Purification and characterization of a Bacillus subtilis 168 nuclease, YokF, involved in chromosomal DNA degradation and cell death caused by thermal shock treatments.
We purified and characterized a 39-kDa Bacillus subtilis 168 nuclease that has been suggested in this laboratory to be involved in chromosomal DNA degradation induced by lethal heat and cold shock treatments in vivo. The nuclease activity was inhibited in vitro by aurintricalboxylic acid but not by Zn(2+). By the mutant analysis, we identified the 39-kDa nuclease as a product of yokF gene. The yokF gene contained a putative lipoprotein signal peptide motif. After in vivo exposure to lethal heat and cold stresses, the chromosomal DNA fragmentation was reduced in the yokF mutant, which demonstrated about a 2-10-fold higher survival rate than the wild type. The yokF mutant was found to be more sensitive to mitomycin C than the wild type. The transformation efficiency of the yokF mutant was about 10 times higher than that of the wild type. It is suggested that when B. subtilis cells are exposed to a stressful thermal shock resulting in membrane perturbation, YokF nuclease consequently dislocates into the cytoplasm and then attacks DNA.
Sakamoto
J J
JJ
Department of Biotechnology, Faculty of Engineering, Kansai University, Yamate-cho, Suita, Osaka 564-8680, Japan.
Sasaki
M
M
Tsuchido
T
T
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001312:Aurintricarboxylic Acid / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins; D002462:Cell Membrane / Q000201:enzymology; D002875:Chromosomes / Q000378:metabolism; D003080:Cold Temperature; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D016685:Mitomycin / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014170:Transformation, Genetic; D019076:Transgenes; D015032:Zinc / Q000494:pharmacology
D001426:Bacterial Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D011994:Recombinant Proteins; D001312:Aurintricarboxylic Acid; D016685:Mitomycin; D004247:DNA; C444658:YncB protein, Bacillus subtilis; D003851:Deoxyribonucleases; D004720:Endonucleases; C444657:YokF protein, Bacillus subtilis; D015032:Zinc