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. 1990 Mar;172(3):1509–1515. doi: 10.1128/jb.172.3.1509-1515.1990

Molecular cloning of the C-terminal domain of Escherichia coli D-mannitol permease: expression, phosphorylation, and complementation with C-terminal permease deletion proteins.

D W White 1, G R Jacobson 1
PMCID: PMC208627  PMID: 2407724

Abstract

We have subcloned a portion of the Escherichia coli mtlA gene encoding the hydrophilic, C-terminal domain of the mannitol-specific enzyme II (mannitol permease; molecular mass, 68 kilodaltons [kDa]) of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system. This mtlA fragment, encoding residues 379 to 637 (residue 637 = C terminus), was cloned in frame into the expression vector pCQV2 immediately downstream from the lambda pr promoter of the vector, which also encodes a temperature-sensitive lambda repressor. E. coli cells carrying a chromosomal deletion in mtlA (strain LGS322) and harboring this recombinant plasmid, pDW1, expressed a 28-kDa protein cross-reacting with antipermease antibody when grown at 42 degrees C but not when grown at 32 degrees C. This protein was relatively stable and could be phosphorylated in vitro by the general phospho-carrier protein of the phosphotransferase system, phospho-HPr. Thus, this fragment of the permease, when expressed in the absence of the hydrophobic, membrane-bound N-terminal domain, can apparently fold into a conformation resembling that of the C-terminal domain of the intact permease. When transformed into LGS322 cells harboring plasmid pGJ9-delta 137, which encodes a C-terminally truncated and inactive permease (residues 1 to ca. 480; molecular mass, 51 kDa), pDW1 conferred a mannitol-positive phenotype to this strain when grown at 42 degrees C but not when grown at 32 degrees C. This strain also exhibited phosphoenolpyruvate-dependent mannitol phosphorylation activity only when grown at the higher temperature. In contrast, pDW1 could not complement a plasmid encoding the complementary N-terminal part of the permease (residues 1 to 377). The pathway of phosphorylation of mannitol by the combined protein products of pGJ9-delta 137 and pDPW1 was also investigated by using N-ethylmaleimide to inactivate the second phosphorylation sites of these permease fragments (proposed to be Cys-384). These results are discussed with respect to the domain structure of the permease and its mechanism of transport and phosphorylation.

