1887

Abstract

Previous sequence analysis of the glucose-specific PTS gene locus from revealed the unexpected finding of two adjacent, highly similar ORFs, and ,each encoding a glucose-specific membrane permease EIICBA. and show 73% identity at the nucleotide level and is located 131 bp downstream from . Each of the genes is flanked by putative regulatory elements such as a termination stem–loop, promoter and ribosome-binding site, suggesting independent regulation. The finding of putative -active operator sequences, CRE (catabolite-responsive elements) suggests additional regulation by carbon catabolite repression. As described previously by the authors, both genes can be expressed in under control of their own promoters. Two putative promoters are located upstream of , and both were found to initiate transcription in . Although the two permeases EIICBA1 and EIICBA2 show 69% identity at the protein level, and despite the common primary substrate glucose, they have different specificities towards glucosides as substrate. EIICBA1 phosphorylates glucose in a PEP-dependent reaction with a of 12 μM; the reaction can be inhibited by 2-deoxyglucose and methyl β-D-glucoside. EIICBA2 phosphorylates glucose with a of 19 μM and this reaction is inhibited by methyl α-D-glucoside, methyl β-D-glucoside, -nitrophenyl α-D-glucoside, -nitrophenyl β-D-glucoside and salicin, but unlike other glucose permeases, including EIICBA1, not by 2-deoxyglucose. Natural mono- or disaccharides, such as mannose or -acetylglucosamine, that are transported by other glucose transporters are not phosphorylated by either EIICBA1 nor EIICBA2, indicating a high specificity for glucose. Together, these findings support the suggestion of evolutionary development of different members of a protein family, by gene duplication and subsequent differentiation. C-terminal fusion of a histidine hexapeptide to both gene products did not affect the activity of the enzymes and allowed their purification by Ni-NTA affinity chromatography after expression in a (EIICB) deletion mutant of . Upstream of , the 3’ end of a further ORF encoding 138 amino acid residues of a putative antiterminator of the BglG family was found, as well as a putative target DNA sequence (RAT), which indicates a further regulation by glucose specific antitermination.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-145-10-2881
1999-10-01
2020-01-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/145/10/1452881a.html?itemId=/content/journal/micro/10.1099/00221287-145-10-2881&mimeType=html&fmt=ahah

References

  1. Begley, G. S., Warner, K. A., Arents, J. C., Postma, P. W. & Jacobson, G. R. ( 1996; ). Isolation and characterization of a mutation that alters the substrate specificity of the Escherichia coli glucose permease. J Bacteriol 178, 940-942.
    [Google Scholar]
  2. Bramley, H. F. & Kornberg, L. ( 1987; ). Nucleotide sequence of bglC, the gene specifying enzyme IIBgl of the PEP:sugar phosphotransferase system in Escherichia coli K12, and overexpression of the gene product. J Gen Microbiol 133, 563-573.
    [Google Scholar]
  3. Buhr, A. & Erni, B. ( 1993; ). Membrane topology of the glucose transporter of Escherichia coli. J Biol Chem 268, 11599-11603.
    [Google Scholar]
  4. Buhr, A., Daniels, G. A. & Erni, B. ( 1992; ). The glucose transporter of Escherichia coli. Mutants with impaired translocation activity that retain phosphorylation activity. J Biol Chem 267, 3847-3851.
    [Google Scholar]
  5. Buhr, A., Flükiger, K. & Erni, B. ( 1994; ). The glucose transporter of Escherichia coli – overexpression, purification, and characterization of functional domains. J Biol Chem 269, 23427-23443.
    [Google Scholar]
  6. Christiansen, I. & Hengstenberg, W. ( 1996; ). Cloning and sequencing of two genes from Staphylococcus carnosus coding for glucose-specific PTS and their expression in Escherichia coli K-12. Mol Gen Genet 250, 375-379.
    [Google Scholar]
  7. Crutz, A.-M., Steinmetz, M., Aymerich, S., Richter, R. & Le Coq, D. ( 1990; ). Induction of levansucrase in Bacillus subtilis: an antitermination mechanism negatively controlled by the phosphotransferase system. J Bacteriol 172, 1043-1050.
    [Google Scholar]
  8. Débarbouillé, M., Arnaud, M., Fouet, A., Klier, A. & Rapoport, G. ( 1990; ). The sacT gene regulating the sacPA operon in Bacillus subtilis shares strong homology with transcriptional antiterminators. J Bacteriol 172, 3966-3973.
