    [Google Scholar]
  25. Humphrey W., Dalke A., Schulten K. ( 1996). VMD: visual molecular dynamics. J Mol Graph 14:33–38, 27–28 [View Article][PubMed]
    [Google Scholar]
  26. Ignoul S., Eggermont J. ( 2005). CBS domains: structure, function, and pathology in human proteins. Am J Physiol Cell Physiol 289:C1369–C1378 [View Article][PubMed]
    [Google Scholar]
  27. Jain M., Ramirez D., Seshadri R., Cullina J. F., Powers C. A., Schulert G. S., Bar-Meir M., Sullivan C. L., McColley S. A., Hauser A. R. ( 2004). Type III secretion phenotypes of Pseudomonas aeruginosa strains change during infection of individuals with cystic fibrosis. J Clin Microbiol 42:5229–5237 [View Article][PubMed]
    [Google Scholar]
  28. Janert P. K. ( 2010). Gnuplot in Action: Understanding Data with Graphs Greenwich, CT: Manning;
    [Google Scholar]
  29. Kakuda T., DiRita V. J. ( 2006). Cj1496c encodes a Campylobacter jejuni glycoprotein that influences invasion of human epithelial cells and colonization of the chick gastrointestinal tract. Infect Immun 74:4715–4723 [View Article][PubMed]
    [Google Scholar]
  30. Kolisek M., Launay P., Beck A., Sponder G., Serafini N., Brenkus M., Froschauer E. M., Martens H., Fleig A., Schweigel M. ( 2008). SLC41A1 is a novel mammalian Mg2+ carrier. J Biol Chem 283:16235–16247 [View Article][PubMed]
    [Google Scholar]
  31. Kopp J., Schwede T. ( 2004). Automated protein structure homology modeling: a progress report. Pharmacogenomics 5:405–416 [View Article][PubMed]
    [Google Scholar]
  32. Kuchma S. L., Connolly J. P., O’Toole G. A. ( 2005). A three-component regulatory system regulates biofilm maturation and type III secretion in Pseudomonas aeruginosa. J Bacteriol 187:1441–1454 [View Article][PubMed]
    [Google Scholar]
  33. Kushwaha H. R., Singh A. K., Sopory S. K., Singla-Pareek S. L., Pareek A. ( 2009). Genome wide expression analysis of CBS domain containing proteins in Arabidopsis thaliana (L.) Heynh and Oryza sativa L. reveals their developmental and stress regulation. BMC Genomics 10:200 [View Article][PubMed]
    [Google Scholar]
  34. Laskowski R. A. ( 2003). Structural quality assurance. Methods Biochem Anal 44:273–303[PubMed]
    [Google Scholar]
  35. Laskowski R. A., Rullmannn J. A. C., MacArthur M. W., Kaptein R., Thornton J. M. ( 1996). aqua and procheck-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8:477–486 [View Article][PubMed]
    [Google Scholar]
  36. Lee D. G., Urbach J. M., Wu G., Liberati N. T., Feinbaum R. L., Miyata S., Diggins L. T., He J., Saucier M. & other authors ( 2006). Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial. Genome Biol 7:R90 [View Article][PubMed]
    [Google Scholar]
  37. Li L., Tutone A. F., Drummond R. S., Gardner R. C., Luan S. ( 2001). A novel family of magnesium transport genes in Arabidopsis. Plant Cell 13:2761–2775 [View Article][PubMed]
    [Google Scholar]
  38. Merino S., Gavín R., Altarriba M., Izquierdo L., Maguire M. E., Tomás J. M. ( 2001). The MgtE Mg2+ transport protein is involved in Aeromonas hydrophila adherence. FEMS Microbiol Lett 198:189–195 [View Article][PubMed]
    [Google Scholar]
  39. Miao E. A., Freeman J. A., Miller S. I. ( 2002). Transcription of the SsrAB regulon is repressed by alkaline pH and is independent of PhoPQ and magnesium concentration. J Bacteriol 184:1493–1497 [View Article][PubMed]
    [Google Scholar]
  40. Mulcahy H., Lewenza S. ( 2011). Magnesium limitation is an environmental trigger of the Pseudomonas aeruginosa biofilm lifestyle. PLoS ONE 6:e23307 [View Article][PubMed]
    [Google Scholar]
  41. Müsken M., Di Fiore S., Dötsch A., Fischer R., Häussler S. ( 2010). Genetic determinants of Pseudomonas aeruginosa biofilm establishment. Microbiology 156:431–441 [View Article][PubMed]
    [Google Scholar]
  42. O’Connor K., Fletcher S. A., Csonka L. N. ( 2009). Increased expression of Mg2+ transport proteins enhances the survival of Salmonella enterica at high temperature. Proc Natl Acad Sci U S A 106:17522–17527 [View Article][PubMed]
    [Google Scholar]
  43. O’Toole G. A. ( 2011). Microtiter dish biofilm formation assay. J Vis Exp47e2437 [View Article][PubMed]
    [Google Scholar]
