Dennis G. Searcy

Sulfur reduction by human erythrocytes

Washed human erythrocytes incubated with glucose and S8 and purged with N2 produced H2S at a nearly constant rate of 170 mumol (L cells)-1 min-1, which continued for several hours. In sealed vials up to 25 mM HS- accumulated. Glucose caused the fastest H2S production, although either lactate or glycerol could support slower rates. When glucose was added without S8, anoxic H2S production nonetheless occurred at about 1.5% of the maximum rate, after 24 hr totaling 0.5 mmol H2S (L cells)-1, suggesting the presence of endogenous reducible sulfur. Anaerobic conditions were not required, since oxygenated cells produced H2S from S8 at 80% of the anoxic rate. Using cell lysates, production of H2S occurred after addition of either glutathione, NADH, or NADPH. The observations suggest possible physiological roles for H2S as an electron carrier, and are consistent with an evolutionary relationship between eukaryotic cytoplasm and sulfur-reducing Archaea.

Metabolic integration during the evolutionary origin of mitochondria

ABSTRACTAlthough mitochondria provide eukaryotic cells with certain metabolic advantages, in other ways they may be disadvantageous. For example, mitochondria produce reactive oxygen species that damage both nucleocytoplasm and mitochondria, resulting in mutations, diseases, and aging. The relationship of mitochondria to the cytoplasm is best understood in the context of evolutionary history. Although it is clear that mitochondria evolved from symbiotic bacteria, the exact nature of the initial symbiosis is a matter of continuing debate. The exchange of nutrients between host and symbiont may have differed from that between the cytoplasm and mitochondria in modern cells. Speculations about the initial relationships include the following. (1) The pre-mitochondrion may have been an invasive, parasitic bacterium. The host did not benefit. (2) The relationship was a nutritional syntrophy based upon transfer of organic acids from host to symbiont. (3) The relationship was a syntrophy based upon H2 transfer from symbiont to host, where the host was a methanogen. (4) There was a syntrophy based upon reciprocal exchange of sulfur compounds. The last conjecture receives support from our detection in eukaryotic cells of substantial H2S-oxidizing activity in mitochondria, and sulfur-reducing activity in the cytoplasm.

Sulfide oxidation coupled to ATP synthesis in chicken liver mitochondria

Chicken liver mitochondria consumed O2 at an accelerated rate when supplied with low concentrations of hydrogen sulfide. Maximum respiration occurred in 10 microM sulfide, and continued more slowly up to concentrations as high as 60 microM. Sulfide oxidation was coupled to adenosine triphosphate (ATP) synthesis, as shown by firefly luciferase luminescence and by measurement of the mitochondrial membrane electrochemical gradient. Synthesis of ATP required low, steady-state concentrations of sulfide (< 5 microM), which were maintained by use of a syringe pump. The ratio of consumed O2 to sulfide changed at low sulfide and O2 concentrations, indicating alternative metabolic reactions and products. In low concentrations of sulfide, presumably most similar to physiological, the O2/sulfide ratio was 0.75. This is the first report of sulfide oxidation linked to ATP synthesis in any organism not specifically adapted to a sulfide-rich environment. We suggest that this may be a widespread mitochondrial trait, and that it is consistent with the hypothesis that mitochondria originated from sulfide-oxidizing symbionts.

Histone-like protein in the prokaryote Thermoplasma acidophilum

The DNA of the prokaryote Thermoplasma acidophilum is associated with a histone-like protein that has the following properties: it has a high content (23%) of basic amino acids, is positively charged at neutral pH, is soluble in acid, and can stabilize DNA against thermal denaturation. In polyacrylamide gel electrophoresis, in the presence of either sodium dodecylsulfate or urea, it migrates at the same rate as histone IV (F2a1) of calf thymus. The amino acid composition, however, it unusually rich in the amides of acidic amino acids (16-20%), and it does not appear to be closely homologous to any of the classes of eukaryotic histones. Escherichia coli DNA, on the other hand, was associated with no detectable acid-soluble proteins, and the nucleoprotein thermally denatured at a lower temperature than pure DNA.