    [Google Scholar]
  9. El Hassouni, M., Henrissat, B., Chippaux, M. & Barras, F. ( 1992; ). Nucleotide sequence of the arb genes, which control β-glucoside utilization in Erwinia chrysanthemi: comparison with the Escherichia coli bgl operon and evidence for a new β-glycohydrolase family including enzymes from eubacteria, archaebacteria and humans. J Bacteriol 174, 765-777.
    [Google Scholar]
  10. Erni, B. ( 1992; ). Group translocation of glucose and other carbohydrates by the bacterial phosphotransferase system. Int Rev Cytol 137A, 127-148.
    [Google Scholar]
  11. Erni, B., Zanolari, B., Graff, P. & Kocher, H. P. ( 1989; ). Mannose permease of Escherichia coli. Domain structure and function of the phosphorylating subunit. J Biol Chem 264, 18733-18741.
    [Google Scholar]
  12. Galinier, A., Kravanja, M., Engelmann, R., Hengstenberg, W., Kilhoffer, M.-C., Deutscher, J. & Haiech, J. ( 1998; ). New protein kinase and protein phosphatase families mediate signal transduction in bacterial catabolite repression. Proc Natl Acad Sci USA 95, 1823-1828.[CrossRef]
    [Google Scholar]
  13. Heller, K. B. ( 1978; ). Apparent molecular weights of a heat-modifiable protein from the outer membrane of Escherichia coli in gels with different acrylamide concentrations. J Bacteriol 134, 1181-1183.
    [Google Scholar]
  14. Hengstenberg, W., Kohlbrecher, D., Witt, E. & 7 other authors ( 1993; ). Structure and function of proteins of the phosphotransferase system and of 6-phospho-β-glycosidases in Gram-positive bacteria. FEMS Microbiol Rev 12, 149–164.[CrossRef]
    [Google Scholar]
  15. Hewick, R. H., Hunkapiller, M. W., Hood, L. E. & Dreyer, W. J. ( 1981; ). A gas–liquid solid phase peptide and protein sequenator. J Biol Chem 256, 7990-7997.
    [Google Scholar]
  16. Hueck, C. J., Hillen, W. & Saier, M. H.Jr ( 1994; ). Analysis of a cis-active sequence mediating catabolite repression in Gram-positive bacteria. Res Microbiol 145, 503-518.[CrossRef]
    [Google Scholar]
  17. Iobst, S. T. & Drickamer, K. ( 1994; ). Binding of sugar ligands to Ca2+ dependent animal lectins. II. Generation of high-affinity galactose binding by site-directed mutagenesis. J Biol Chem 269, 15512-15519.
    [Google Scholar]
  18. Koebnik, R. & Kr mer, L. ( 1995; ). Membrane assembly of circularly permuted variants of the E. coli outer membrane protein OmpA. J Mol Biol 250, 617–626.[CrossRef]
    [Google Scholar]
  19. Köster, W. & Braun, V. ( 1986; ). Iron hydroxamate transport of Escherichia coli: nucleotide sequence of the fhuB gene and identification of the protein. Mol Gen Genet 204, 435-442.[CrossRef]
    [Google Scholar]
  20. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[CrossRef]
    [Google Scholar]
  21. Lee, C. A. & Saier, M. H.Jr ( 1983; ). Mannitol-specific enzyme II of the bacterial phosphotransferase system. III. The nucleotide sequence of the permease gene. J Biol Chem 258, 10761-10767.
