The heat-shock DnaK protein is required for plasmid R1 replication and it is dispensable for plasmid ColE1 replication.
Plasmid R1 replication in vitro is inactive in extracts prepared from a dnaK756 strain but is restored to normal levels upon addition of purified DnaK protein. Replication of R1 in extracts of a dnaKwt strain can be specifically inhibited with polyclonal antibodies against DnaK. RepA-dependent replication of R1 in dnaK756 extracts supplemented with DnaKwt protein at maximum concentration is partially inhibited by rifampicin and it is severely inhibited at sub-optimal concentrations of DnaK protein. The copy number of a run-away R1 vector is reduced in a dnaK756 background at 30 degrees C and at 42 degrees C the amplification of the run-away R1 vector is prevented. However a runaway R1 vector containing dnaK gene allows the amplification of the plasmid at high temperature. These data indicate that DnaK is required for both in vitro and in vivo replication of plasmid R1 and show a partial compensation for the low level of DnaK by RNA polymerase. In contrast ColE1 replication is not affected by DnaK as indicated by the fact that ColE1 replicates with the same efficiency in extracts from dnaKwt and dnaK756 strains.
Giraldo-Suárez
R
R
Centro de Investigaciones Biolgicas (CSIC), Madrid, Spain.
Fernández-Tresguerres
E
E
Díaz-Orejas
R
R
Malki
A
A
Kohiyama
M
M
D001426:Bacterial Proteins / Q000378:metabolism; D004265:DNA Helicases; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D010957:Plasmids; D011506:Proteins; D015534:Trans-Activators; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; C063842:dnaK protein, E coli; D004265:DNA Helicases