Phylogenetic affinities between eukaryotic cells and a thermophilic mycoplasma

Thermoplasma acidophilum, a thermophilic mycoplasma, has several unusual features suggesting a possible relationship to eukaryotic cells. One feature is a histone-like protein that is associated with the DNA, condensing it into subunits similar to those in eukaryotic chromatin. A second feature is an association of cytoplasmic proteins that resembles eukaryotic actin and myosin. These two components are widely distributed in different groups of eukaryotic cells, but are typically lacking in prokaryotic cells. Furthermore, T. acidophilum lacks cytochromes and respires by enzymes that apparently are not coupled to oxidative phosphorylation. This primitive type of respiration resembles that of microbodies, another feature which is represented in the cytoplasm of all groups of eukaryotic cells. Furthermore, since T. acidophilum lacks a cell wall and appears to have a primitive correlate of endocytosis, it would appear to be mechanically capable of acquiring a symbiotic mitochondrion. Thus, our observations are consistent with the symbiotic hypothesis for the origin of eukaryotic cells. We suggest that an organism similar to T. acidophilum was the host cell for the original symbiosis, becoming the nucleus and cytoplasm of modern eukaryotic cells.

Thermoplasma acidophilum: intracellular pH and potassium concentration.

Thermoplasma acidophilum is a free-living thermophilic mycoplasma. Although the organism lacks a cell wall, it can grow in medium as dilute as 66 mosM. The intracellular K+ concentration can be as low as 17 mM, but varies according to the osmolality of the culture medium. The internal pH can be measured by taking advantage of the fact that T. acidophilum undergoes lysis when the pH is adjusted to neutrality. Thus, by appropriate analysis of titration curves, it is possible to conclude that the internal pH is near 5.5. This result was confirmed by a second type of experiment in which the internal pH was analyzed by rupturing the cells in a French Pressure Cell.

Nucleoprotein subunit structure in an unusual prokaryotic organism: Thermoplasma acidophilum

The freeliving thermophilic mycoplasma, Thermoplasma acidophilum, has a small acid-soluble protein tightly bound to its DNA. This protein is similar to eukaryotic histones in both size and amino acid composition. Here we report that the protein condenses DNA into globular particles that are about half the size of eukaryotic nucleosomes. Our conclusions are based primarily upon the following observations: (1) Nuclease digestion produced DNA fragments of 40 base-pairs. (2) The ratio of protein to DNA was such that 4--5 molecules of protein were associated with each 40 base-pair segment of DNA. (3) Protein crosslinking reagents produced tetramers of the histone-like protein. (4) Electron microscopy revealed globular particles 5--6 nm in diameter. (5) Each globular particle reduced the apparent contour length of the DNA by 40 base-pairs. Thus, each nucleoprotein particle is apparently composed of 40 base-pairs of DNA coiled around four molecules of proteins.

Histone-like protein in the Archaebacterium Sulfolobus acidocaldarius

The Archaebacterium Thermoplasma acidophilum contains a basic chromosomal protein remarkably similar to the histones of eukaryotes. Therefore, it was of interest to examine a different Archaebacterium for similar proteins. We chose to examine Sulfolobus acidocaldarius because it is thermophilic, like T. acidophilum, but nevertheless the two organisms are not particularly closely related. Two major chromosomal proteins were found in S. acidocaldarius. The smaller of these was soluble in 0.2 M H2SO4 and had a molecular weight of 14500. The larger was acid-insoluble and had a molecular weight of about 36000. Together, the proteins protected about 5% of the DNA against nuclease digestion and stabilized about 50% against thermal denaturation. Overall, the properties of these proteins were intermediate between those of the Escherichia coli protein HU and T. acidophilum protein HTa.

Characterization of Thermoplasma acidophilum Deoxyribonucleic Acid

The deoxyribonucleic acid (DNA) of the mycoplasma Thermoplasma acidophilum has a base composition of 46 mol% guanine plus cytosine (G+C). Depurination can occur to the DNA in the culture conditions, giving rise to a product of lower buoyant density. This effect probably accounts for earlier descriptions of a much lower G+C content in the DNA. The size of the T. acidophilum genome, as measured by DNA renaturation, was 30% the size of the Escherichia coli genome.

Thermoplasma-Acidophilum Histone-Like Protein - Partial Amino-Acid-Sequence Suggestive of Homology to Eukaryotic Histones

Thermoplasma acidophilum is a freeliving mycoplasma-like organism that has a small basic protein tightly bound to its DNA. The N-terminal sequence of this protein has been determined. It has a distanct but statistically significant homology to eukaryotic histones H2A, H3, and to Escherichia coli protein HU.