• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Sep 1993; 175(17): 5375–5383.
PMCID: PMC206592

Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12.

Abstract

We isolated a transposon Tn10 insertion mutant of Escherichia coli K-12 which could not grow on MacConkey plates containing D-ribose. Characterization of the mutant revealed that the level of the transketolase activity was reduced to one-third of that of the wild type. The mutation was mapped at 63.5 min on the E. coli genetic map, in which the transketolase gene (tkt) had been mapped. A multicopy suppressor gene which complemented the tkt mutation was cloned on a 7.8-kb PstI fragment. The cloned gene was located at 53 min on the chromosome. Subcloning and sequencing of a 2.7-kb fragment containing the suppressor gene identified an open reading frame encoding a polypeptide of 667 amino acids with a calculated molecular weight of 72,973. Overexpression of the protein and determination of its N-terminal amino acid sequence defined unambiguously the translational start site of the gene. The deduced amino acid sequence showed similarity to sequences of transketolases from Saccharomyces cerevisiae and Rhodobacter sphaeroides. In addition, the level of the transketolase activity increased in strains carrying the gene in multicopy. Therefore, the gene encoding this transketolase was designated tktB and the gene formerly called tkt was renamed tktA. Analysis of the phenotypes of the strains containing tktA, tktB, or tktA tktB mutations indicated that tktA and tktB were responsible for major and minor activities, respectively, of transketolase in E. coli.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.9M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Chen JH, Gibson JL, McCue LA, Tabita FR. Identification, expression, and deduced primary structure of transketolase and other enzymes encoded within the form II CO2 fixation operon of Rhodobacter sphaeroides. J Biol Chem. 1991 Oct 25;266(30):20447–20452. [PubMed]
  • Edman P, Begg G. A protein sequenator. Eur J Biochem. 1967 Mar;1(1):80–91. [PubMed]
  • Eidels L, Osborn MJ. Lipopolysaccharide and aldoheptose biosynthesis in transketolase mutants of Salmonella typhimurium. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1673–1677. [PMC free article] [PubMed]
  • Fletcher TS, Kwee IL, Nakada T, Largman C, Martin BM. DNA sequence of the yeast transketolase gene. Biochemistry. 1992 Feb 18;31(6):1892–1896. [PubMed]
  • Goad WB, Kanehisa MI. Pattern recognition in nucleic acid sequences. I. A general method for finding local homologies and symmetries. Nucleic Acids Res. 1982 Jan 11;10(1):247–263. [PMC free article] [PubMed]
  • Harayama S, Palva ET, Hazelbauer GL. Transposon-insertion mutants of Escherichia coli K12 defective in a component common to galactose and ribose chemotaxis. Mol Gen Genet. 1979 Mar 20;171(2):193–203. [PubMed]
  • Hawkins CF, Borges A, Perham RN. A common structural motif in thiamin pyrophosphate-binding enzymes. FEBS Lett. 1989 Sep 11;255(1):77–82. [PubMed]
  • Hewick RM, Hunkapiller MW, Hood LE, Dreyer WJ. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed]
  • Icho T, Iino T. Isolation and characterization of motile Escherichia coli mutants resistant to bacteriophage chi. J Bacteriol. 1978 Jun;134(3):854–860. [PMC free article] [PubMed]
  • Iida A, Groarke JM, Park S, Thom J, Zabicky JH, Hazelbauer GL, Randall LL. A signal sequence mutant defective in export of ribose-binding protein and a corresponding pseudorevertant isolated without imposed selection. EMBO J. 1985 Jul;4(7):1875–1880. [PMC free article] [PubMed]
  • Iida A, Harayama S, Iino T, Hazelbauer GL. Molecular cloning and characterization of genes required for ribose transport and utilization in Escherichia coli K-12. J Bacteriol. 1984 May;158(2):674–682. [PMC free article] [PubMed]
