Mechanism of reversion of the gal3 mutation of Escherichia coli

Abstract

The gal3 mutation is an insertion of a DNA sequence in the operator-promoter region of the galactose operon of E. coli. It reverts spontaneously to produce three kinds of gal+ revertants, which are: (i) stable and inducible, (ii) stable and constitutive, and (iii) unstable and constitutive. The constitutive revertants also show drastically reduced frequencies of transduction with lambda. The mechanism by which these reversions occur has remained unknown. It is proposed that the stable and inducible revertants arise by accurate excision of the insertion sequence. The unstable and constitutive revertants arise by tandem duplications of the gal operon in such a way that the structural genes of the extra copy of gal operon become connected to a different promoter. The resulting tandem configuration (gal3 ETK...P'E'T'K') permits constitutive expression and gal3 segregation (by internal recombination) simultaneously. The proposal was tested by comparison of the buoyant densities in CsCl of derivatives of a lambdagal phage carrying gal+, gal3, and the inducible and constitutive revertants. The densities of the inducible revertants were identical to the wild type, and the slight increase in density found to be associated with the gal3 insertion was missing. It was concluded that inducible revertants arise by excision of the inserted sequence. In contrast, lysates of a constitutive revertant exhibited several anomalous properties. The lysates contained a small quantity of phage whose density was identical to lambdagal3, produced few gal+ transductants (10(-3)-10(-4) of a normal HFT lysate), and the transductants were stable and constitutive. In turn, these abnormal transductants produced lysates which showed no lambdagal particles on centrifugation, and no transducing activity whatsoever. These anomalous properties of the constitutive revertant were attributed to the failure of lambda to package the DNA duplication efficiently. Transduction experiments with P1 (which can package more DNA than lambda) show that the unstable, constitutive reversions were located adjacent to prophage lambda. Segregation of the gal and lambda markers among the gal+ transductants was in accordance with the pattern expected for a duplication. Introduction of a recA marker resulted in stabilization of the reversion without affecting its constitutive expression. It was concluded that the unstable, constitutive reversion was a tandem duplication. It is further proposed that the stable, constitutive class of revertants might represent inverted (gal3 ETK...K'T'E'P') or partial tandem (gal3 ET...E'T'K') duplications of the gal operon.