Plasmid-mediated transfer of nitrogen-fixing capability to bacteria from the rhizosphere of grasses

Summary

Cells of a non-nitrogen-fixing, drug-sensitive Enterobacter cloacae strain, isolated from the rhizosphere of Festuca heterophylla, were mated to Escherichia coli cells harboring plasmid pRD1. This plasmid carries the nitrogen-fixation (nif⁺) genes as well as three markers of drug resistance. After mating, triple-resistant Enterobacter transferants could be selected. These were screened for plasmids, acetylene reduction, and stability of the transferred markers.

Transferants contained plasmid pRD1. Of 48, 43 were acetylene-reducing and therefore carried the nif⁺ genes. Triple-resistance was stable upon passage in liquid minimal medium, but the number of cells with nif⁺ genes decreased. Both the triple-resistant and the nif⁺ genotypes decreased in complete medium, although by different rates, depending on the particular line. The most stable line, M14, was chosen and checked further.

Samples taken after 8–14 passages in minimal medium contained cells with different genotypes, plasmid sizes smaller than the original plasmid pRD1 and no free plasmids. Progeny of the latter cells, in addition to being triple-resistant, were the best acetylene reducers. It is concluded that in these cells the plasmid pRD1 with all its relevant genes had become integrated into the recipients' chromosome.

Grass seedlings were inoculated with the bacteria containing integrated plasmid pRD1. They were then planted into pots with sterile ash and watered with a nutrient salt solution of limited nitrogen content. Sampling after 8 weeks showed that the inoculated bacteria were preserved, as demonstrated by their triple-resistance. They could also still fix nitrogen.