  • Janowicz ZA, Eckart MR, Drewke C, Roggenkamp RO, Hollenberg CP, Maat J, Ledeboer AM, Visser C, Verrips CT. Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula polymorpha. Nucleic Acids Res. 1985 May 10;13(9):3043–3062. [PMC free article] [PubMed]
  • Josephson BL, Fraenkel DG. Transketolase mutants of Escherichia coli. J Bacteriol. 1969 Dec;100(3):1289–1295. [PMC free article] [PubMed]
  • Josephson BL, Fraenkel DG. Sugar metabolism in transketolase mutants of Escherichia coli. J Bacteriol. 1974 Jun;118(3):1082–1089. [PMC free article] [PubMed]
  • Kleckner N, Barker DF, Ross DG, Botstein D. Properties of the translocatable tetracycline-resistance element Tn10 in Escherichia coli and bacteriophage lambda. Genetics. 1978 Nov;90(3):427–461. [PMC free article] [PubMed]
  • Kleckner N, Bender J, Gottesman S. Uses of transposons with emphasis on Tn10. Methods Enzymol. 1991;204:139–180. [PubMed]
  • Kohara Y, Akiyama K, Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. [PubMed]
  • Kushner SR, Nagaishi H, Templin A, Clark AJ. Genetic recombination in Escherichia coli: the role of exonuclease I. Proc Natl Acad Sci U S A. 1971 Apr;68(4):824–827. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lambden PR, Drabble WT. The gua operon of Escherichia coli K-12: evidence for polarity from guaB to guaA. J Bacteriol. 1973 Sep;115(3):992–1002. [PMC free article] [PubMed]
  • Lindqvist Y, Schneider G, Ermler U, Sundström M. Three-dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution. EMBO J. 1992 Jul;11(7):2373–2379. [PMC free article] [PubMed]
  • Mead DA, Szczesna-Skorupa E, Kemper B. Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986 Oct-Nov;1(1):67–74. [PubMed]
  • Radnis BA, Rhee DK, Morrison DA. Genetic transformation in Streptococcus pneumoniae: nucleotide sequence and predicted amino acid sequence of recP. J Bacteriol. 1990 Jul;172(7):3669–3674. [PMC free article] [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Satishchandran C, Markham GD, Moore RC, Boyle SM. Locations of the speA, speB, speC, and metK genes on the physical map of Escherichia coli. J Bacteriol. 1990 Sep;172(9):4748–4748. [PMC free article] [PubMed]
  • Schaaff I, Hohmann S, Zimmermann FK. Molecular analysis of the structural gene for yeast transaldolase. Eur J Biochem. 1990 Mar 30;188(3):597–603. [PubMed]
  • Schellenberg GD, Wilson NM, Copeland BR, Furlong CE. Transketolase from human red blood cells. Methods Enzymol. 1982;90(Pt E):223–228. [PubMed]
  • Shine J, Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. [PMC free article] [PubMed]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
  • Sprenger GA. Location of the transketolase (tkt) gene on the Escherichia coli physical map. J Bacteriol. 1992 Mar;174(5):1707–1708. [PMC free article] [PubMed]
  • Stern MJ, Ames GF, Smith NH, Robinson EC, Higgins CF. Repetitive extragenic palindromic sequences: a major component of the bacterial genome. Cell. 1984 Jul;37(3):1015–1026. [PubMed]
  • Szybalski EH, Szybalski W. A comprehensive molecular map of bacteriophage lambda. Gene. 1979 Nov;7(3-4):217–270. [PubMed]
  • Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. [PubMed]
  • Winans SC, Elledge SJ, Krueger JH, Walker GC. Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli. J Bacteriol. 1985 Mar;161(3):1219–1221. [PMC free article] [PubMed]
  • Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed]
  • Yura T, Mori H, Nagai H, Nagata T, Ishihama A, Fujita N, Isono K, Mizobuchi K, Nakata A. Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region. Nucleic Acids Res. 1992 Jul 11;20(13):3305–3308. [PMC free article] [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links