Heat sensitivity of Azotobacter vinelandii genetic transformation

Abstract

Heating competent Azotobacter vinelandii at 37 or 42°C resulted in a total loss of competence with no loss of viability. The transformation process was relatively insensitive to heating at either temperature once DNase-resistant DNA binding was nearly complete. Although competent and 42° C-treated cells bound equivalent amounts of [32P]DNA in a DNase-resistant state, no donor DNA marker (nif) or radioactivity was detected in the envelope-free cell lysate of heated cells, suggesting that DNA transport across the cell envelope was a heat-sensitive event. Competence was reacquired in a 42°C-treated culture after 2 h of incubation at 30°C by a process which required RNA and protein syntheses. The release of a surface glycoprotein, required for competence, from cells treated at 42°C occurred in an insufficient amount to account for the total loss of competence. Recovery of competence in 42°C-treated cells and further transformation of competent cells were prevented by the exposure of cells to saturating amounts of transforming DNA. Further DNase-resistant DNA binding, however, still occurred, suggesting that there were two types of receptors for DNase-resistant DNA binding to competent A. vinelandii. DNase-resistant DNA binding was dependent on magnesium ions, and at least one receptor type did not discriminate against heterologous DNA.