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Volume 162, Issue 8

Effect of differing +2 amino acids on export of a heterologous PhoA lipoprotein in Free

Abstract

The significance of the amino acid adjacent to the amino terminal cysteine of lipoproteins, the +2 amino acid, has been well documented in and there have also been limited studies on Gram-positive bacteria. In this study we investigated whether there was any preference for specific residues and any targeting role attributable to different residues following the cysteine at the amino terminus in lipoproteins of . There were found to be distinct preferences in this position that vary considerably from the preferences seen in Gram-positive and Gram-negative bacteria. The effect of different amino acids at the +2 position was studied using the pTAP vector, which has been shown to express PhoA as a lipoprotein. Replacement of the threonine at the +2 position in the PhoA lipoprotein with hydrophobic amino acids resulted in higher levels of expression of alkaline phosphatase, while replacement with hydrophilic amino acids resulted in lower levels of expression of alkaline phosphatase. Changes in the +2 amino acid did not appear to alter export of the PhoA lipoprotein to the membrane fraction, but a difference was seen in susceptibility to proteolysis in PhoA lipoproteins with differing +2 amino acids. This is the first study to examine the role of the +2 amino acid in mycoplasma lipoproteins and establish a difference between and Gram-positive and Gram-negative bacteria and will assist in optimization of the design of recombinant lipoprotein genes in mycoplasmas for maximal levels of expression and stability on the cell surface.

  • Received:
  • Accepted:
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Keyword(s): C+2 amino acid , heterologous protein expression , Lipoprotein , membrane protein and Mycoplasma gallisepticum
© 2016 The Authors
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2016-08-01
2024-04-18
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References

