1887

Abstract

For osmoprotection, can synthesize glycine betaine from externally supplied choline by the Bet system ( products). The major carrier of choline is the high-affinity, proton-driven, secondary transporter BetT, which belongs to the BCCT family of transporters. Fusion proteins consisting of N-terminal fragments of BetT linked to -galactosidase (LacZ) or alkaline phosphatase (PhoA) were constructed. By analysis of 51 fusion proteins with 37 unique fusion-points, the predictions that BetT comprised 12 membrane-spanning regions and that its N- and C-terminal extensions of about 12 and 180 amino acid residues, respectively, were situated in the cytoplasm were confirmed. This is believed to represent the first experimental examination of the membrane topology of a BCCT family protein. Osmotic upshock experiments were performed with spectinomycin-treated cells that had expressed the wild-type or a mutant BetT protein during growth at low osmolality (160 mosmol kg). The choline transport activity of wild-type BetT increased tenfold when the cells were stressed with 0.4 M NaCl (total osmolality 780 mosmol kg). The peak activity was recorded 5 min after the upshock and higher or lower concentrations of NaCl reduced the activity. Deletions of 1–12 C-terminal residues of BetT caused a gradual reduction in the degree of osmotic activation from ten- to twofold. Mutant proteins with deletion of 18–101 residues displayed a background transport activity, but they could not be osmotically activated. The data showed that the cytoplasmic C-terminal domain of BetT plays an important role in the regulation of BetT activity and that C-terminal truncations can cause BetT to be permanently locked in a low-transport-activity mode.

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2007-03-01
2020-02-27
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References

  1. Adams C. W., Forrest M. E., Cohen S. N., Beatty J. T.. 1989; Structural and functional analysis of transcriptional control of the Rhodobacter capsulatus puf operon. J Bacteriol171:473–482
    [Google Scholar]
  2. Andresen P. A., Kaasen I., Styrvold O. B., Boulnois G., Strøm A. R.. 1988; Molecular cloning, physical mapping and expression of the bet genes governing the osmoregulatory choline-glycine betaine pathway of Escherichia coli . J Gen Microbiol134:1737–1746
    [Google Scholar]
  3. Bochner B. R., Huang H. C., Schieven G. L., Ames B. N.. 1980; Positive selection for loss of tetracycline resistance. J Bacteriol143:926–933
    [Google Scholar]
  4. Boscari A., Mandon K., Dupont L., Poggi M. C., Le Rudulier D.. 2002; BetS is a major glycine betaine/proline betaine transporter required for early osmotic adjustment in Sinorizobium meliloti . J Bacteriol184:2654–2663[CrossRef]
    [Google Scholar]
  5. Calamia J., Manoil C.. 1990; lac permease of Escherichia coli : topology and sequence elements promoting membrane insertion. Proc Natl Acad Sci U S A87:4937–4941[CrossRef]
    [Google Scholar]
  6. Chung C. T., Niemela S. L., Miller R. H.. 1989; One-step preparation of competent Escherichia coli : transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A86:2172–2175[CrossRef]
    [Google Scholar]
  7. Culham D. E., Tripet B., Racher K. I., Voegele R. T., Hodges R. S., Wood J. M.. 2000; The role of the carboxyl terminal α -helical coiled-coil domain in osmosensing by transporter ProP of Escherichia coli . J Mol Recognit13:309–322[CrossRef]
    [Google Scholar]
  8. Culham D. E., Henderson J., Crane R. A., Wood J. M.. 2003; Osmosensor ProP of Escherichia coli responds to the concentration, chemistry, and molecular size of osmolytes in the proteoliposome lumen. Biochemistry42:410–420[CrossRef]
    [Google Scholar]
  9. Danielsen S., Boyd D., Neuhard J.. 1995; Membrane topology analysis of the Escherichia coli cytosine permease. Microbiology141:2905–2913[CrossRef]
    [Google Scholar]
  10. Derman A. I., Beckwith J.. 1995; Escherichia coli alkaline phosphatase localized to the cytoplasm slowly acquires enzymatic activity in cells whose growth has been suspended: a caution for gene fusion studies. J Bacteriol177:3764–3770
    [Google Scholar]
  11. Fan X., Pericone C. D., Lysenko E., Goldfine H., Weiser J. N.. 2003; Multiple mechanisms for choline transport and utilization in Haemophilus influenzae . Mol Microbiol50:537–548[CrossRef]
    [Google Scholar]
  12. Farwick M., Siewe R. M., Krämer R.. 1995; Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum . J Bacteriol177:4690–4695
    [Google Scholar]
  13. Froshauer S., Green G. N., Boyd D., McGovern K., Beckwith J.. 1988; Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli . J Mol Biol200:501–511[CrossRef]
    [Google Scholar]
  14. Giæver H. M., Styrvold O. B., Kaasen I., Strøm A. R.. 1988; Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli . J Bacteriol170:2841–2849
    [Google Scholar]
  15. Hanahan D., Jessee J., Bloom F. R.. 1991; Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol204:63–113
    [Google Scholar]
  16. Hillar A., Culham D. E., Vernikovska Y. I., Wood J. M., Boggs J. M.. 2005; Formation of an antiparallel, intermolecular coiled coil is associated with in vivo dimerization of osmosensor and osmoprotectant transporter ProP in Escherichia coli . Biochemistry44:10170–10180[CrossRef]
    [Google Scholar]
  17. Jung K., Veen M., Altendorf K.. 2000; K+ and ionic strength directly influence the autophosphorylation activity of the putative turgor sensor KdpD of Escherichia coli . J Biol Chem275:40142–40147[CrossRef]
    [Google Scholar]
  18. Jung K., Hamann K., Revermann A.. 2001; K+ stimulates specifically the autokinase activity of purified and reconstituted EnvZ of Escherichia coli . J Biol Chem276:40896–40902[CrossRef]
    [Google Scholar]
  19. Jung H., Buchholz M., Clausen J., Nietschke M., Revermann A., Schmid R., Jung K.. 2002; CaiT of Escherichia coli , a new transporter catalysing l-carnitine/ γ -butyrobetaine exchange. J Biol Chem277:39251–39258[CrossRef]
    [Google Scholar]
  20. Kappes R. M., Kempf B., Bremer E.. 1996; Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis : characterization of OpuD. J Bacteriol178:5071–5079
    [Google Scholar]
  21. Krogh A., Larsson B., von Heijne G., Sonnhammer E. L. L.. 2001; Predicting transmembrane protein topology with a Hidden Markov Model: application to complete genomes. J Mol Biol305:567–580[CrossRef]
    [Google Scholar]
  22. Lamark T., Kaasen I., Eshoo M. W., Falkenberg P., McDougall J., Strøm A. R.. 1991; DNA sequence and analysis of the bet genes encoding the osmoregulatory choline-glycine betaine pathway of Escherichia coli . Mol Microbiol5:1049–1064[CrossRef]
    [Google Scholar]
  23. Lamark T., Røkenes T. P., McDougall J., Strøm A. R.. 1996; The complex bet promoters of Escherichia coli : regulation by oxygen (ArcA). choline (BetI), and osmotic stress. J Bacteriol178:1655–1662
    [Google Scholar]
  24. Landfald B., Strøm A. R.. 1986; Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli . J Bacteriol165:849–855
    [Google Scholar]
  25. Manoil C.. 1991; Analysis of membrane protein topology using alkaline phosphatase and β -galactosidase gene fusions. Methods Cell Biol34:61–75
    [Google Scholar]
  26. Manoil C., Beckwith J.. 1986; A genetic approach to analyzing membrane protein topology. Science233:1403–1408[CrossRef]
    [Google Scholar]
  27. May G., Faatz E., Villarejo M., Bremer E.. 1986; Binding protein dependent transport of glycine betaine and its osmotic regulation in Escherichia coli K12. Mol Gen Genet205:225–233[CrossRef]
    [Google Scholar]
  28. Miller J. H.. 1972; Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  29. Milner J. L., Grothe S., Wood J. M.. 1988; Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12. J Biol Chem263:14900–14905
    [Google Scholar]