    [Google Scholar]
  22. Lengeler, J. W., Jahreis, K. & Wehmeier, U. F. ( 1994; ). Enzymes II of the phosphoenolpyruvate-dependent phosphotransferase systems: their structure and function in carbohydrate transport. Biochim Biophys Acta 1188, 1-28.[CrossRef]
    [Google Scholar]
  23. Lengsfeld, A. M., Alexander, E. T., Hengstenberg, W. & Korte, T. ( 1973; ). Morphological changes in staphylococcal cytoplasmic membrane due to action of non-ionic detergent Triton X-100. Exp Cell Res 76, 159-169.[CrossRef]
    [Google Scholar]
  24. Locher, K. P. & Rosenbusch, J. P. ( 1997; ). Oligomeric states and siderophore binding of the ligand-gated FhuA protein that forms channels across Escherichia coli outer membranes. Eur J Biochem 247, 770-775.[CrossRef]
    [Google Scholar]
  25. Martin-Verstraete, I., Débarbouillé, M., Klier, A. & Rapoport, G. ( 1990; ). Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon. J Mol Biol 214, 657-671.[CrossRef]
    [Google Scholar]
  26. Peters, D., Frank, R. & Hengstenberg, W. ( 1995; ). Lactose-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. Purification of the histidine-tagged transmembrane component IICBLac and its hydrophilic IIB domain by metal-affinity chromatography, and its functional characterization. Eur J Biochem 228, 798-804.[CrossRef]
    [Google Scholar]
  27. Peterson, G. L. ( 1977; ). A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83, 346-356.[CrossRef]
    [Google Scholar]
  28. Postma, P. W., Lengeler, J. W. & Jacobson, G. R. ( 1993; ). Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. Microbiol Rev 57, 543-594.
    [Google Scholar]
  29. Reizer, J., Hoischen, C., Titgemeyer, F., Rivolta, C., Rabus, R., Stülke, J., Karamata, D., Saier, M. H.Jr & Hillen, W. ( 1998; ). A novel protein kinase that controls catabolite repression in bacteria. Mol Microbiol 27, 1157-1169.[CrossRef]
    [Google Scholar]
  30. Ried, G., Koebnik, R., Hindennach, I., Mutschler, B. & Henning, U. ( 1994; ). Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12. Mol Gen Genet 243, 127-135.
    [Google Scholar]
  31. Ruijter, G. J. G., van Meurs, G., Verwey, M. A., Postma, P. W. & van Dam, K. ( 1992; ). Analysis of mutations that uncouple transport from phosphorylation in Enzyme IIGlc of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system. J Bacteriol 174, 2843-2850.
    [Google Scholar]
  32. Saier, M. H.Jr & Reizer, J. ( 1994; ). The bacterial phosphotransferase system: new frontiers 30 years later. Mol Microbiol 13, 755-764.[CrossRef]
    [Google Scholar]
  33. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  34. Schleifer, K. H. & Fischer, U. ( 1982; ). Description of a new species of the genus Staphylococcus: Staphylococcus carnosus. Int J Syst Bacteriol 32, 153-156.[CrossRef]
    [Google Scholar]
  35. Schnetz, K. & Rak, B. ( 1990; ). β-Glucoside permease represses the bgl operon of Escherichia coli by phosphorylation of the antiterminator protein and also interacts with glucose-specific enzyme IIIGlc, the key element in catabolite control. Proc Natl Acad Sci USA 87, 5074-5078.[CrossRef]
    [Google Scholar]
  36. Schnetz, K., Stülke, J., Gertz, S., Krüger, S., Krieg, M., Hecker, M. & Rak, B. ( 1996; ). LicT, a Bacillus subtilis transcriptional antiterminator of the BglG family. J Bacteriol 178, 1971-1979.
    [Google Scholar]
  37. Strokopytov, B., Penninga, D., Rozeboom, H. J., Kalk, K. H., Dijkhuizen, L. & Dijkstra, B. W. ( 1994; ). X-ray structure of cyclodextrin glycosyltransferase complexed with acarbose. Implications for the catalytic mechanism of glycosidases. Biochemistry 34, 2234-2240.
    [Google Scholar]
  38. Stülke, J., Martin-Verstraete, I., Zagorec, M., Rose, M., Klier, A. & Rapoport, G. ( 1997; ). Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT. Mol Microbiol 25, 65-78.[CrossRef]
    [Google Scholar]
  39. Sugiyama, J. E., Mahmoodian, S. M. & Jacobson, G. R. ( 1991; ). Membrane topology analysis of the Escherichia coli mannitol permease by using a nested-deletion method to create mtlA–phoA fusions. Proc Natl Acad Sci USA 88, 9603-9607.[CrossRef]
    [Google Scholar]
  40. Vieira, J. & Messing, J. ( 1985; ). The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with the synthetic universal primers. Gene 19, 259-268.
    [Google Scholar]
  41. Wöhrl, B. M. & Lengeler, J. W. ( 1990; ). Cloning and physical mapping of the sor genes for l-sorbose transport and metabolism from Klebsiella pneumoniae. Mol Microbiol 4, 1557-1565.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-145-10-2881
Loading
/content/journal/micro/10.1099/00221287-145-10-2881
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