Reversion of the gal3 mutation of Escherichia coli: Partial deletion of the insertion sequence

Summary

The gal3 mutation of E. coli is an insertion of a DNA sequence, 1,100 base pairs in length, into the operator-promoter region of the galactose operon. This mutation reverts spontaneously to gal⁺ by excision of the insertion to produce stable, inducible revertants, or by tandem duplications of the gal operon to produce unstable, constitutive revertants. The nature of a third class of revertants, which are stable and constitutive, is the subject of the present study.

The stable, constitutive class of revertants included approximately 30% of all gal⁺ revertants obtained from a gal3(λ) strain. Although the constitutive reversions could be transduced by λ, the efficiency was found to be extremely poor and the rare transductants which did appear seemed to originate from abnormal transducing particles. It was concluded that these reversions were not normally packaged by λ.

In order to facilitate the packaging of these reversions, the chlD-pgl region was deleted from the parent gal3(λ) strain. Unexpectedly, the gal3 mutation in the majority of these deletions reverted to produce stable, constitutive reversions exclusively. The explanation proposed was that the chlD-pgl deletions had also removed part of the gal operator-promoter up to the gal3 insertion, so that simple excisions of the insertion yielded stable, constitutive revertants by connecting the gal structural genes to a different promoter. These revertants were not considered to be true representatives of the stable, constitutive class. The specificity of deletion end-points at the insertion was found only in the gal3(λ) strain, and not in gal ⁺, gal ⁺(λ), or gal3 strains. Moreover, the frequency of spontaneous chlD-pgl deletions increased 10- to 15-fold in presence of the gal3 insertion.

A λgal phage bearing a true stable, constitutive reversion (gal ^c 200) was isolated from the revertant strain by subsequent deletion of the chlD-pgl segment (Δ31). Electron micrographs of λgal ⁺ and λgal ^c 200 Δ31(chlD pgl) DNA heteroduplexes were interpreted to indicate that the stable, constitutive reversion had arisen by a deletion of 3/4 of the gal3 insertion sequence.

The main conclusions are: (i) the stable, constitutive reversions of gal3 can arise by partial deletions of the insertion sequence, apparently by elimination of the nucleotide sequence which causes polarity; (ii) the chlD-pgl deletions may exhibit preferential termination at the right extremity of the gal3 insertion in presence of prophage λ; and (iii) the gal3 insertion appears to inhibit the production of λgal particles by providing a nucleotide sequence which is recognized and degraded by a specific endonuclease. It is suggested that inhibition of transducing particle formation by gal3 and the preferred termination of deletions at gal3 might represent related phenomena.