Replication of oriJ-based plasmid DNA during the stringent and relaxed responses of Escherichia coli.
The oriJ-based plasmids contain the origin of DNA replication from the cryptic Rac prophage, present in the chromosomes of most Escherichia coli K-12 strains. The organization of the oriJ replication region resembles that of the bacteriophage lambda, although sequence similarity is small. Here we investigated the regulation of replication of the oriJ-based plasmid in E. coli relA(+) and relA(-) hosts during amino acid starvation and limitation, i.e., during the stringent and relaxed responses. We found that, contrary to plasmids derived from phage lambda, replication of the oriJ-based plasmid proceeds efficiently during both stringent and relaxed responses. On the other hand, density shift experiments and measurement of the stability of a putative replication initiator protein (the lambda O protein homologue) suggest that this replication may be carried out by the heritable replication complex, as previously demonstrated for lambda plasmids. We demonstrate that contrary to bacteriophage lambda p(R) promoter, an analogous promoter from the oriJ region is activated rather than inhibited at increased ppGpp levels. We propose that various responses of these promoters (p(R) and p(R-Rac), which are necessary for transcriptional activation of orilambda and perhaps oriJ, respectively) to ppGpp are responsible for differences in the replication regulation between orilambda- and oriJ-based plasmids during the stringent response.
Potrykus
K
K
Department of Molecular Biology, University of Gdańsk, Kladki 24, Gdańsk, 80-822, Poland.
Wróbel
B
B
Wegrzyn
A
A
Wegrzyn
G
G
D019677:Artificial Gene Fusion; D001483:Base Sequence; D004261:DNA Replication; D004269:DNA, Bacterial / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification; D018741:Replication Origin; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D004269:DNA, Bacterial; D001616:beta-Galactosidase