Population ecology of Sulfolobus acidocaldarius

Ecology of Sulfolobus acidocaldarius was studied in situ by the use of the immunofluorescence and immunodiffusion techniques. The fluorescent antibodies (FA) prepared against four strains of Sulfolobus were highly reactive against their homologous antigens. Two of the FA's were strain specific and the other two exhibited reciprocal corssreactions against each other's antigens, but immunodiffusion patterns showed that the two strains were not identical. The growth of a serologically distinct isolate in a hot spring was measured by immunofluorescence staining of immersion slides. On glass immersion slides Sulfolobus grew and formed colonies with a mean-doubling time of approximately 36 h. Immunofluorescence was applied to study the geographical distribution of two serologically different strains and to establish population composition of individual springs. One strain was found in all sites studied, and most springs contained more than one serologic type. Immunodiffusion was capable of detecting specific Sulfolobus antigens in hot springs which contained a high population of FA-reactive cells.     

Growth of the extreme thermophile Sulfolobus acidocaldarius in a hyperbaric helium bioreactor

The relationship between pressure and temperature as it affects microbial growth and metabolism has been examined only for a limited number of bacterial species. Because many newly-discovered, extremely thermophilic bacteria have been isolated from pressurized environments, this relationship merits closer scrutiny. In this study, the extremely thermophilic bacterium, Sulfolobus acidocaldarius, was cultured successfully in a hyperbaric chamber containing helium and air enriched with 5% carbon dioxide. Over a pressure range of approximately 1-120 bar and a temperature range of 67-80 degrees C, growth was achieved in a heterotrophic medium with the air mixture at partial pressures up to 3.5 bar. Helium was used to obtain the final, desired incubation pressure. No significant growth was noted above 80 degrees C over the same range of hyperbaric pressures, or at 70 degrees C when pressure was applied hydrostatically. Growth experiments conducted under hyperbaric conditions may provide a means to study these bacteria under simulated in situ conditions and simultaneously avoid the complications associated with hydrostatic experiments. Results indicate that hyperbaric helium bioreactors will be important in the study of extremely thermophilic bacteria that are isolated from pressurized environments.     

Subunit Cell Wall of Sulfolobus acidocaldarius

The cell wall of Sulfolobus acidocaldarius has been isolated. Cells were mechanically disrupted with a French press, and the cytoplasmic membrane was removed by extracting cell-envelope fragments with Triton X-100. The Triton-insoluble cell wall material retained the characteristic subunit structure when examined in the electron microscope. Isolated cell wall fragments formed in open sheets that were easily separated from cytoplasmic contamination. Chemical studies showed that the Triton-insoluble cell wall fragments consisted of lipoprotein with small amounts of carbohydrate and hexosamine. The amino acid composition indicated a highly charged hydrophobic cell surface. The presence of diaminopimelic acid with only traces of muramic acid indicates that the cell envelope does not have a rigid peptidoglycan layer. The results of chemical analyses and electron microscopy suggest a wall-membrane interaction stabilizing the cell envelope. The chemical and physical properties of this type of cell envelope would appear to form the basis for a new major division of bacteria with the definitive characteristics of a morphologically distinct subunit cell wall devoid of peptidoglycan.     

A plasmid in the archaebacterium Sulfolobus acidocaldarius

A plasmid of mol. wt. ~9 × 10⁶ has been isolated from the archaebacterium Sulfolobus acidocaldarius strain B12. Plasmid production is induced by u.v. radiation. A copy of the plasmid is probably carried by the chromosome, integrated at a specific site. The entire plasmid, and also restriction fragments of it, has been cloned into Escherichia coli plasmid vectors, and the cleavage sites on the plasmid DNA of three restriction endonucleases have been mapped.     

Oxidation of Elemental Sulfur by Sulfolobus acidocaldarius

Oxidation of elemental sulfur by Sulfolobus acidocaldarius, an autotroph which grows at high temperatures and low pH, was examined by use of (35)S-labeled elemental sulfur. When cultured at pH 3.2 and 70 C, S. acidocaldarius oxidized elemental sulfur essentially quantitatively to sulfuric acid. Oxidation rate paralleled growth rate and decrease in pH of the culture medium. Elemental sulfur was not oxidized under these conditions if the culture was poisoned with formaldehyde. During the growth phase, the proportion of cells attached to the sulfur crystals increased progressively, and in the later phases of growth over 10 times more cells were attached to sulfur than were free. Doubling times for eight strains growing on elemental sulfur varied from 37 to 55 h. The organism grows much more rapidly on yeast extract than on sulfur. In a medium containing both sulfur and yeast extract, sulfur oxidation was partially inhibited, although growth was excellent.     

