Segregation of Genes for Nitrogen Fixation into Minicells of Escherichiacoll

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Abstract
An E. coli strain containing N2 fixation (nif) genes on a transferable plasmid (RP41) was conjugated with an E. coli minicell-producing strain. The nif genes were expressed in the new strain as measured by acetylene reduction assays. Segregation of the RP41 plasmid into minicells was proved by the incorporation of acid-precipitable [3H]thymidine into minicells and also by the conjugal transfer of acetylene reduction ability during matings using minicell donors. Protein synthesis was measured by the time-dependent increase of acid-precipitable [3H]leucine in minicell suspensions. Minicells which contained the plasmid had greater synthetic ability than those which did not. Technical contributions to minicell purification were made. Sucrose gradients made by a new freeze-thaw procedure allowed easy preparations of sterile gradients which were used to obtain purity of 105 minicells/contaminating cell. A Dap- mutant was constructed to prevent outgrowth of contaminating cells during long incubations.

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