1887

Abstract

XpsD is an outer-membrane protein required for extracellular protein secretion in Cross-linking and gel-filtration chromatography analyses have suggested that it forms a multimer. To determine its structure-function relationship, linker-insertion mutants were constructed in an gene carried on a plasmid. To assay for secretion function, each mutant gene was introduced into an ::Tn5 mutant strain (XC1708) and assayed for α-amylase secretion on starch plates. To test whether the mutant genes exerted a dominant-negative effect, each was introduced into the parental strain XC1701 and examined for secretion interference. Nine functional, one semi-functional and eleven non-functional mutants were obtained. All the non-functional mutants, except two for which the mutant proteins were undetectable on immunoblots, showed interference of normal secretion. The insertion sites in the different mutant proteins are randomly distributed throughout the entire sequence of the XpsD protein. All the permissive insertion sites are located where β-turn or coiled secondary structure is predicted. Over half of the non-permissive sites are located within predicted helical or β-sheet regions. By pretreating total membranes of XC1701 in SDS at 50 °C, an immunoreactive band with high molecular mass (HMM) could be detected that remained in the stacking gel during SDS-PAGE. The semi-functional and all functional mutant proteins formed HMM complexes that were as SDS-resistant as those of the wild-type, whereas all except three of the non-functional mutant proteins formed HMM structures that were less resistant to SDS than the wild-type. By analysing the appearance of SDS-resistant HMM complexes, we were able to detect conformational alterations in XpsD that are too subtle to be detected by other assays.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-144-6-1479
1998-06-01
2021-05-08
Loading full text...

Full text loading...

/deliver/fulltext/micro/144/6/mic-144-6-1479.html?itemId=/content/journal/micro/10.1099/00221287-144-6-1479&mimeType=html&fmt=ahah

