The hydrogenosomal malic enzyme from the anaerobic fungus neocallimastix frontalis is targeted to mitochondria of the methylotrophic yeast hansenula polymorpha.

Hydrogenosomal proteins always contain an amino-terminal extension which is believed to be a hydrogenosomal targeting signal. In the anaerobic fungus Neocallimastix frontalis these putative targeting signals are 27 amino acids long, are enriched in Ala, Leu, Ser and Arg, and have an Arg at position -2 relative to amino-acid 1 of the mature protein. These features are typically observed in mitochondrial targeting signals. Here we show that the 27 amino-acid leader sequence of the hydrogenosomal malic enzyme of N. frontalis was capable of targeting the enzyme to mitochondria of the methylotrophic ascomycete yeast Hansenula polymorpha. The same protein without this leader sequence remained cytosolic. These data suggest a close relationship between the protein import machineries of mitochondria and hydrogenosomes in fungi and provide further support for the notion that these two organelles share a common evolutionary origin.

Hydrogenosomes in the anaerobic fungus Neocallimastix frontalis have a double membrane but lack an associated organelle genome.

The presence of hydrogenosomes in phylogenetically distinct anaerobic eukaryotes implies that they have been acquired independently, and previously reported differences in ultrastructure among taxa have suggested that some hydrogenosomes have different origins. Of particular interest are reports that Neocallimastix frontalis hydrogenosomes resemble microbodies in possessing a single membrane, in contrast to those in ciliates and trichomonads which have two and thus resemble mitochondria. In this investigation we have clearly demonstrated that N. frontalis hydrogenosomes possess two, rather than one, closely apposed membranes and in some preparations cristae-like structures were observed. These observations have led us to reject the microbody hypothesis and provide some indirect support for a possible mitochondrion origin as proposed for other hydrogenosomes. N. frontalis hydrogenosomes were shown to lack an associated genome as previously demonstrated for trichomonad hydrogenosomes. This might be explained by assuming that a mitochondrial genome encoding proteins for aerobic function is no longer necessary for either organelle.

Structural organization of the Streptococcus pneumoniae chromosome and relatedness of penicillin-sensitive and -resistant strains in type 9V.

Fragmentation of Streptococcus pneumoniae genomic DNA with low-frequency-cleavage restriction endonucleases and separation of the fragments by field-inversion gel electrophoresis (FIGE) provides a DNA-fingerprint of a strain. This method enables us to construct a physical and genetic map of the R6 laboratory strain what will be presented. The origin of replication containing several Dna boxes was located in the dnaA region. It was of interest to compare the profiles of subclones. Two clones of strain R36A (R6 and C13) were cultivated separately for more than 15,000 generations in two laboratories. FIGE profiles differed by only one band. Another R36A descendant, isolated in 1958 by Ravin, strain Rx was of interest since it was deficient in Dpn restriction enzymes and methylases and in the hex B function. Its origin was questionable; its profile is identical to others R6 descendants, demonstrating that Rx is derived from R36A. FIGE analysis was carried out on several penicillin-resistant strains of type 9V because penicillin-resistance in this type increased recently. The profiles of a collection of a number of these resistant isolates were very similar, showing that they result from a clone. The profiles of penicillin sensitive isolates of the same type are very similar to the resistant isolates. This suggests that the 9V type has spread recently from a clone, and the resistance genes have mutated and were selected when penicillin was extensively used.

A mitochondrial-like targeting signal on the hydrogenosomal malic enzyme from the anaerobic fungus Neocallimastix frontalis: support for the hypothesis that hydrogenosomes are modified mitochondria.

The hydrogenosomal malic enzyme (ME) was purified from the anaerobic fungus Neocallimastix frontalis. Using reverse genetics, the corresponding cDNA was isolated and characterized. The deduced amino acid sequence of the ME showed high similarity to ME from metazoa, plants and protists. Putative functional domains for malate and NAD+/NADP+ binding were identified. Phylogenetic analysis of the deduced amino acid sequence of the new ME suggests that it is homologous to reference bacterial and eukaryotic ME. Most interestingly, the cDNA codes for a protein which contains a 27-amino-acid N-terminus which is not present on the purified mature protein. This presequence shares features with known mitochondrial targeting signals, including an enrichment in Ala, Leu, Ser, and Arg, and the presence of an Arg at position-2 relative to amino acid 1 of the mature protein. This is the first report of a mitochondrial-like targeting signal on a hydrogenosomal enzyme from an anaerobic fungus and provides support for the hypothesis that hydrogenosomes in Neocallimastix frontalis might be modified mitochondria.

scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis.

A clone containing a Neocallimastix frontalis cDNA assumed to encode the beta subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counter-parts. An open reading frame of 1311 bp was found. The deduced 437 amino acid sequence showed a high degree of identity to the beta-succinyl-CoA synthetase of Escherichia coli (46%), the mitochondrial beta-succinyl-CoA synthetase from pig (48%) and the hydrogenosomal beta-succinyl-CoA synthetase from Trichomonas vaginalis (49%). The G + C content of the succinyl-CoA synthetase coding sequence (43.8%) was considerably higher than that of the 5' (14.8%) and 3' (13.3%) non-translated flanking sequences, as has been observed for other genes from N. frontalis. The codon usage pattern was biased, with only 34 codons used and a strong preference for a pyrimidine (T) in the third positions of the codons. The coding sequence of the beta-succinyl-CoA synthetase cDNA was cloned in an E. coli expression vector encoding a 6(His) tag. The recombinant protein was purified by affinity binding and used to produce polyclonal antibodies. The anti-succinyl-CoA synthetase serum recognized a 45 kDa protein from a N. frontalis fraction enriched for hydrogenosomes and similar polypeptides in two related anaerobic fungi, Piromyces rhizinflata (45 kDa) and Caecomyces communis (47 kDa). Immunocytochemical experiments suggest that succinyl-CoA synthetase is located in the hydrogenosomal matrix. Staining for SCS activity in native electrophoretic gels revealed a band with an apparent molecular weight of approximately 330 kDa. The C-terminus of the succinyl-CoA synthetase sequence was devoid of the typical targeting signals identified so far in microbody proteins, indicating that N. frontalis uses a different signal for sorting SCSB into hydrogenosomes. Based on comparisons with other proteins we propose a putative N-terminal targeting signal for succinyl-CoA synthetase of N. frontalis that shows some of the features of mitochondrial targeting sequences.