Abstract
Mutations that uncouple glucose transport from phosphorylation were isolated in plasmid-encoded Escherichia coli enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The uncoupled enzymes IIGlc were able to transport glucose in the absence of the general phosphoryl-carrying proteins of the PTS, enzyme I and HPr, although with relatively low affinity. Km values of the uncoupled enzymes IIGlc for glucose ranged from 0.5 to 2.5 mM, 2 orders of magnitude higher than the value of normal IIGlc. Most of the mutant proteins were still able to phosphorylate glucose and methyl alpha-glucoside (a non-metabolizable glucose analog specific for IIGlc), indicating that transport and phosphorylation are separable functions of the enzyme. Some of the uncoupled enzymes IIGlc transported glucose with a higher rate and lower apparent Km in a pts+ strain than in a delta ptsHI strain lacking the general proteins enzyme I and HPr. Since the properties of these uncoupled enzymes IIGlc in the presence of PTS-mediated phosphoryl transfer resembled those of wild-type IIGlc, these mutants appeared to be conditionally uncoupled. Sequencing of the mutated ptsG genes revealed that all amino acid substitutions occurred in a hydrophilic segment within the hydrophobic N-terminal part of IIGlc. These results suggest that this hydrophilic loop is involved in binding and translocation of the sugar substrate.
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Selected References
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- ARBER W. Transduction of chromosomal genes and episomes in Escherichia coli. Virology. 1960 May;11:273–288. doi: 10.1016/0042-6822(60)90066-0. [DOI] [PubMed] [Google Scholar]
- Adler J., Epstein W. Phosphotransferase-system enzymes as chemoreceptors for certain sugars in Escherichia coli chemotaxis. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2895–2899. doi: 10.1073/pnas.71.7.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bochner B. R., Huang H. C., Schieven G. L., Ames B. N. Positive selection for loss of tetracycline resistance. J Bacteriol. 1980 Aug;143(2):926–933. doi: 10.1128/jb.143.2.926-933.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cordaro J. C., Roseman S. Deletion mapping of the genes coding for HPr and enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium. J Bacteriol. 1972 Oct;112(1):17–29. doi: 10.1128/jb.112.1.17-29.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Curtis S. J., Epstein W. Phosphorylation of D-glucose in Escherichia coli mutants defective in glucosephosphotransferase, mannosephosphotransferase, and glucokinase. J Bacteriol. 1975 Jun;122(3):1189–1199. doi: 10.1128/jb.122.3.1189-1199.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Erni B. Glucose transport in Escherichia coli. FEMS Microbiol Rev. 1989 Jun;5(1-2):13–23. doi: 10.1016/0168-6445(89)90004-1. [DOI] [PubMed] [Google Scholar]
- Erni B. Glucose-specific permease of the bacterial phosphotransferase system: phosphorylation and oligomeric structure of the glucose-specific IIGlc-IIIGlc complex of Salmonella typhimurium. Biochemistry. 1986 Jan 28;25(2):305–312. doi: 10.1021/bi00350a004. [DOI] [PubMed] [Google Scholar]
- Erni B., Zanolari B. Glucose-permease of the bacterial phosphotransferase system. Gene cloning, overproduction, and amino acid sequence of enzyme IIGlc. J Biol Chem. 1986 Dec 15;261(35):16398–16403. [PubMed] [Google Scholar]
- Erni B., Zanolari B., Graff P., Kocher H. P. Mannose permease of Escherichia coli. Domain structure and function of the phosphorylating subunit. J Biol Chem. 1989 Nov 5;264(31):18733–18741. [PubMed] [Google Scholar]
- 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]
- Hartman P. E. Some improved methods in P22 transduction. Genetics. 1974 Apr;76(4):625–631. doi: 10.1093/genetics/76.4.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
- King S. C., Wilson T. H. Characterization of Escherichia coli lactose carrier mutants that transport protons without a cosubstrate. Probes for the energy barrier to uncoupled transport. J Biol Chem. 1990 Jun 15;265(17):9645–9651. [PubMed] [Google Scholar]
- King S. C., Wilson T. H. Identification of valine 177 as a mutation altering specificity for transport of sugars by the Escherichia coli lactose carrier. Enhanced specificity for sucrose and maltose. J Biol Chem. 1990 Jun 15;265(17):9638–9644. [PubMed] [Google Scholar]
- Kornberg H. L., Riordan C. Uptake of galactose into Escherichia coli by facilitated diffusion. J Gen Microbiol. 1976 May;94(1):75–89. doi: 10.1099/00221287-94-1-75. [DOI] [PubMed] [Google Scholar]
- Kundig W., Roseman S. Sugar transport. II. Characterization of constitutive membrane-bound enzymes II of the Escherichia coli phosphotransferase system. J Biol Chem. 1971 Mar 10;246(5):1407–1418. [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lengeler J. W., Titgemeyer F., Vogler A. P., Wöhrl B. M. Structures and homologies of carbohydrate: phosphotransferase system (PTS) proteins. Philos Trans R Soc Lond B Biol Sci. 1990 Jan 30;326(1236):489–504. doi: 10.1098/rstb.1990.0027. [DOI] [PubMed] [Google Scholar]
- Lolkema J. S., Dijkstra D. S., ten Hoeve-Duurkens R. H., Robillard G. T. Interaction between the cytoplasmic and membrane-bound domains of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system. Biochemistry. 1991 Jul 9;30(27):6721–6726. doi: 10.1021/bi00241a013. [DOI] [PubMed] [Google Scholar]