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Selected References

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  1. Begley G. S., Hansen D. E., Jacobson G. R., Knowles J. R. Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate:glucose phosphotransferase system. Biochemistry. 1982 Oct 26;21(22):5552–5556. doi: 10.1021/bi00265a026. [DOI] [PubMed] [Google Scholar]
  2. Grisafi P. L., Scholle A., Sugiyama J., Briggs C., Jacobson G. R., Lengeler J. W. Deletion mutants of the Escherichia coli K-12 mannitol permease: dissection of transport-phosphorylation, phospho-exchange, and mannitol-binding activities. J Bacteriol. 1989 May;171(5):2719–2727. doi: 10.1128/jb.171.5.2719-2727.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Jacobson G. R., Lee C. A., Saier M. H., Jr Purification of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system. J Biol Chem. 1979 Jan 25;254(2):249–252. [PubMed] [Google Scholar]
  4. Jacobson G. R., Stephan M. M. Structural and functional domains of the mannitol-specific enzyme II of the E. coli phosphoenolpyruvate-dependent phosphotransferase system. FEMS Microbiol Rev. 1989 Jun;5(1-2):25–34. doi: 10.1016/0168-6445(89)90005-3. [DOI] [PubMed] [Google Scholar]
  5. Khandekar S. S., Jacobson G. R. Evidence for two distinct conformations of the Escherichia coli mannitol permease that are important for its transport and phosphorylation functions. J Cell Biochem. 1989 Feb;39(2):207–216. doi: 10.1002/jcb.240390212. [DOI] [PubMed] [Google Scholar]
  6. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  7. Lee C. A., Jacobson G. R., Saier M. H., Jr Plasmid-directed synthesis of enzymes required for D-mannitol transport and utilization in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7336–7340. doi: 10.1073/pnas.78.12.7336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lee C. A., Saier M. H., Jr Mannitol-specific enzyme II of the bacterial phosphotransferase system. III. The nucleotide sequence of the permease gene. J Biol Chem. 1983 Sep 10;258(17):10761–10767. [PubMed] [Google Scholar]
  9. Lengeler J. W., Vogler A. P. Molecular mechanisms of bacterial chemotaxis towards PTS-carbohydrates. FEMS Microbiol Rev. 1989 Jun;5(1-2):81–92. doi: 10.1016/0168-6445(89)90011-9. [DOI] [PubMed] [Google Scholar]
  10. Leonard J. E., Saier M. H., Jr Mannitol-specific enzyme II of the bacterial phosphotransferase system. II. Reconstitution of vectorial transphosphorylation in phospholipid vesicles. J Biol Chem. 1983 Sep 10;258(17):10757–10760. [PubMed] [Google Scholar]
  11. Pas H. H., Ellory J. C., Robillard G. T. Bacterial phosphoenolpyruvate-dependent phosphotransferase system: association state of membrane-bound mannitol-specific enzyme II demonstrated by inactivation. Biochemistry. 1987 Oct 20;26(21):6689–6696. doi: 10.1021/bi00395a019. [DOI] [PubMed] [Google Scholar]
  12. Pas H. H., Robillard G. T. Enzyme IIMtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: identification of the activity-linked cysteine on the mannitol carrier. Biochemistry. 1988 Jul 26;27(15):5515–5519. doi: 10.1021/bi00415a019. [DOI] [PubMed] [Google Scholar]
  13. Pas H. H., Robillard G. T. S-phosphocysteine and phosphohistidine are intermediates in the phosphoenolpyruvate-dependent mannitol transport catalyzed by Escherichia coli EIIMtl. Biochemistry. 1988 Aug 9;27(16):5835–5839. doi: 10.1021/bi00416a002. [DOI] [PubMed] [Google Scholar]
  14. Pas H. H., ten Hoeve-Duurkens R. H., Robillard G. T. Bacterial phosphoenolpyruvate-dependent phosphotransferase system: mannitol-specific EII contains two phosphoryl binding sites per monomer and one high-affinity mannitol binding site per dimer. Biochemistry. 1988 Jul 26;27(15):5520–5525. doi: 10.1021/bi00415a020. [DOI] [PubMed] [Google Scholar]
  15. Postma P. W., Lengeler J. W. Phosphoenolpyruvate:carbohydrate phosphotransferase system of bacteria. Microbiol Rev. 1985 Sep;49(3):232–269. doi: 10.1128/mr.49.3.232-269.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Queen C. A vector that uses phage signals for efficient synthesis of proteins in Escherichia coli. J Mol Appl Genet. 1983;2(1):1–10. [PubMed] [Google Scholar]
  17. Robillard G. T., Blaauw M. Enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: protein-protein and protein-phospholipid interactions. Biochemistry. 1987 Sep 8;26(18):5796–5803. doi: 10.1021/bi00392a032. [DOI] [PubMed] [Google Scholar]
  18. Robillard G. T., Lolkema J. S. Enzymes II of the phosphoenolpyruvate-dependent sugar transport systems: a review of their structure and mechanism of sugar transport. Biochim Biophys Acta. 1988 Oct 11;947(3):493–519. doi: 10.1016/0304-4157(88)90005-6. [DOI] [PubMed] [Google Scholar]
  19. Roossien F. F., Blaauw M., Robillard G. T. Kinetics and subunit interaction of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system. Biochemistry. 1984 Oct 9;23(21):4934–4939. doi: 10.1021/bi00316a017. [DOI] [PubMed] [Google Scholar]
  20. Roossien F. F., Brink J., Robillard G. T. A simple procedure for the synthesis of [32P]phosphoenolpyruvate via the pyruvate kinase exchange reaction at equilibrium. Biochim Biophys Acta. 1983 Oct 4;760(1):185–187. doi: 10.1016/0304-4165(83)90141-1. [DOI] [PubMed] [Google Scholar]
  21. Roossien F. F., van Es-Spiekman W., Robillard G. T. Dimeric enzyme IImtl of the E. coli phosphoenolpyruvate-dependent phosphotransferase system. Cross-linking studies with bifunctional sulfhydryl reagents. FEBS Lett. 1986 Feb 17;196(2):284–290. doi: 10.1016/0014-5793(86)80264-2. [DOI] [PubMed] [Google Scholar]
  22. Saier M. H., Jr Protein phosphorylation and allosteric control of inducer exclusion and catabolite repression by the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Microbiol Rev. 1989 Mar;53(1):109–120. doi: 10.1128/mr.53.1.109-120.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stephan M. M., Jacobson G. R. Membrane disposition of the Escherichia coli mannitol permease: identification of membrane-bound and cytoplasmic domains. Biochemistry. 1986 Dec 16;25(25):8230–8234. doi: 10.1021/bi00373a016. [DOI] [PubMed] [Google Scholar]
  24. Stephan M. M., Jacobson G. R. Subunit interactions of the Escherichia coli mannitol permease: correlation with enzymic activities. Biochemistry. 1986 Jul 15;25(14):4046–4051. doi: 10.1021/bi00362a009. [DOI] [PubMed] [Google Scholar]
  25. Stephan M. M., Khandekar S. S., Jacobson G. R. Hydrophilic C-terminal domain of the Escherichia coli mannitol permease: phosphorylation, functional independence, and evidence for intersubunit phosphotransfer. Biochemistry. 1989 Sep 19;28(19):7941–7946. doi: 10.1021/bi00445a058. [DOI] [PubMed] [Google Scholar]
  26. Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

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