544 Biochimica et Biophysica Acta, 1183 (1994) 544-546 © 1994 Elsevier Science B.V. All rights reserved 0005-2728/94/$07.00 BBABIO 40286 Short Sequence-Paper Molecular cloning of hydrogenosomal ferredoxin cDNA from the anaerobic amoeboflagellate Psalteriomonas lanterna i Stanley Brul *, Rob H. Veltman, Maria C.P. Lombardo 2 and Godfried D. Vogels Department of Microbiology and Evolutionary Biology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen (The Netherlands) (Received 9 September 1993) Key words: Hydrogenosome; Ferredoxin; cDNA; Protein sequence; Anaerobic free living protozoon; Protozoon; (P. lanterna) Anaerobic parasitic and free living protozoa and anaerobic rumen fungi often contain a characteristic organelle, the hydrogeno- some. Recently obtained molecular data show that hydrogenosomes in parasitic protozoa probably use a mitochondria-like protein targeting mechanism, whereas for hydrogenosomes in fungi a microbody-likemechanism is inferred. Here we present, to our knowledge, the first sequence data of a hydrogenosomal protein in a free-living anaerobic protozoan. It is shown that ferredoxin of the amoeboflagellate Psalteriomonas lanterna is similar to hydrogenosomal ferredoxin of the parasite Trichomonas vaginalis. We suggest that the two ferredoxins use similar organelle targeting mechanisms. Ferredoxins act as proximal electron donor for hy- drogenosomal hydrogenase and form as such one of the characteristic compounds of hydrogenosomes, or- ganelles present in many anaerobic eukaryotic microor- ganisms. Whereas much is known about the biochem- istry of hydrogenosomes [1-4], only recently has molec- ular information on their biogenesis been gathered through studies on hydrogenosomal protein import [5- 7]. Johnson et al. [5,6] identified a putative N-terminal cleavable topogenic signal in hydrogenosomal ferre- doxin and the /3-subunit of succinate thiokinase of T. vaginalis. Although much shorter, the amino acid stretch resembles mitochondrial targeting sequences. On the other hand, Marvin-Sikkema et al. [7] inferred in the anaerobic fungus Neocallimastix sp. L2 a typical microbody targeting signal (SKL) on hydrogenosomal hydrogenase. On the free-living anaerobic protozoa, an assembly of phylogenetically distant hydrogenosome- containing eukaryotes, no molecular information is yet available. Here, we report on the isolation and se- quencing of the cDNA coding for ferredoxin from the free-living anaerobic amoeboflagellate Psalteriomonas lanterna [8] 1. The deduced protein sequence is com- pared to those of ferredoxins from T. vaginalis and other organisms. Using PCR amplification (program: 2 min, 94°C; 1.5 min,. 55°C and 2 rain 72°C for 35 cycles) with primers 5'-acaatcacagccgtcaagg-3' (positions 27 to 47; sense primer) and 5'-ttttcaccactgagtgtgat-3' (positions 251 to 231; antisense primer) a 224 bp fragment of T. vagi- nalis hydrogenosomal ferredoxin was obtained ('fd probe'). The fd probe was tested on dot blots for cross-hybridization with gDNA of P. lanterna and Bac- teroides sp. The latter acted as control, since our P. lanterna cultures are grown with this prokaryote as food bacterium. Hybridization was performed at 55°C in 5 x SSC, 0.1% (w/v) N-laurylsarcosine sodium salt (Sigma, St. Louis, USA), 0.02% (w/v) SDS, 1% (w/v) blocking reagent and 100 ~g/ml herring sperm DNA (Boehringer, Mannheim, Germany). The blot was washed under low stringency (final wash was twice with 2 × SSC 0.1% SDS at 55°C) and developed according to the digoxigenin detection protocol of Boehringer. The heterologous fd probe hybridized only with P. lanterna gDNA (Fig. 1) and was next used to screen a lambda gtll cDNA library of the amoeboflagellate. The library was constructed from oligo(dT) purified and thus bacterial RNA-free mRNA. Hybridization and wash conditions were identical to those used for the dot blot. Two positive reacting recombinants, phage 313 and 312, contained inserts of 350 and 250 bp, respectively, and were used for sequencing studies. * Corresponding author. Fax:+ 31 80 553450; e-mail: stan@ sci.kun.nl. 2 Current address: Department of Genetics, Erasmus University, 3015 GE Rotterdam, The Netherlands. 1 The DNA sequence data reported in this paper are deposited in the EMBL data base under accession number X74556. SSDI 0005-2728(93)E01 75-P