We have previously described a system of experimental evolution in which many of the mutants of Klebsiella aerogenes selected for faster growth on xylitol (evolvants) synthesized elevated levels of ribitol dehydrogenase and have presented genetic evidence implicating gene duplication in the enzyme superproduction in some of the evolvants. Here we describe a physical approach to the screening for gene duplications and subsequent structure determination. Nick-translated, cloned ribitol operon (rbt) DNA was used as a hybridization probe to identify fragments containing rbt operon sequences in restriction digests of total bacterial DNA. Whilst several of the evolvants probably harbour duplications spanning the entire rbt operon, one of the spontaneously arising evolvants (strain A3) was shown to harbour a small (5·8 kilobase pairs) direct DNA repeat which encodes the dehydrogenase (but not the kinase) of the closely linked d-arabitol operon as well as the dehydrogenase (but not the kinase) of the rbt operon. The hybridization data suggest that there are 4 to 5 copies of the repeat arranged contiguously on the chromosome. The genetic instability of strain A3, the rbt fragment hybridization pattern of an A3 segregant and the activities of the pentitol catabolic enzymes in A3 are all consistent with the proposed gene duplication structure.
AndersonR.P., RothJ.1978; Tandem genetic duplication in Salmonella typhimurium: amplification of the histidine operon. Journal of Molecular Biology 126:53–71
BeeftinckF., CuninR., GlansdorffN.1974; Arginine gene duplication in recombination proficient and deficient strains of Escherichia coli. . Molecular and General Genetics 161:185–196
BergquistP.L., JamiesonA.J.1977; Genetic inversion in the formation of an Hfr strain from a temperature-sensitive F ´gal strain. Journal of Bacteriology 129:282–290
CharlierD., CrabeelM., CuninR., GlansdorffN.1979; Tandem and inverted repeats of the arginine genes in Escherichia coli. Structural and evolutionary considerations. Molecular and General Genetics 174:75–88
CharnetzkyW.T., MortlockR.P.1974; Close genetic linkage of the determinants of the ribitol and d-arabitol catabolic pathways in Klebsiella aerogenes. . Journal of Bacteriology 119:176–182
EmmonsS.W., ThomasJ.O.1975; Tandem genetic duplications in phage λ: the locations of spontaneously arising duplications. Journal of Molecular Biology 91:147–152
HaggertyD.M., SchleifR.F.1976; Locations on phage lambda of the cleavage sites of the site-specific endonuclease from Bacillus amyloliquefaciens H. Journal of Virology 18:659–663
HartleyB.S., AltosaarI., DothieJ.M., NeubergerM.S.1976; Experimental evolution of a xylitol dehydrogenase. In Structure-Function Relationships in Proteins pp. 191–200 Edited by MarkhamR., HorneR.W. Amsterdam: North Holland;
HillC.W., GrafstromR.H., HarnishB.W., HillmanB.S.1977; Tandem duplications resulting from recombination between the ribosomal RNA genes in Escherichia coli. . Journal of Molecular Biology 116:407–428
LaskeyR.A., MillsA.D.1977; Enhanced autoradiographic detection of 32P and 125I using intensifying screens and hypersensitized film. FEBS Letters 82:314–316
MorrisH.R., WilliamsD.H., MidwinterG.G., HartleyB.S.1974; A mass spectrometric sequence study of the enzyme ribitol dehydrogenase from Klebsiella aerogenes. . Biochemical Journal 141:701–713
NeubergerM.S., HartleyB.S.1979; Investigations into the Klebsiella aerogenes pentitol operons using specialised transducing phages kprbt and λprbt dal. . Journal of Molecular Biology 132:435–470
NeubergerM.S., HartleyB.S., WalkerJ.E.1980; Purification and properties of D-ribulokinase and d-xylulokinase from Klebsiella aerogenes. . Biochemical Journal in the Press
SutcliffeJ.G.1978; pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long. Nucleic Acids Research 5:2721–2728
TrowsdaleJ., AnagnostopoulosC.1975; Evidence for the translocation of a chromosome segment in Bacillus subtilis strains carrying the trpE26 mutation. Journal of Bacteriology 122:886–898
WuT.T., LinE.C.C., TanakaS.1968; Mutants of Aerobacter aerogenes capable of utilizing xylitol as a novel carbon source. Journal of Bacteriology 96:447–456