THE DISCOVERY AND STUDY OF A SELECTIVE ADVANTAGE CONFERRED BY TN5 IN CARBON SOURCE LIMITED CHEMOSTAT CULTURES

SUSAN WURSTER BIEL, Purdue University

Abstract

This work encompasses the discovery and study of an unusual in vivo effect of the transposable element Tn5 on Escherichia coli K-12. When isogenic strains, one of which contains Tn5, were grown together in carbon source limited chemostats, in the absence of kanamycin, the strain containing Tn5 increased at a rate of 3-6 percent per hour. Preadaptation of strains in monoculture to chemostat conditions for 72 hours before being placed in competition erased the selective advantage. The advantage was also independent of the carbon source used for growth and could be demonstrated to occur in the presence of lactose, glycerol, glucose, and proline. The selection was found to be independent of the insertion site, as lac and ilvD insertions have selection coefficients of the same magnitude. The kinetics of selection and the effect of preadaptation do not fit the expected results if the advantage is caused by the selection of rare, advantageous mutations. Three different, unrelated strains of E. coli K-12 have been used, and one of the three did not show the advantage. This seems to indicate that the genetic background is an important factor. Experiments with strains that carry a wild-type Tn5 on either an F'lac or in the lac operon of the chromosome show that transposition of the element is not required for the Tn5-containing strain to be favored. Experiments with various mutants of Tn5 were conducted to determine what gene(s) are involved in mediating the advantage. Tn5-410 is kanamycin sensitive and transposition defective and does not confer an advantage upon the strain carrying it. An identical result is obtained with a mutant that is kanamycin resistant and transposition defective. When the kanamycin resistant and kanamycin sensitive, transposition defective strains are placed in competition, neither has an advantage. This result shows that the antibiotic resistance gene is not responsible for the advantage. The evidence presented shows that the advantage is not due to actual transposition, but that the right-hand IS50 element which encodes the transposition-mediating proteins(s) is required. It is postulated that these protein(s) interact with proteins that are normal cellular constituents to enhance the growth rate of the Tn5-containing strain.

Degree

Ph.D.

Subject Area

Genetics

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