Evidence of the involvement of an outer membrane protein in DNA initiation.

Protein D of molecular weight 80,000 is incorporated into the membrane of Escherichia coli B/r for a brief fraction of the cell cycle. The protein is incorporated into the outer membrane; its incorporation seems to reflect de novo synthesis and, once incorporated, it is stable. In synchronous acetate-grown cultures a 10-fold increase in the rate of protein D synthesis occurs toward the end of the cell cycle, near the end of one round of DNA synthesis and the start of the next. In synchronous glucose-grown cultures the protein appears approximately halfway through the cell cycle, shortly before DNA synthesis begins. The observed correlation between protein D synthesis and DNA initiation, approximately 15 min later, is strengthened by experiments with inhibitors and mutants. Inhibition of protein synthesis at any time during DNA synthesis delays the subsequent appearance of protein D and the initiation of the next round of DNA synthesis. The synthesis of both protein D and DNA is prevented by nalidixic acid; after its removal a sharp spike of protein D synthesis is followed by a wave of DNA synthesis. Experiments with DNA and DNAB mutants, with amino acid starvation of an auxotroph and with nutritional shift-up, all supported the coupling of protein D synthesis with the initiation of DNA synthesis. The addition of beta-lactam antibiotics which affect murein metabolism dramatically increases the amount of protein D. The cells are prevented from terminating protein D synthesis; different antibiotics have different kinetics of action. Protein D preferentially binds to double-stranded DNA in vitro. We suggest that this protein provides a metabolic link between murein synthesis, protein synthesis, and DNA initiation, and that it acts at a specific time in the cell cycle as an attachment site for DNA to the cell envelope.