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.     

Comparative lipid composition of heterotrophically and autotrophically grown Sulfolobus acidocaldarius.

Complex lipids from the thermoacidophilic facultative autotroph Sulfolobus acidocaldarius, as well as a strictly autotrophic isolate, were compared between cells grown on yeast extract and elemental sulfur. Lipids from both organisms grown autotrophically were nearly identical. Each contained about 15% neutral lipids, 35% glycolipids, and 50% acidic lipids. Glycolipids and acidic lipids contained C40H82-76-derived glycerol ether residues. Major glycolipids included the glycerol ether analogues of glucosyl galactosyl diglyceride (5%) and glucosyl polyol diglyceride (75%). Acidic lipids were comprised mainly of the glycerol ether analogues of phosphatidyl inositol (7%), inositolphosphoryl glucosyl polyol diglyceride (72%), and a partially characterized sulfate- and phosphate-containing derivative of glucosyl polyol diglyceride (13%). The lipids from cells grown heterotrophically were similar to those from autotrophically grown cells, except that the partially characterized acidic lipid was absent. In addition, the two glycolipids as well as the respective inositolphosphoryl derivatives were each present in nearly equal proportions.     

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 Rates of Sulfolobus acidocaldarius in Nature

Turnover times for water passing through several Sulfolobus acidocaldarius-containing springs were determined by measuring the dilution rates of small amounts of sodium chloride that were added to the springs. Chloride was diluted out exponentially, while concentrations of the bacteria remained constant. Additionally, temperature, pH, and chemical composition of the springs also remained constant during the time that the chloride was being diluted. The springs are thus steady-state systems, and since the rates of bacterial growth must be at least equal to the chloride dilution rates, minimal doubling times for the bacterial populations can be calculated. Half-times for chloride dilution, equivalent to bacterial doubling times, were on the order of 10 to 20 h for springs ranging in volume from about 20 to 2,000 liters, but approximately 30 days for two larger springs of about 1 million liters. Formaldehyde-fixed cells of a serologically distinguishable strain of S. acidocaldarius were also added as markers to four of the smaller springs, and the dilution rates of these bacteria were compared with the chloride dilution rates. The rates agreed reasonably well, thus verifying the growth rates obtained from the chloride dilution rates. In three springs, exponential growth was studied by draining the springs and allowing them to refill with bacteria-free water. Exponential doubling times were on the order of a few hours, much more rapid than steady-state doubling times. The methods used in this work may have wider utility in aquatic environments.     

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.     

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.     

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     

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.     

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.     

Electromechanical stability of planar lipid membranes from bipolar lipids of the thermoacidophilic archebacterium Sulfolobus acidocaldarius

Stable planar membranes have been obtained from the bipolar lipid glycerol dialkyl nonitol tetraether (GDNT) extracted from the thermoacidophilic archebacterium Sulfolobus acidocaldarius. The electric capacity Cm, the resistance Rm and tension sigma of these membranes were measured. The dependence of the bipolar lipid membranes mean life time tau 1 on voltage was investigated. It was shown that the irreversible electric breakdown of membranes from GDNT and usual phospholipids is due to the same mechanism, viz., due to formation of a hydrophilic pore with an overcritical radius. Under electric field the GDNT molecules take U-shape, and the polar headgroups of such molecules cover the pore's interior.     

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.     

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     

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.     

Circular chromosomal DNA in the sulfur-dependent archaebacterium Sulfolobus acidocaldarius.

The shape of the chromosomal DNA of the sulfur-dependent archaebacterium Sulfolobus acidocaldarius was analyzed by the pulsed-field gel electrophoresis(PFGE). S.acidocaldarius DNA digested with Notl showed two DNA bands at around 1.0 Mbp and 2.1 Mbp. Notl-linking clones were isolated from the library of S.acidocaldarius chromosomal DNA. It contained two Notl sites. Both 1.0 and 2.1 Mbp DNA band separated by PFGE were hybridized with the two independent Notl-linking fragment. Each right and left arms of two Notl-linking fragments were hybridized with one of the two DNA bands separated by PFGE. The results indicated that the chromosomal DNA of S.acidocaldarius is circular.     

Glycogen-bound polyphosphate kinase from the archaebacterium Sulfolobus acidocaldarius.

Glycogen-bound polyphosphate kinase has been isolated from a crude extract of Sulfolobus acidocaldarius by isopycnic centrifugation in CsCl. Divalent cations (Mn2+ greater than Mg2+) stimulated the reaction. The enzyme does not require the presence of histones for its activity; it is inhibited strongly by phosphate and slightly by fluoride. The protein from the glycogen complex migrated in a sodium dodecyl sulfate-polyacrylamide gel as a 57-kilodalton protein band; after isoelectric focusing it separated into several spots in the pH range of 5.6 to 6.7.     

Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius

Central to genetic work in any organism are the availability of a range of inducible and constitutive promoters. In this work we studied several promoters for use in the hyperthermophilic archaeon Sulfolobus acidocaldarius. The promoters were tested with the aid of an E. coli–Sulfolobus shuttle vector in reporter gene experiments. As the most suitable inducible promoter a maltose inducible promoter was identified. It comprises 266 bp of the sequence upstream of the gene coding for the maltose/maltotriose binding protein (mbp, Saci_1165). Induction is feasible with either maltose or dextrin at concentrations of 0.2–0.4%. The highest increase in expression (up to 17-fold) was observed in late exponential and stationary phase around 30–50 h after addition of dextrin. Whereas in the presence of glucose and xylose higher basal activity and reduced inducibility with maltose is observed, sucrose can be used in the growth medium additionally without affecting the basal activity or the inducibility. The minimal promoter region necessary could be narrowed down to 169 bp of the upstream sequence. The ABCE1 protein from S. solfataricus was successfully expressed under control of the inducible promoter with the shuttle vector pC and purified from the S. acidocaldarius culture with a yield of about 1 mg L⁻¹ culture. In addition we also determined the promoter strength of several constitutive promoters.     

Electron transport and energy conservation in the archaebacterium Sulfolobus acidocaldarius

Abstract The bioenergetic properties of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius are reviewed and discussed under the aspect whether this archaebacterium conserved energy by oxidative phosphorylation and how the involved catalysts are related to those from eubacteria and eukaryotes. The thermodynamic parameters contributing to the proton-motive force and the efficiency of proton pumping are presented. The major components of the electron transport system are identified and a novel type of heme- aa ₃ containing terminal oxidase is described, oxidizing reduced caldariella quinone. The properties of an F ₁-analogous ATPase and of a DCCD-binding proteolipid from the plasmamembrane of Sulfolobus are discussed as likely components of an F ₀ F ₁-analogous ATP-synthase. The structural and functional properties of this and other archaebacterial ATPases are compared to each other and with respect to evolutionary relations.