Aspects of outer membrane protein synthesis in Escherichia coli B/r.

The growth and biogenesis of the outer membrane of E.coli B/r has been studied by investigating both the pattern of synthesis of outer membrane protein in the cell cycle, and the kinetics of synthesis and assembly of the major outer membrane protein species, the 36.5K porin. To facilitate this study the detergent sarkosyl was used to separate the outer membrane from the cytoplasmic membrane of E.coli; comparison of these fractions with those obtained by centrifugation revealed that they were qualitatively identical. Analysis of the cell cycle revealed that the rate of synthesis of outer membrane protein was constant through the cycle, with an abrupt doubling in rate occurring late in the cycle. Further experiments revealed that the rate of synthesis of outer membrane protein is not differentially affected by treatments which affect DNA synthesis, and it is therefore concluded that the cell cycle doubling in rate of synthesis is unrelated to the DNA replication cycle. In other experiments the time was measured for the 36.5K porin to be translated, enter the cell envelope, and become associated with the sarkosyl-insoluble outer membrane. This was achieved by a pulse-chase method. The porin associated with the envelope as soon as it was completed; however, it took significantly longer to reach the outer membrane, indicating the existence of a sarkosyl-soluble intermediate form of the mature porin. It is concluded that this may be either a true cytoplasmic membrane intermediate, or a nascent form present in atypical regions of the outer membrane. Finally, it was shown that an outer membrane protein whose synthesis has been reported to occur periodically in the cell cycle is, in fact, a protein involved in ferric-enterochelin uptake, and it is concluded that its apparent periodic synthesis is an artefact caused by the methods used to generate synchronous cultures.