Optimization of pyrite bioleaching using Sulfolobus acidocaldarius

Optimization of batch pyrite bioleaching with Sulfolobus acidocaldarius was performed using statistical modelling and experimental design. First a screening design was made followed by response surface modelling. The dominating factors identified were pH, pulp density and particle size. The highest batch leaching rate after optimization was 270 mg iron·l^–1·h^–1 for 6% (w/v) pulp density, pH = 1.5 and particle size <20 m. This represents a 3.5-fold increase from the leaching rate of 80 mg iron·l^–1·h^–1 obtained under our standard laboratory conditions. Correspondence to: E. B. Lindström     

Metabolism of Pentose Sugars in the Hyperthermophilic Archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, d-xylose and l-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of d-xylose and l-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.     

Physiological adsorption of Sulfolobus acidocaldarius on coal surfaces

Summary The adsorption of Sulfolobus acidocaldarius on bituminous coal surfaces and the respiration rate during adsorption at 70° C were enhanced at pH 1.0–2.0, in comparison with those at pH 3.0–5.0. The maximum number of bacterial cells adsorbed per unit area of coal attained a maximum (1.4 × 10¹¹ cells/m²) at pH 2.0. The rate of desulphurization at pH 2.2–2.5 was higher than at other pHs tested. Micrographs of S. acidocaldarius obtained by TEM and SEM indicated that the cells were adsorbed to the coal surfaces by extracellular slime. Specific inhibitors of membrane-bound ATPase (NaF, 20 mm) and respiration (NaN₃, 1 mm; KCN, 1 mm) had pronounced effects on suppressing adsorption. The amount of S. acidocaldarius adsorbed decreased when the coal particles were leached in advance with 2.0 m HNO₃. These facts lead to the conclusion that the adsorption of S. acidocaldarius on coal surfaces requires physiological activity relatd to respiration or energy conversion. Offprint requests to: V. B. Vitaya     

Cell cycle regulation in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius

The regulation and co-ordination of the cell cycle of the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius was investigated with antibiotics. We provide evidence for a core regulation involving alternating rounds of chromosome replication and genome segregation. In contrast, multiple rounds of replication of the chromosome could occur in the absence of an intervening cell division event. Inhibition of the elongation stage of chromosome replication resulted in cell division arrest, indicating that pathways similar to checkpoint mechanisms in eukaryotes, and the SOS system of bacteria, also exist in archaea. Several antibiotics induced cell cycle arrest in the G2 stage. Analysis of the run-out kinetics of chromosome replication during the treatments allowed estimation of the minimal rate of replication fork movement in vivo to 250 bp s-1. An efficient method for the production of synchronized Sulfolobus populations by transient daunomycin treatment is presented, providing opportunities for studies of cell cycle-specific events. Possible targets for the antibiotics are discussed, including topoisomerases and protein glycosylation.     

Energy flux controls tetraether lipid cyclization in Sulfolobus acidocaldarius

Microorganisms regulate the composition of their membranes in response to environmental cues. Many Archaea maintain the fluidity and permeability of their membranes by adjusting the number of cyclic moieties within the cores of their glycerol dibiphytanyl glycerol tetraether (GDGT) lipids. Cyclized GDGTs increase membrane packing and stability, which has been shown to help cells survive shifts in temperature and pH. However, the extent of this cyclization also varies with growth phase and electron acceptor or donor limitation. These observations indicate a relationship between energy metabolism and membrane composition. Here we show that the average degree of GDGT cyclization increases with doubling time in continuous cultures of the thermoacidophile Sulfolobus acidocaldarius (DSM 639). This is consistent with the behavior of a mesoneutrophile, Nitrosopumilus maritimus SCM1. Together, these results demonstrate that archaeal GDGT distributions can shift in response to electron donor flux and energy availability, independent of pH or temperature. Paleoenvironmental reconstructions based on GDGTs thus capture the energy available to microbes, which encompasses fluctuations in temperature and pH, as well as electron donor and acceptor availability. The ability of Archaea to adjust membrane composition and packing may be an important strategy that enables survival during episodes of energy stress.     

Multiple environmental parameters impact lipid cyclization in Sulfolobus acidocaldarius

Adaptation of lipid membrane composition is an important component of archaeal homeostatic response. Historically, the number of cyclopentyl and cyclohexyl rings in the glycerol dibiphytanyl glycerol tetraether (GDGT) Archaeal lipids has been linked to variation in environmental temperature. However, recent work with GDGT-making archaea highlight the roles of other factors, such as pH or energy availability, in influencing the degree of GDGT cyclization. To better understand the role of multiple variables in a consistent experimental framework and organism, we cultivated the model Crenarchaeon Sulfolobus acidocaldarius DSM639 at different combinations of temperature, pH, oxygen flux, or agitation speed. We quantified responses in growth rate, biomass yield, and core lipid compositions, specifically the degree of core GDGT cyclization. The degree of GDGT cyclization correlated with growth rate under most conditions. The results suggest the degree of cyclization in archaeal lipids records a universal response to energy availability at the cellular level, both in thermoacidophiles, and in other recent findings in the mesoneutrophilic Thaumarchaea. Although we isolated the effects of key individual parameters, there remains a need for multi-factor experiments (e.g., pH + temperature + redox) in order to more robustly establish a framework to better understand homeostatic membrane responses.     

