Synthesis of Protein and Nucleic Acid by Disrupted Spheroplasts of Pseudomonas schuylkilliensis1

Shigeki Mizuno, Hiroshi Matsuzawa, Yoshiho Nagata, Isao Shibuya, Hajime Takahashi, Bunji Maruo
1971 Journal of Bacteriology  
Osmotically shocked spheroplasts obtained from Pseudomonas schuylkilliensis strain P contained about 54, 32, 28, and 82% of the total cellular protein, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and phospholipid, respectively. This preparation was capable of incorporating 3apP-orthophosphate into RNA and DNA, aH-adenosine or 3H-uridine into RNA, and 3H-leucine or 14C-phenylalanine into protein. These activities were not found in the cytoplasmic fraction which contained most of the
more » ... ose-6-phosphate dehydrogenase activity. The synthesis of RNA by intact and disrupted spheroplast preparations was sensitive to actinomycin D, chromomycin Aa, streptovaricin, rifampin, Lubrol W, Triton X-100, and sodium deoxycholate, whereas RNA synthesis by intact cells was insensitive to these agents. Ethylenediaminetetraacetic acid, porcine pancreatic lipase, the protoplast-bursting factor, high concentrations of salts, and washing the preparation inhibited the synthesis of RNA by disrupted spheroplasts but had little or no effect on intact spheroplasts. Most of the newly synthesized RNA made by disrupted spheroplasts had the characteristics of messenger RNA. The DNA present in this preparation functioned as a template for RNA synthesis; continued protein synthesis was dependent on concomitant RNA synthesis. An unusual feature of the preparation was the finding that the synthesis of macromolecules was completely dependent on oxidative phosphorylation. found to be dependent on oxidative phosphorylation (28, 41). We are now interested in analyzing the relationship between the production of energy and its utilization for the synthesis of macromolecules. However, special care must be taken in any experiments with subcellular preparations derived from spheroplasts to avoid contamination with intact cells and intact spheroplasts (38). The first part of this paper demonstrates that the RNA-snthesizing activity of our disrupted spheroplast preparation can be distinguished from that of intact cells and intact spheroplasts by differences in sensitivity to detergents, antibiotics, enzymes, and high concentrations of salts, and by differences in stability. The second part describes the conditions required for the synthesis of RNA, especially the absolute dependency on oxidative phosphorylation and the stimulation observed when amino acids and chloramphenicol were added. Lastly, the newly synthesized nucleic acids are characterized with respect to base ratios, chromatography on methylated albumin-kieselguhr (MAK) columns, and distribution into ribosome and soluble fractions. 538 on May 9, 2020 by guest
doi:10.1128/jb.105.2.538-552.1971 fatcat:2qulrpokobc3hjefpgljulcnry