plasmid questions -8

8. Temperature sensitive mutations that prevent the initiation of replication of the E. coli chromosome are lethal at 42 C. However, Hfr strains that are known to contain such mutations survive at 42 C. Why is the temperature sensitive phenotype not observed in Hfr strains?

ANSWER: When the chromosomal origin is inactive, replication can be initiated at the integrated F-plasmid origin and proceed around the chromosome. This process is called "integrative suppression". How could you prove that a Hfr strain still contains the original temperature sensitive mutation?

ANSWER:

(i) Loss of the Hfr would restore the original replication mutant phenotype. (For example, it is possible to select for loss of the Hfr, by recombination with adjacent markers but such transductants are rare due to the ccd plasmid addiction system of the F-plasmid.)

(ii) Transfer of the chromosome origin into a new strain would confer the replication mutant phenotype. (For example, this could be done by selecting for an adjacent marker in an Hfr cross.)

(iii) If the Hfr was integrated at a site distinguishable from the chromosomal replication origin, you could determine where replication initiates using molecular approaches. If replication initiates at the Hfr under nonpermissive conditions but at the chromosomal origin under permissive conditions, then the original replication mutation is present in this strain.