Purification of glycerol dialkyl nonitol tetraether from Sulfolobus acidocaldarius

A modified procedure for extraction and purification of hydrolyzed archaebacterial lipids is described. Lipids were extracted from Sulfolobus acidocaldarius using a Soxhlet extraction procedure followed by trichloroacetic acid solvent-extraction of the residue. The yield of total extractable material by this protocol was 14% which, after a two-phase wash, yielded 10% lipid. Modifications to the published steps for purifying the subsequently hydrolyzed lipids were developed to purify glycerol dialkyl nonitol tetraether (GDNT). The nearly colorless final macrocyclic product was characterized by TLC, IR, NMR, and mass spectrometry.     

Aminoacylation in Sulfolobus acidocaldarius and in methanogenic and halophilic archaebacteria

Abstract Phenylalanyl-tRNA synthetase (PRS) from the sulphur-metabolizing thermoacidophilic archaebacterium Sulfolobus acidocaldarius has been purified 150-fold using different chromatographic steps. The enzyme has a M _r of 270 000 and exhibits considerable thermostability in a temperature range up to 90°C with optimal activity at 70°C. Conservation of antigenic determinants could not be detected by antibodies against various PRS of all primary kingdoms. As a further means to detect traits of phylogenetic relationship, the cross-species reactivity between PRS and tRNAs of organisms from the three branches of archaebacteria and from all primary kingdoms reveals the group character of all 3 branches of the archaebacterial domain, the sulphur-metabolizing, methanogenic and halophilic archaebacteria.     

Energy metabolism of a thermoacidophilic archaebacterium,Sulfolobus acidocaldarius

To elucidate the phylogenic status of the archaebacterium and mechanisms of acidophily, membrane bound ATPase, cytochromes and NADH dehydrogenase of a thermoacidophilic archaebacterium,Sulfolobus acidocaldarius, were studied. Typea cytochrome was found in the membrane. The organism was sensitive to cyanide and azide, and though cytochromec is lacking in this organism, these respiratory poisons inhibited a terminal oxidase, when assayed with cytochromec from other sources. NADH dehydrogenase was highly purified from the crude extract of the cells. The enzyme was able to transfer electrons from NADH to caldariellaquinone, a unique benzothiophenequinone in the genusSulfolobus. Thus, the enzyme is a possible member of the respiratory chain. Membrane fraction contained two types of ATPase, one was active at neutral pH and slightly activated by sulfate; the other was an acid apyrase and inhibited by sulfate. Typical characteristics of F₀F₁ATPase could not be found in these enzymes. These results suggest that (1) the thermoacidophilic archaebacteria are phylogenically distant from both eubacteria and eukaryotes, (2) the archaebacterial thermoacidophiles can be classified in a different subgroup from methanogens and extreme halophiles, and (3) in spite of the aerobic nature of the organism, the energy yielding mechanisms appear quite unique, when compared to those of other aerobes and mitochondria.     

Membrane-bound ATPase of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius

The membranes of Sulfolobus, a thermoacidophilic archaebacterium showed two types of ATP hydrolyzing activity. One was that of a neutral ATPase at an optimum pH around 6.5. This enzyme was activated by 10 mM sulfate with a shift of optimum pH to 5. In these respects, the enzyme was similar to membrane-bound ATPase of Thermoplasma, another thermoacidophilic archaebacterium, reported by Searcy and Whatley [1982) Zbl. Bakt. Hyg., I. Abt. Orig. C3, 245-257). The enzyme hydrolyzed ATP and other NTPs, but not ADP or AMP. It was highly thermostable, but irreversibly inactivated in 0.1 M HCl. The other activity was that of an acidic apyrase at an optimum pH around 2.5. This enzyme was extremely stable toward high temperature and acid and inhibited by sulfate. Both of these ATP hydrolyzing enzymes were resistant to N,N'-dicyclohexylcarbodiimide (DCCD), azide, oligomycin, N'-ethylmaleimide, p-chloromercuribenzoate, orthovanadate, or ouabain. Sulfolobus ATPases differ from F1 and other transport ATPases so far described.