- Lolkema J. S., Dijkstra D. S., ten Hoeve-Duurkens R. H., Robillard G. T. The membrane-bound domain of the phosphotransferase enzyme IImtl of Escherichia coli constitutes a mannitol translocating unit. Biochemistry. 1990 Nov 27;29(47):10659–10663. doi: 10.1021/bi00499a012. [DOI] [PubMed] [Google Scholar]
- Lolkema J. S., ten Hoeve-Duurkens R. H., Dijkstra D. S., Robillard G. T. Mechanistic coupling of transport and phosphorylation activity by enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system. Biochemistry. 1991 Jul 9;30(27):6716–6721. doi: 10.1021/bi00241a012. [DOI] [PubMed] [Google Scholar]
- Manayan R., Tenn G., Yee H. B., Desai J. D., Yamada M., Saier M. H., Jr Genetic analyses of the mannitol permease of Escherichia coli: isolation and characterization of a transport-deficient mutant which retains phosphorylation activity. J Bacteriol. 1988 Mar;170(3):1290–1296. doi: 10.1128/jb.170.3.1290-1296.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marshall-Carlson L., Celenza J. L., Laurent B. C., Carlson M. Mutational analysis of the SNF3 glucose transporter of Saccharomyces cerevisiae. Mol Cell Biol. 1990 Mar;10(3):1105–1115. doi: 10.1128/mcb.10.3.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meadow N. D., Fox D. K., Roseman S. The bacterial phosphoenolpyruvate: glycose phosphotransferase system. Annu Rev Biochem. 1990;59:497–542. doi: 10.1146/annurev.bi.59.070190.002433. [DOI] [PubMed] [Google Scholar]
- Meins M., Zanolari B., Rosenbusch J. P., Erni B. Glucose permease of Escherichia coli. Purification of the IIGlc subunit and functional characterization of its oligomeric forms. J Biol Chem. 1988 Sep 15;263(26):12986–12993. [PubMed] [Google Scholar]
- Nuoffer C., Zanolari B., Erni B. Glucose permease of Escherichia coli. The effect of cysteine to serine mutations on the function, stability, and regulation of transport and phosphorylation. J Biol Chem. 1988 May 15;263(14):6647–6655. [PubMed] [Google Scholar]
- Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
- Postma P. W. Defective enzyme II-BGlc of the phosphoenolpyruvate:sugar phosphotransferase system leading to uncoupling of transport and phosphorylation in Salmonella typhimurium. J Bacteriol. 1981 Aug;147(2):382–389. doi: 10.1128/jb.147.2.382-389.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Postma P. W. Galactose transport in Salmonella typhimurium. J Bacteriol. 1977 Feb;129(2):630–639. doi: 10.1128/jb.129.2.630-639.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Postma P. W. Involvement of the phosphotransferase system in galactose transport in Salmonella typhimurium. FEBS Lett. 1976 Jan 1;61(1):49–53. doi: 10.1016/0014-5793(76)80169-x. [DOI] [PubMed] [Google Scholar]
- Postma P. W., Keizer H. G., Koolwijk P. Transport of trehalose in Salmonella typhimurium. J Bacteriol. 1986 Dec;168(3):1107–1111. doi: 10.1128/jb.168.3.1107-1111.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Postma P. W., Schuitema A., Kwa C. Regulation of methyl beta-galactoside permease activity in pts and crr mutants of Salmonella typhimurium. Mol Gen Genet. 1981;181(4):448–453. doi: 10.1007/BF00428734. [DOI] [PubMed] [Google Scholar]
- Postma P. W., Stock J. B. Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation. J Bacteriol. 1980 Feb;141(2):476–484. doi: 10.1128/jb.141.2.476-484.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Ruijter G. J., Postma P. W., van Dam K. Adaptation of Salmonella typhimurium mutants containing uncoupled enzyme IIGlc to glucose-limited conditions. J Bacteriol. 1990 Sep;172(9):4783–4789. doi: 10.1128/jb.172.9.4783-4789.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruijter G. J., Postma P. W., van Dam K. Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc. Arch Microbiol. 1991;155(3):234–237. doi: 10.1007/BF00252206. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schnetz K., Sutrina S. L., Saier M. H., Jr, Rak B. Identification of catalytic residues in the beta-glucoside permease of Escherichia coli by site-specific mutagenesis and demonstration of interdomain cross-reactivity between the beta-glucoside and glucose systems. J Biol Chem. 1990 Aug 15;265(23):13464–13471. [PubMed] [Google Scholar]
- 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]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- White D. W., Jacobson G. R. Molecular cloning of the C-terminal domain of Escherichia coli D-mannitol permease: expression, phosphorylation, and complementation with C-terminal permease deletion proteins. J Bacteriol. 1990 Mar;172(3):1509–1515. doi: 10.1128/jb.172.3.1509-1515.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- van Weeghel R. P., Meyer G. H., Keck W., Robillard G. T. Phosphoenolpyruvate-dependent mannitol phosphotransferase system of Escherichia coli: overexpression, purification, and characterization of the enzymatically active C-terminal domain of enzyme IImtl equivalent to enzyme IIImtl. Biochemistry. 1991 Feb 19;30(7):1774–1779. doi: 10.1021/bi00221a007. [DOI] [PubMed] [Google Scholar]
- van Weeghel R. P., Meyer G., Pas H. H., Keck W., Robillard G. T. Cytoplasmic phosphorylating domain of the mannitol-specific transport protein of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli: overexpression, purification, and functional complementation with the mannitol binding domain. Biochemistry. 1991 Oct 1;30(39):9478–9485. doi: 10.1021/bi00103a013. [DOI] [PubMed] [Google Scholar]