UBC Theses and Dissertations
Cell division in a temperature-sensitive mutant of Escherichia coli Reeve, John N.
A temperature-sensitive division mutant, Escherichia coli BUG-6, has been investigated. This organism divides normally when grown at 30 C but fails to divide at 42 C. Growth continues at 42 C to produce very long, multinucleate filamentous cells. When returned to 30 C, or a high osmotic environment is added at 42 C, the filamentous cells divide rapidly to produce cells of normal size. The kinetics of cell division of the filaments at 30 C depends on the period at 42 C. During filament formation, DNA, RNA and protein synthesis continue as measured by radio-isotopic incorporation and chemical cell fractionation. DNA segregation occurs as shown by autoradiography. The rapid division of filaments replaced at 30 C cannot be prevented by novobiocin or cycloserine but is prevented by vancomycin and penicillin suggesting that de novo synthesis of cell wall precursors is not required for division but that positioning and cross-linking of the precursors is required. Filaments growing at 42 C were treated with nalidixic acid for different lengths of time. On returning these filaments to 30 C in nalidixic acid the number of divisions was proportional to the length of time at 42 C in the absence of nalidixic acid, ie. proportional to the amount of DNA synthesized at 42 C. Inhibition of protein synthesis, by chloramphenicol, does not prevent the division of filaments on replacing at 30 C provided that the period of filamentation at 42 C was greater than 6 minutes and less than 110 minutes. The maximum amount of division in the absence of protein synthesis occurred after a longer lag and slower than in non-inhibited control cultures. If protein synthesis was inhibited in filaments at 42 C the ability of such treated cells to divide at 30 C was rapidly lost. This loss of 'division potential’ has a half-life of about 0.5 minutes, ie. 0.5 minutes of protein inhibition at 42 C reduces the subsequent division at 30 C by 50%. The normal presence of 'division potential', therefore requires the synthetic doubling rate to be in excess of 0.5 minutes. Very short periods at 42 C indicate that 10 minutes incubation at 42 C is required to produce this extremely fast synthetic rate. A model for the production and expression of 'division potential' is presented. A biochemical analysis of the cell envelope of the filamentous cells and of normally dividing cells is presented. The major phospholipid compositions are the same. However, the fatty acid contents differ especially with regard to the cyclic fatty acids. When the filaments are allowed to divide by replacing at 30 C their fatty acid composition very rapidly reverts to that of normally dividing cells. The rates of individual phospholipid syntheses appear to change during the rapid cell division phase, however this may be an artifact resulting from an overall increase in the rate of phospholipid synthesis during this period. An analysis of the proteins within the cell envelope by radio-active double labelling techniques and followed by gel electrophoresis indicates that a protein(s) of molecular weight 80,000 - 90,000 exists in the envelope of filamentous cells which is not in the envelope of normal cells or is made at a much slower rate in normal cells. The protein is not incorporated into septa during the division of filaments at 30 C and little turnover occurs in the major proteins synthesized at 42 C when these cells are placed at 30 C. The possibility exists, however, that this protein is a product of filamentation and not the temperature sensitive gene product.
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