  44. O’Toole G. A., Kolter R. ( 1998). Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30:295–304 [View Article][PubMed]
    [Google Scholar]
  45. Palmer K. L., Aye L. M., Whiteley M. ( 2007). Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum. J Bacteriol 189:8079–8087 [View Article][PubMed]
    [Google Scholar]
  46. Parkins M. D., Rendall J. C., Elborn J. S. ( 2012). Incidence and risk factors for pulmonary exacerbation treatment failures in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa. Chest 141:485–493 [View Article][PubMed]
    [Google Scholar]
  47. Pettersen E. F., Goddard T. D., Huang C. C., Couch G. S., Greenblatt D. M., Meng E. C., Ferrin T. E. ( 2004). UCSF Chimera – a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612 [View Article][PubMed]
    [Google Scholar]
  48. Ragumani S., Sauder J. M., Burley S. K., Swaminathan S. ( 2010). Structural studies on cytosolic domain of magnesium transporter MgtE from Enterococcus faecalis. Proteins 78:487–491 [View Article][PubMed]
    [Google Scholar]
  49. Rahme L. G., Stevens E. J., Wolfort S. F., Shao J., Tompkins R. G., Ausubel F. M. ( 1995). Common virulence factors for bacterial pathogenicity in plants and animals. Science 268:1899–1902 [View Article][PubMed]
    [Google Scholar]
  50. Rosenbluth D. B., Wilson K., Ferkol T., Schuster D. P. ( 2004). Lung function decline in cystic fibrosis patients and timing for lung transplantation referral. Chest 126:412–419 [View Article][PubMed]
    [Google Scholar]
  51. Sanders N. N., Franckx H., De Boeck K., Haustraete J., De Smedt S. C., Demeester J. ( 2006). Role of magnesium in the failure of rhDNase therapy in patients with cystic fibrosis. Thorax 61:962–966 [View Article][PubMed]
    [Google Scholar]
  52. Shanks R. M., Caiazza N. C., Hinsa S. M., Toutain C. M., O’Toole G. A. ( 2006). Saccharomyces cerevisiae-based molecular tool kit for manipulation of genes from gram-negative bacteria. Appl Environ Microbiol 72:5027–5036 [View Article][PubMed]
    [Google Scholar]
  53. Shanks R. M., Kadouri D. E., MacEachran D. P., O’Toole G. A. ( 2009). New yeast recombineering tools for bacteria. Plasmid 62:88–97 [View Article][PubMed]
    [Google Scholar]
  54. Shen M. Y., Sali A. ( 2006). Statistical potential for assessment and prediction of protein structures. Protein Sci 15:2507–2524 [View Article][PubMed]
    [Google Scholar]
  55. Sievers F., Wilm A., Dineen D., Gibson T. J., Karplus K., Li W., Lopez R., McWilliam H., Remmert M. & other authors ( 2011). Fast, scalable generation of high-quality protein multiple sequence alignments using clustal Omega. Mol Syst Biol 7:539 [View Article][PubMed]
    [Google Scholar]
  56. Simon R., Priefer U., Puhler A. ( 1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nat Biotech 1:784 [View Article][PubMed]
    [Google Scholar]
  57. Smith R. L., Thompson L. J., Maguire M. E. ( 1995). Cloning and characterization of MgtE, a putative new class of Mg2+ transporter from Bacillus firmus OF4. J Bacteriol 177:1233–1238[PubMed]
    [Google Scholar]
  58. Tanaka Y., Hattori M., Fukai S., Ishitania R., Nureki O. ( 2007). Crystallization and preliminary X-ray diffraction analysis of the cytosolic domain of the Mg2+ transporter MgtE. Acta Crystallogr Sect F Struct Biol Cryst Commun 63:678–681 [View Article][PubMed]
    [Google Scholar]
  59. Townsend D. E., Esenwine A. J., George J. III, Bross D., Maguire M. E., Smith R. L. ( 1995). Cloning of the mgtE Mg2+ transporter from Providencia stuartii and the distribution of mgtE in gram-negative and gram-positive bacteria. J Bacteriol 177:5350–5354[PubMed]
    [Google Scholar]
  60. Winsor G. L., Lam D. K., Fleming L., Lo R., Whiteside M. D., Yu N. Y., Hancock R. E., Brinkman F. S. ( 2011). Pseudomonas Genome Database: improved comparative analysis and population genomics capability for Pseudomonas genomes. Nucleic Acids Res 39:Database issueD596–D600 [View Article][PubMed]
    [Google Scholar]
  61. Yahr T. L., Wolfgang M. C. ( 2006). Transcriptional regulation of the Pseudomonas aeruginosa type III secretion system. Mol Microbiol 62:631–640 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.075275-0
Loading
/content/journal/micro/10.1099/mic.0.075275-0
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