JOURNAL OF BACrERIOLOGY, Mar., 1966 Copyright © 1966 American Society for Microbiology Vol. 91, No. 3 Printed in U.S.A. Cellular Site in Bacillus subtilis of a Nuclease Which Preferentially Degrades Single-Stranded Nucleic Acids H. C. BIRNBOIM Department of Biochemistry, Albert Einstein College of Medicine, New York, New York Received for publication 8 November 1965 ABSTRACT BIRNBOIM, H. C. (Albert Einstein College of Medicine, New York, N.Y.). Cellular site in Bacillus subtilis of a nuclease which preferentially degrades single-stranded nucleic acids. J. Bacteriol. 91:1004-1011. 1966.-A nuclease, identified by a marked preference for single-stranded nucleic acids, has been demonstrated in extracts of Bacillus subtilis. The enzyme was associated with the cell wall-membrane fraction of mechanically disrupted cells and was released from cells which had been con- verted to protoplasts by lysozyme. The nuclease activity prepared by the latter procedure was found to be activated and solubilized by treatment with trypsin. The enzyme had about 2% activity on native deoxyribonucleic acid (DNA) as com- pared with denatured DNA. By use of CsCl analytical density gradient ultracen- trifugation, this preparation was shown to degrade denatured DNA selectively in mixtures of native and denatured DNA. The cellular location of certain enzymes in bacteria has been the subject of several recent investigations. In Escherichia coli, several phos- phatases and nucleases (6, 10, 11, 12) have been shown to be released from bacterial cells when converted to spheroplasts. The localization of these enzymes at or near the cell surface has been proposed. In Bacillus licheniformis (previously classified as B. subtilis), a penicillinase has been found attached to the protoplast membrane (3, 4). In B. subtilis, Momose et al. (8) demon- strated a 5'-nucleotidase, the major portion of which could be released from cells upon conver- sion to protoplasts with lysozyme. In strains of Mycoplasma, several enzymes, including a ribo- nuclease and a deoxyribonuclease have been found associated with cell membranes (13). In this paper, we present evidence for the exist- ence in B. subtilis of a nuclease characterized by a marked preference for single-stranded deoxy- ribonucleic acid (DNA), as compared with native, double-stranded DNA, as a substrate. In mechan- ically disrupted cells, about one-half of the enzyme was found associated with the "cell wall- membrane" pellet. When cells were converted to protoplasts by treatment with lysozyme, the en- zyme was released and recovered in the super- natant fraction. The association of the nuclease with the cell wall-membrane of B. subtilis was studied, and the partially purified enzyme was characterized with respect to substrate and re- requirements for optimal activity. MATERIALS AND METHODS Bacterial strain. B. subtilis 168-2, a transformable strain requiring tryptophan and leucine, was used for studies on enzyme distribution and purification. B. subtilis 1681>, which requires thymine and trypto- phan, was used for the preparation of radioactive DNA. Media and growth conditions. V-Y broth [Difco Veal Infusion broth, 2.5% (w/v); Difco yeast extract, 0.5% (w/v)] was the routine medium used for grow- ing B. subtilis for enzyme isolation. Volumes of 1 liter were inoculated from a single colony and incu- bated in 2-liter flasks at 37 C in a New Brunswick gyrotory floor shaker for 16 hr. Under these condi- tions, the culture reached the early stationary phase of growth, about 3 g (wet weight) of bacteria per liter. Reagents. Lysozyme (egg white), three-times crystallized, B grade, was a product of Calbiochem. Tris(hydroxymethyl)aminomethane (Tris) was Trizma of the Sigma Chemical Co., St. Louis, Mo. Analytical reagent-grade sucrose was obtained from Mallinckrodt Chemical Works, New York, N.Y. Trypsin (Worth- ington Biochemical Corp., Freehold, N.J.) was used without further purification. Diethylaminoethyl (DEAE) cellulose (Carl Schleicher and Schuell Co., Keene, N.H.) was prepared for use by washing with 1 N NaOH, water, 1 N HCI, 1 N NaOH, and water, and 1004 on July 24, 2020 by guest http://jb.asm.org/ Downloaded from