Reconstitution of binding protein dependent active transport of glutamine in spheroplasts of Escherichia coli

Abstract

In order to directly prove that the periplasmic glutamine binding protein is an essential component of the osmotic shock sensitive active transport system for glutamine in Escherichia coli, we demonstrated the reconstitution of binding protein dependent glutamine transport in spheroplasts of that organism. It was shown by arsenate inhibition that the reconstituted transport system was energy dependent, and the use of azaserine, an inhibitor of glutamine-utilizing enzymes, indicated that the restoration of transport by binding protein did not require the metabolizing of the transport substrate. Furthermore, the binding protein dependent transport of glutamine was shown to require at least one other macromolecular component, presumably membrane bound, which was absent in a strain containing a deletion of the genes coding for the glutamine transport system but was present in a strain carrying a mutation only in the structural gene for the glutamine binding protein.