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

Abstract

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 M_r 74,000 and M_r 83,000, the methanogenic ones from M_r 83,000 up to M_r 89,000, the halophilic ones between M_r 101,000 and M_r 111,000. With the same technique eukaryotic factors gave values of Mr 96,000.