Synergism of mutations in bacteriophage Qbeta RNA affecting host factor dependence of Qbeta replicase.
We have recently shown that Escherichia coli cells deficient in Hfq protein (i.e. the Qbeta "host factor") support bacteriophage Qbeta replication inefficiently, but that the phage evolves rapidly in the mutant host to become essentially host factor independent. An identical set of four point mutations was identified as being responsible for the adapted phenotype in each of three independent adaptation experiments. Here we report the effects of the single mutations and of some of their combinations on host factor dependence of phage multiplication in vivo and of phage RNA replication by Qbeta replicase in vitro. We find that each single substitution produces only small effects, but that in combination the four mutations synergistically account for most of the observed adaptation of the evolved phages. Surprisingly, a reanalysis of the 3'-terminal sequence of the adapted phages resulted in the discovery of a fifth mutation in all three independently evolved phage populations, namely, a C to U residue transition at nucleotide 4214. This mutation had been missed previously because of its location only three nucleotides from the 3'-end. It appears to contribute little to the Hfq independence but may enhance RNA stability by re-establishing the possibility of forming a long-range base-pairing interaction involving the immediate 3'-terminal sequence.
Schuppli
D
D
Universit]at Z]urich, Z]urich, 8057, Switzerland.
Georgijevic
J
J
Weber
H
H
D017908:Allolevivirus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D035001:Host Factor 1 Protein; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011777:Q beta Replicase / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*
D002352:Carrier Proteins; D035001:Host Factor 1 Protein; D034982:Integration Host Factors; D012367:RNA, Viral; D011777:Q beta Replicase