Macrofibers in steady-state growth at one temperature were subjected to pulses of various durations at a temperature at which the opposite helix hand would form and then returned to the initial temperature. In an upshift pulse (20 to 48 degrees C), at least 3 min of incubation was required to induce a transient inversion that occurred later after return to 20 degrees C. Longer pulses resulted in shorter delays in onset of the transient inversion. This "memory" of a brief high-temperature pulse suggests that even a small amount of material can influence the twist of the entire macrofiber. Similar results were found for temperature downshift pulses corresponding to the opposite inversion. Adding chloramphenicol during the temperature pulse blocked the establishment of memory associated with the right-to-left inversion but not that associated with left-to-right inversion. In contrast, inhibiting peptidoglycan synthesis with D-cycloserine during the temperature pulse did not prevent establishment of memory. Inhibiting protein synthesis in mutants fixed as left-handed structures over the entire temperature range induced conversion to right-handedness but did not affect mutants fixed as right-handed structures. Adding protease to either live or formaldehyde-killed macrofibers always induced rotations of right-handed orientation. Steady-state growth in the presence of protease was found to shift the initial macrofiber twist towards the right-hand end of the twist spectrum. The phenomenon was observed in several mutants with different initial twists
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