Identification and Comparison of Eighteen Archaebacteria by Means of the Diphtheria Toxin Reaction

Abstract

Summary Cell-free extracts were prepared from seven extremely halophilic, six methanogenic and five thermoacidophilic archaebacteria covering nearly all known subgroups. The 150,000 X g supernatants were incubated with diphtheria toxin (DT) and NAD [adenine-14C]. TCA-precipitable radioactivity was compared with that from toxin-free controls. All eighteen archaebacterial strains gave significant, DT-dependent protein labelling, as had done all eukaryotic extracts previously assayed. No eubacterial nor mitochondrial proteins were substrates for DT. Therefore the DT reaction in this simple form provides a general method for identifying a procaryote as an archaebacterium. SDS-gel electrophoresis of the reaction products followed by autoradiography revealed in many but not all cases one predominant labelled protein band; nearly all halophilic gels and several methanogenic ones showed minor bands. Direct evidence from H. cutirubrum (Kessel and Klink, 1980) and the known specifity of DT for eukaryotic EF-2 led to the conclusion that only the analogous archaebacterial EF was ADP-ribosylated, minor bands being products of proteolysis. The apparent molecular weights showed a considerable diversity and formed three groups: The thermoacidophilic elongation factors banded between Mr 74,000 and Mr 83,000, the methanogenic ones from Mr 83,000 up to Mr 89,000, the halophilic ones between Mr 101,000 and Mr 111,000. With the same technique eukaryotic factors gave values of Mr 96,000.