References

  1. Brissette, J. L., Russel, M. (1990); Secretion and membrane integration of a filamentous phage-encoded morphogenetic protein.. Journal of Molecular Biology, 211,:565–580
    [Google Scholar]
  2. Chen, L.-Y., Chen, D.-Y., Miaw, J., Hu, N.-T. (1996); XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer.. ] Biol Chem, 271,:2703–2708
    [Google Scholar]
  3. Chou, P. Y., Fasman, G. D. (1978); Prediction of the secondary structure of proteins from their amino acid sequence.. Adv Enzymol Relat Areas Mol Biol, 47,:45–148
    [Google Scholar]
  4. Daefler, S., Russel, M., Model, P. (1997); Module swaps between related translocator proteins pIVn, pIVIKe and PulD: identification of a specificity domain.. Journal of Molecular Biology, 266,:978–992
    [Google Scholar]
  5. Daniels, M. J., Barber, C. E., Turner, P. C., Cleary, W. G., Sawczyc, M. K. (1984); Isolation of mutants of Xanthomonas campestris pv. campestris showing altered pathogenicity.. Journal of General Microbiology, 130,:2447–2455
    [Google Scholar]
  6. Drake, S. L., Koomey M. (1995); The product of the pilQ gene is essential for the biogenesis of type IV pili in Neisseria gonorrhoeae.. Molecular Microbiology, 18,:975–986
    [Google Scholar]
  7. Genin, S., Boucher C.A. (1994); A superfamily of proteins involved in different secretion pathways in gram-negative bacteria : modular structure and specificity of the N-terminal domain.. Molecular & General Genetics, 243,:112–118
    [Google Scholar]
  8. Gough, C. L., Dow, J. M., Barber, C. E., Daniels, M. J. (1988); Cloning of two endoglucanase genes of Xanthomonas campestris pv. campestris: analysis of the role of the major endoglucanase in pathogenesis.. Mol Plant-Microbe Interact, 1,:275–281
    [Google Scholar]
  9. Hardie, K. R., Lory, S., Pugsley, A. P. (1996); Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein.. Embo Journal, 15,:978–988
    [Google Scholar]
  10. Hill, D. F., Petersen, G. B. (1982); Nucleotide sequence of bacteriophage fl DNA.. Journal of Virology, 44,:32–46
    [Google Scholar]
  11. Hu, N.-T., Hung, M.-N., Chiou, S.-J., Tang, F., Chiang, D.-C. et al. (1992a); Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris.. Journal of Bacteriology, 174,:2679–2687
    [Google Scholar]
  12. Hu, N.-T., Hung, M.-N., Huang, A.-M., Tsai, H.-F., Yang, B.-Y. et al. (1992b); Molecular cloning, characterization and nucleotide sequence of the gene for secreted a- amylase from Xanthomonas campestris pv. campestris.. Journal of General Microbiology, 138,:1647–1655
    [Google Scholar]
  13. Hu, N.-T., Hung, M.-N., Liao, C.-T., Lin, M.-H. (1995); Subcellular location of XpsD, a protein required for extracellular protein secretion by Xanthomonas campestris pv. campestris.. Microbiology, 141,:1395–1406
    [Google Scholar]
  14. Kazmierczak, B. I., Mielke, D. L., Russel, M., Model, P. (1994); pIV, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer.. Journal of Molecular Biology, 238,:187–198
    [Google Scholar]
  15. Lathe, R., Kieny, M. P., Skory, S., Lecocq J. P. (1984); Linker tailing: unphosphorylated linker oligonucleotides for joining DNA termini.. DNA, 3,:173–182
    [Google Scholar]
  16. Lindeberg, M., Collmer, A. (1992); Analysis of eight out genes in a cluster required for pectic enzyme secretion by Erwinia chrysanthemi: sequence comparison with secretion genes from other gram-negative bacteria.. Journal of Bacteriology, 174,:7385–7397
    [Google Scholar]
  17. Linderoth, N. A., Model, P., Russel M. (1996); Essential role of a sodium dodecyl sulfate-resistant protein IV multimer in assembly-export of filamentous phage.. Journal of Bacteriology, 178,:1962–1970
    [Google Scholar]
  18. Lory, S. (1992); Determinants of extracellular protein secretion in gram-negative bacteria.. Journal of Bacteriology, 174,:3423–3428
    [Google Scholar]
  19. D., G., Schoenmakers, J. G., Konings, R. N., Day, L. A. (1985); Nucleotide sequence of the genome of Pf3, an IncP-1 plasmid-specific filamentous bacteriophage of Pseudomonas aeruginosa.. Journal of Virology, 56,:268–276
    [Google Scholar]
  20. Martin, P. R., Hobbs, M., Free, P. D., Jeske, Y., Mattick, J. S. (1993); Characterization of pilQ, a new gene required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa.. Molecular Microbiology, 9,:857–868
    [Google Scholar]
  21. Mead, D. A., Szczesna-Skorupa, E., Kemper, B. (1986); Singlestranded DNA ‘blue’ T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering.. Protein Engineering, 1,:67–76
    [Google Scholar]
  22. Miller, J. H. (1972) Experiments in Molecular Genetics. 433. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory;
    [Google Scholar]
  23. Newhall, W.j, Wilde, C. E., Sawyer, W. D., Haak, R. A. (1980)
  24. High-molecular-weight antigenic protein complex in the outer membrane of Neisseria gonorrhoeae Infection and Immunity, 27,:475–182
    [Google Scholar]
  25. Pugsley, A. P. (1993); The complete general secretory pathway in gram-negative bacteria.. Microbiological Reviews, 57,:50–108
    [Google Scholar]
  26. Ramer, S. W., Bieber, D., Schoolnik, G. K. (1996); BfpB, an outer membrane lipoprotein required for the biogenesis of bundleforming pili in enteropathogenic Escherichia coli.. Journal of Bacteriology, 178,:6555–6563
    [Google Scholar]
  27. Russel, M. (1994a); Phage assembly: a paradigm for bacterial virulence factor export.. Science, 265,:612–614
    [Google Scholar]
  28. Russel, M. (1994b); Mutants at conserved positions in gene IV, a gene required for assembly and secretion of filamentous phage.. Molecular Microbiology, 14,:357–369
    [Google Scholar]
  29. Salmond, G. P. C., Reeves P. J. (1993); Membrane traffic wardens and protein secretion in gram-negative bacteria.. Trends in Biochemical Sciences, 18,:7–12
    [Google Scholar]
  30. Sanger, F., Nicklen, S., Coulson A. R. (1977); DNA sequencing with chain-terminating inhibitors.. Proc Natl Acad Sci USA, 74,:5463–5467
    [Google Scholar]
  31. Shevchik, V. E., Robert-Baudouy, J., Condemine, G. (1997); Specific interaction between OutD, an Erwinia chrysanthemi outer membrane protein of the general secretory pathway, and secreted proteins.. Embo Journal, 11,:3007–3016
    [Google Scholar]
  32. Tang, J. L., Gough, C. L., Barber, G. E., Dow, J. M., Daniels, M. J. (1987); Molecular cloning of protease gene(s) from Xanthomonas campestris pv. campestris: expression in Escherichia coli and role in pathogenicity.. Molecular & General Genetics, 210,:443–448
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-144-6-1479
Loading
/content/journal/micro/10.1099/00221287-144-6-1479
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