Identification of a new deoxyribonuclease and DNA gyrase in P̲s̲e̲u̲ḏo̲m̲o̲ṉa̲s̲ a̲e̲ṟu̲g̲i̲ṉo̲s̲a̲ strain PAO

A new deoxyribonuclease, PaeExoIx, has been purified to electrophoretic homogeneity from extracts of P. Aeruginosa by using two DEAE-cellulose columns, a Sepharose 6B column, and a glycerol gradient. This enzyme, which was active in the presence of EDTA, was equally efficient in hydrolyzing native and heat-denatured DNA to acid-soluble products. The enzyme was partially or totally inhibited by the presence of several divalent cations. Optimal activity was demonstrated between pH 8.0 and 9.0. The active protein has a molecular weight of 1.6 ± 0.1 x 10⁵ and is composed of two nonidentical polypeptides with molecular weights of 78,000 and 69,000. Preliminary data indicate that both the 3' end and the 5' end of DNA are rapidly degraded by this enzyme.

The data indicate that this enzyme has several unique biochemical properties when compared to other deoxyribonucleases identified in other bacteria. However, the enzyme has activities which are similar to those of several deoxyribonucleases which are involved in genetic recombination in other bacteria.

The second part of this study is a report of DNA gyrase in P. Aeruginosa. This enzyme catalyzes the supercoiling of DNA in the presence of ATP. Its activity was detected by using a relaxed closed-circular DNA substrate in the reaction mixture followed by analysis with agarose gel electrophoresis. DNA gyrase has also been identified in E. Coli, Micrococcus luteus, and Bacillus subtilis by other investigators. DNA gyrase from these species is inhibited by the drugs nalidixic acid and novobiocin.

DNA gyrase from P. Aeruginosa, like DNA gyrase identified in other bacteria, requires ATP and was inhibited by the drugs novobiocin and nalidixic acid. The native enzyme has a molecular weight of 360,000 when estimated by Sephadex G-200 chromatography. Double-stranded cleavage or relaxing activity could not be demonstrated in P. Aeruginosa DNA gyrase as is found in E. Coli gyrase. However, a relaxing activity was separated from P. aeruginosa DNA gyrase with a Sephadex G-200 column. Therefore, P. Aeruginosa does have a DNA gyrase type enzyme, but there are several differences in its properties when compared with DNA gyrase from other bacteria.