  30. Morbach S., Krämer R.. 2003; Impact of transport processes in the osmotic response of Corynebacterium glutamicum . J Biotechnol104:69–75[CrossRef]
    [Google Scholar]
  31. Peter H., Burkovski A., Krämer R.. 1996; Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine. J Bacteriol178:5229–5234
    [Google Scholar]
  32. Peter H., Burkovski A., Krämer R.. 1998; Osmo-sensing by N- and C-terminal extensions of the glycine betaine uptake system BetP of Corynebacterium glutamicum . J Biol Chem273:2567–2574[CrossRef]
    [Google Scholar]
  33. Racher K. I., Voegele R. T., Marshall E. V., Culham D. E., Wood J. M., Jung H., Bacon M., Cairns M. T., Ferguson S. M.. & other authors 1999; Purification and reconstitution of an osmosensor: transporter ProP of Escherichia coli senses and responds to osmotic shifts. Biochemistry38:1676–1684[CrossRef]
    [Google Scholar]
  34. Røkenes T. P., Lamark T., Strøm A. R.. 1996; DNA-binding properties of the BetI repressor protein of Escherichia coli : the inducer choline stimulates BetI-DNA complex formation. J Bacteriol178:1663–1670
    [Google Scholar]
  35. Rübenhagen R., Rönsch H., Jung H., Krämer R., Morbach S.. 2000; Osmosensor and osmoregulator properties of the betaine carrier BetP from Corynebacterium glutamicum in proteoliposomes. J Biol Chem275:735–741[CrossRef]
    [Google Scholar]
  36. Rübenhagen R., Morbach S., Krämer R.. 2001; The osmoreactive betaine carrier BetP from Corynebacterium glutamicum is a sensor for cytoplasmic K+. EMBO J20:5412–5420[CrossRef]
    [Google Scholar]
  37. Saier M. H., Eng B. H., Fard S., Garg J., Haggerty D. A., Hutchinson W. J., Jack D. L., Lai E. C., Liu H. J.. & other authors 1999; Phylogenetic characterization of novel transport protein families revealed by genome analyses. Biochim Biophys Acta1422:1–56[CrossRef]
    [Google Scholar]
  38. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  39. Schiller D., Ott V., Morbach S., Krämer R.. 2006; Influence of membrane composition on osmosensing by the betaine carrier BetP from Corynebacterium glutamicum . J Biol Chem281:7737–7746[CrossRef]
    [Google Scholar]
  40. Schweizer H., Boos W.. 1983; Transfer of the Δ( argF-lac ) U169 mutation between Escherichia coli strains by selection for a closely linked Tn 10 insertion. Mol Gen Genet192:293–294[CrossRef]
    [Google Scholar]
  41. Styrvold O. B., Falkenberg P., Landfald B., Eshoo M. W., Bjornsen T., Strøm A. R.. 1986; Selection, mapping, and characterisation of osmoregulatory mutants of Escherichia coli blocked in the choline-glycine betaine pathway. J Bacteriol165:856–863
    [Google Scholar]
  42. van der Heide T., Poolman B.. 2000; Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane. Proc Natl Acad Sci U S A97:7102–7106[CrossRef]
    [Google Scholar]
  43. van der Heide T., Stuart M. C. A., Poolman B.. 2001; On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine. EMBO J20:7022–7032[CrossRef]
    [Google Scholar]
  44. Varga A. R., Kaplan S.. 1989; Construction, expression, and localization of a CycA : : PhoA fusion protein in Rhodobacter sphaeriodes and Escherichia coli . J Bacteriol171:5830–5839
    [Google Scholar]
  45. Vinothkumar K. R., Raunser S., Jung H., Kühlbrandt W.. 2006; Oligomeric structure of the carnitine transporter CaiT from Escherichia coli . J Biol Chem281:4795–4801[CrossRef]
    [Google Scholar]
  46. Ziegler C., Morbach S., Schiller D., Schubert D., Krämer R., Tziatzios C., Kühlbrandt W.. 2004; Projection structure and oligomeric state of the osmoregulated sodium/glycine betaine synporter BetP of Corynebacterium glutamicum . J Mol Biol337:1137–1147[CrossRef]
    [Google Scholar]
  47. Zoetewey D. L., Tripet B. P., Kutateladze T. G., Overduin M. J., Wood J. M., Hodges R. S.. 2003; Solution structure of the C-terminal antiparallel coiled-coil domain from Escherichia coli osmosensor ProP. J Mol Biol334:1063–1076[CrossRef]
    [Google Scholar]
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