A 39 kDa DNase Cleaves Chromosomal DNA of Bacillus subtilis Cells Exposed to Lethal Cold and Heat Shock Treatments

Abstract

When the log-phase cells of Bacillus subtilis 168 were exposed to a cold shock drop in temperature from 37 to 0°C followed by incubation at 37°C or a heat shock rise in temperature from 37 to 55°C, their chromosomal DNA was found to be cleaved, when detected by agarose electrophoresis. The DNA fragmentation in vivo was observed in Spizizen minimal medium containing Ca²⁺ or Mn²⁺ but not in Mg²⁺. DNA fragmentation was inhibited by the addition of an endonuclease inhibitor, aurintricalboxylic acid (ATA), but not by Zn²⁺. By using the DNase ymographic analysis of the cell-free extract, Ca²⁺ or Mn²⁺-dependent DNase bands having molecular masses of 60, 39, 28 and 17 kDa were detected. Among those, the 17 kDa DNase was confirmed to be produced from the nucA gene by the nucA deletion mutant analysis.The extent of DNA cleavage and total DNase activity in the nucA mutant were similar to those of the parent strain, indicating no involvement of this enzyme in the DNA fragmentation caused by thermal shock treatments. On the other hand, since the 39 kDa DNase was found to be a Zn 2⁺-resistant type different from other DNases detected, this DNase is suggested to be a major factor involvedin the DNA fragmentation observed here.