  1. Babu M. M., Priya M. L., Selvan A. T., Madera M., Gough J., Aravind L., Sankaran K. 2006; A database of bacterial lipoproteins (DOLOP) with functional assignments to predicted lipoproteins. J Bacteriol 188:2761–2773 [View Article][PubMed]
     [Google Scholar]
  2. Bagos P. G., Tsirigos K. D., Liakopoulos T. D., Hamodrakas S. J. 2008; Prediction of lipoprotein signal peptides in Gram-positive bacteria with a hidden Markov model. J Proteome Res 7:5082–5093 [View Article][PubMed]
     [Google Scholar]
  3. Barré A., de Daruvar A., Blanchard A. 2004; MolliGen, a database dedicated to the comparative genomics of Mollicutes. Nucleic Acids Res 32:D307–D310 [View Article][PubMed]
     [Google Scholar]
  4. Bessler W. G., Jung G. 1992; Synthetic lipopeptides as novel adjuvants. Res Immunol 143:548–553 [View Article][PubMed]
     [Google Scholar]
  5. Bordier C. 1981; Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 256:1604–1607[PubMed]
     [Google Scholar]
  6. Cao T. B., Saier M. H., Jr. 2003; The general protein secretory pathway: phylogenetic analyses leading to evolutionary conclusions. Biochim Biophys Acta 1609:115–125 [View Article][PubMed]
     [Google Scholar]
  7. Chambaud I., Wróblewski H., Blanchard A. 1999; Interactions between mycoplasma lipoproteins and the host immune system. Trends Microbiol 7:493–499 [View Article][PubMed]
     [Google Scholar]
  8. de Castro E., Sigrist C. J., Gattiker A., Bulliard V., Langendijk-Genevaux P. S., Gasteiger E., Bairoch A., Hulo N. 2006; ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res 34:W362–W365 [View Article][PubMed]
     [Google Scholar]
  9. Hallamaa K. M., Browning G. F., Tang S. L. 2006; Lipoprotein multigene families in Mycoplasma pneumoniae. J Bacteriol 188:5393–5399 [View Article][PubMed]
     [Google Scholar]
  10. Harwood C. R., Cranenburgh R. 2008; Bacillus protein secretion: an unfolding story. Trends Microbiol 16:73–79 [View Article][PubMed]
     [Google Scholar]
  11. Konovalova A., Silhavy T. J. 2015; Outer membrane lipoprotein biogenesis: Lol is not the end. Philos Trans R Soc Lond B Biol Sci 370: pii: 20150030 [View Article][PubMed]
     [Google Scholar]
  12. Lewenza S., Mhlanga M. M., Pugsley A. P. 2008; Novel inner membrane retention signals in Pseudomonas aeruginosa lipoproteins. J Bacteriol 190:6119–6125 [View Article][PubMed]
     [Google Scholar]
  13. Ley D. H., Yoder Jr H. W. 1997; Mycoplasma gallisepticum infection. In Diseases of Poultry , pp. 194–207 Edited by Calnek B. W., Barnes H. J., Beard C. W., McDougald L. R., Saif Y. M. Ames, IA: Iowa State University Press;
     [Google Scholar]
  14. Liu Y. C., Lin I. H., Chung W. J., Hu W. S., Ng W. V., Lu C. Y., Huang T. Y., Shu H. W., Hsiao K. J. et al. 2012; Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64. PLoS One 7:e35304 [View Article][PubMed]
     [Google Scholar]
  15. Markham P. F., Glew M. D., Brandon M. R., Walker I. D., Whithear K. G. 1992; Characterization of a major hemagglutinin protein from Mycoplasma gallisepticum. Infect Immun 60:3885–3891[PubMed]
     [Google Scholar]
  16. Masuda K., Matsuyama S., Tokuda H. 2002; Elucidation of the function of lipoprotein-sorting signals that determine membrane localization. Proc Natl Acad Sci U S A 99:7390–7395 [View Article][PubMed]
     [Google Scholar]
  17. Minion F. C., Lefkowitz E. J., Madsen M. L., Cleary B. J., Swartzell S. M., Mahairas G. G. 2004; The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis. J Bacteriol 186:7123–7133 [View Article][PubMed]
     [Google Scholar]
  18. Narita S., Tokuda H. 2007; Amino acids at positions 3 and 4 determine the membrane specificity of Pseudomonas aeruginosa lipoproteins. J Biol Chem 282:13372–13378 [View Article][PubMed]
     [Google Scholar]
  19. Okuda S., Tokuda H. 2011; Lipoprotein sorting in bacteria. Annu Rev Microbiol 65:239–259 [View Article][PubMed]
     [Google Scholar]
  20. Panicker I. S., Browning G. F., Markham P. F. 2015; The effect of an alternate start codon on heterologous expression of a PhoA fusion protein in Mycoplasma gallisepticum. PLoS One 10:e0127911 [View Article][PubMed]
     [Google Scholar]
  21. Panicker I. S., Kanci A., Chiu C. J., Veith P. D., Glew M. D., Browning G. F., Markham P. F. 2012; A novel transposon construct expressing PhoA with potential for studying protein expression and translocation in Mycoplasma gallisepticum. BMC Microbiol 12:138 [View Article][PubMed]
     [Google Scholar]
  22. Proft T., Herrmann R. 1994; Identification and characterization of hitherto unknown Mycoplasma pneumoniae proteins. Mol Microbiol 13:337–385 [View Article][PubMed]
     [Google Scholar]
  23. Pugsley A. P., Kornacker M. G. 1991; Secretion of the cell surface lipoprotein pullulanase in Escherichia coli. Cooperation or competition between the specific secretion pathway and the lipoprotein sorting pathway. J Biol Chem 266:13640–13645[PubMed]
     [Google Scholar]
  24. Razin S., Yogev D., Naot Y. 1998; Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 62:1094–1156[PubMed]
     [Google Scholar]
  25. Schneewind O., Missiakas D. M. 2012; Protein secretion and surface display in Gram-positive bacteria. Philos Trans R Soc Lond B Biol Sci 367:1123–1139 [View Article][PubMed]
     [Google Scholar]
  26. Schneider C. A., Rasband W. S., Eliceiri K. W. 2012; NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675 [View Article][PubMed]
     [Google Scholar]
  27. Seydel A., Gounon P., Pugsley A. P. 1999; Testing the ‘+2 rule’ for lipoprotein sorting in the Escherichia coli cell envelope with a new genetic selection. Mol Microbiol 34:810–821 [View Article][PubMed]
     [Google Scholar]
  28. Silva-Herzog E., Ferracci F., Jackson M. W., Joseph S. S., Plano G. V. 2008; Membrane localization and topology of the Yersinia pestis YscJ lipoprotein. Microbiology 154:593–607 [View Article][PubMed]
     [Google Scholar]
  29. Sutcliffe I. C., Russell R. R. 1995; Lipoproteins of gram-positive bacteria. J Bacteriol 177:1123–1128[PubMed]
     [Google Scholar]
  30. Terada M., Kuroda T., Matsuyama S. I., Tokuda H. 2001; Lipoprotein sorting signals evaluated as the LolA-dependent release of lipoproteins from the cytoplasmic membrane of Escherichia coli. J Biol Chem 276:47690–47694 [View Article][PubMed]
     [Google Scholar]
  31. Tjalsma H., Bolhuis A., Jongbloed J. D., Bron S., van Dijl J. M. 2000; Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev 64:515–547 [View Article][PubMed]
     [Google Scholar]
  32. Tokunaga M., Tokunaga H., Wu H. C. 1982; Post-translational modification and processing of Escherichia coli prolipoprotein in vitro. Proc Natl Acad Sci U S A 79:2255–2259 [View Article][PubMed]
     [Google Scholar]
  33. van Wely K. H., Swaving J., Freudl R., Driessen A. J. 2001; Translocation of proteins across the cell envelope of Gram-positive bacteria. FEMS Microbiol Rev 25:437–454 [View Article][PubMed]
     [Google Scholar]
  34. Yamaguchi K., Yu F., Inouye M. 1988; A single amino acid determinant of the membrane localization of lipoproteins in E. coli. Cell 53:423–432 [View Article][PubMed]
     [Google Scholar]
  35. Yokota N., Kuroda T., Matsuyama S., Tokuda H. 1999; Characterization of the LolA-LolB system as the general lipoprotein localization mechanism of Escherichia coli. J Biol Chem 274:30995–30999 [View Article][PubMed]
     [Google Scholar]
  36. Zückert W. R. 2014; Secretion of bacterial lipoproteins: through the cytoplasmic membrane, the periplasm and beyond. Biochim Biophys Acta 1843:1509–1516 [View Article][PubMed]
     [Google Scholar]
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Effect of differing +2 amino acids on export of a heterologous PhoA lipoprotein in Mycoplasma gallisepticum
Microbiology 162, 1300 (2016); https://doi.org/10.1099/mic.0.000327
/content/journal/micro/10.1099/mic.0.000327
/content/journal/micro/10.1099/mic.0.000327
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