Identification of a high-copy-number plasmid suppressor of a lethal phenotype caused by mutant DnaA protein which has decreased intrinsic ATPase activity.
The induction of a mutant DnaA protein (DnaA E204Q) with decreased intrinsic ATPase activity in cells causes a lethal phenotype. Based on our results that the decreased ATPase activity of DnaA E204Q is activated to the level of a wild-type protein in the presence of partially purified stimulating factors for DnaA ATPase, we tested the hypothesis that genes encoding the stimulating factors can be cloned as high-copy-number plasmid suppressors for the lethality caused by DnaA E204Q. We isolated a number of high copy-number plasmids that suppress the lethal phenotype. The genes responsible for the suppression of the lethal phenotype were revealed to be dnaN, relA, pcnB and cyaA by subcloning, and a site-directed mutational analysis. The mechanisms by which these genes suppressed the lethal phenotype were examined in vivo and in vitro.
Makise
M
M
Faculty of Pharmaceutical Sciences, Okayama University, Tsushimanaka, Japan.
Sakamoto
K
K
Tsuchiya
T
T
Mizushima
T
T
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology; D003001:Cloning, Molecular; D004258:DNA Polymerase III / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D018628:Gene Dosage; D005798:Genes, Bacterial; D005804:Genes, Lethal; D016153:Genes, Suppressor; D008025:Ligases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids; D011062:Polynucleotide Adenylyltransferase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D011498:Protein Precursors; D011062:Polynucleotide Adenylyltransferase; C063643:pcnB protein, E coli; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; C083980:dnaN protein, Bacteria; D000251:Adenosine Triphosphatases; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases