Isolation and characterization of disaggregatase from Methanosarcina mazei LYC

At certain stages in its growth cycle, Methanosarcina mazei produces an enzyme (disaggregatase) that causes aggregates to separate into single cells. M. mazei S-6 and LYC both produce this enzymatic activity, although the specificities of activities differ. The disaggregatase of M. mazei S-6 had little effect on strain LYC cells, but the disaggregatase of M. mazei LYC disaggregated both strain LYC and strain S-6 cells. The disaggregatase of M. mazei LYC was purified by column chromatography, and it apparently consisted of two similar subunits with a combined molecular size of about 180,000 Da. Strain S-6 culture supernatants contained 14 U of activity per liter when activity was measured as uronic acids released from purified cell wall material. When the activity was quantified as the release of uronic acids from boiled M. mazei S-6 cells, the highest activity was found at pH 4.7 and at 35 degrees C.     

Identification of the gene for disaggregatase from Methanosarcina mazei

The gene sequences encoding disaggregatase (Dag), the enzyme responsible for dispersion of cell aggregates of Methanosarcina mazei to single cells, were determined for three strains of M. mazei (S-6^T, LYC and TMA). The dag genes of the three strains were 3234 bp in length and had almost the same sequences with 97% amino acid sequence identities. Dag was predicted to comprise 1077 amino acid residues and to have a molecular mass of 120 kDa containing three repeats of the DNRLRE domain in the C terminus, which is specific to the genus Methanosarcina and may be responsible for structural organization and cell wall function. Recombinant Dag was overexpressed in Escherichia coli and preparations of the expressed protein exhibited enzymatic activity. The RT-PCR analysis showed that dag was transcribed to mRNA in M. mazei LYC and indicated that the gene was expressed in vivo. This is the first time the gene involved in the morphological change of Methanosarcina spp. from aggregate to single cells has been identified.     

Immunochemistry and localization of the enzyme disaggregatase in Methanosarcina mazei.

The enzyme disaggregatase (Dag) from Methanosarcina mazei was studied immunochemically. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified Dag under reducing and nonreducing conditions revealed a single band with a 94-kDa molecular mass. Dag was found to be immunogenic in rabbits; a polyclonal antibody probe was prepared and used to detect the enzyme by slide immunoenzymatic assay, immunofluorescence, and immunoblotting in various species of Methanosarcina known to convert from packets to single cells, including M. mazei. The enzyme could not be detected in other members of the family Methanosarcinaceae that do not convert. By immunogold electron microscopy, Dag was mapped to the cell wall of packets and to the cell membrane of single cells of two M. mazei strains.     

Methanosarcina mazei LYC, a New Methanogenic Isolate Which Produces a Disaggregating Enzyme

A methanogenic coccoid organism, Methanosarcina mazei LYC, was isolated from alkaline sediment obtained from an oil exploration drilling site. The isolate resembled M. mazei S-6 by exhibiting different morphophases during its normal growth cycle. It differed from M. mazei S-6 by undergoint a spontaneous shift from large, irregular aggregates of cells to small, individual, irregular, coccoid units. In batch cultures at pH 7.0, M. mazei LYC grew as aggregates during the early growth stage. As the batch culture began exponential growth, the cell aggregates spontaneously dispersed: the culture liquid became turbid, and myriads of tiny (diameter, 1 to 3 μm) coccoid units were observed under phase-contrast microscopy. Disaggregation apparently was accomplished by the production of an enzyme which hydrolyzed the heteropolysaccharide component of the cell wall; the enzyme was active on other Methanosarcina strains as well. Although the enzyme was active when tested at pH 6.0, it apparently was not produced at that pH: when strain LYC was grown at pH 6.0, only cell aggregates were present throughout batch growth. Individual coccoid cells of M. mazei LYC were sensitive to sodium dodecyl sulfate, but the large aggregates of cells were not. Strain LYC rapidly used H₂-CO₂, in addition to methanol, and mono-, di-, and trimethylamine as methanogenic substrates; acetate was used very slowly. Its optimum growth temperature was 40°C, and its optimum pH was 7.2.     

Spontaneous Disaggregation of Methanosarcina mazei S-6 and Its Use in the Development of Genetic Techniques for Methanosarcina spp

When monomethylamine was the growth substrate, spontaneous disaggregation of Methanosarcina mazei S-6 commenced at the mid-exponential phase and resulted in the formation of a suspension containing 10⁸ to 10⁹ free cells per ml. Free cells were osmotically fragile and amenable to extraction of DNA. Hypertonic media for the manipulation and regeneration of free cells into aggregates were developed, and plating efficiencies of 100% were achieved for M. mazei S-6 and LYC. Free cells of strain S-6 required MgCl₂ (10 mM) for growth, whereas aggregates did not. Specific growth rates of strains S-6 and LYC were increased by MgCl₂. Treatment with pronase caused sphere formation and removal of the protein wall of cells of strain S-6, but protoplasts could not be regenerated. The disaggregating enzyme produced by strain S-6 facilitated the preparation of suspensions of free cells of some strains of Methanosarcina barkeri. Although this provided a means of extracting high-molecular-weight DNA from M. barkeri, less than 0.1% of free cells were viable.     

Life Cycles in the Methanogenic Archaebacterium Methanosarcina mazei

Methanosarcina mazei S6 and LYC were used to study the structure and differentiation of the aggregating methanogens. Cultures harvested under various conditions are described at the ultrastructural level. Cells of strain S6 are enclosed by a layer 12 nm thick in contact with the plasma membrane. In sarcinal colonies, cells are held in close association by a fibrous matrix up to 60 nm thick. Colony maturation was examined in strain S6 over a period of 1 year. Changes occurred in the shape and staining of individual cells. Also, various inclusion bodies were observed that either persist throughout colony maturation or are only found at certain growth stages. Two types of cores that are composed of double membranes in M. mazei S6 are described. One has a 90-nm diameter and contains electron-dense granules similar to those found in the cytoplasm. The other core type does not contain granules, is more numerous, and is found in older cultures. Two life cycles are described for M. mazei based on electron microscope examinations. A complex life cycle involving the release of single cells is described with two variations for strains S6 and LYC. When released cells of strain S6 are placed in fresh medium they can repeat the cycle. In addition, a limited cycle is described for both strains of M. mazei. This limited cycle contains the only sarcinal morphotypes observed in M. barkeri. When M. mazei S6 remains in the limited cycle and does not disaggregate in stationary phase, several types of possible resting forms are found.     

Kinetic study and mathematical modeling of methanogenesis of acetate using pure cultures of methanogens

Kinetics of methanogenesis from acetate was studied using pure cultures of Methanosarcina barkeri and Methanosarcina mazei. Methane formation was found to be associated with cell growth. Nearly equimolar methane was produced from acetate during the methanogenic growth, and about 1.94 g of cells were formed from each mole of acetate consumed. Cell growth can be estimated from methane production. Significant substrate inhibition was found when acetate concentration was higher than 0.12 M. Among the three methanogenic strains studied, M. mazei strain S6 had the highest specific growth rate at all acetate concentrations studied and was least sensitive to environmental factors investigated (e.g., acetate concentration). The maximum specific growth rate found for strain S6 was 0.022 hr(-1) at acetic acid concentration around 7 g/L. The other two strains studied were M. barkeri strain 227 and strain MS. Growth of M. barkeri was completely inhibited at sodium acetate concentrations higher than 0.24 M. The maximum specific growth rate found for strains 227 and MS was 0.019 and 0.021 h(-1) at acetic acid concentrations of 3.6 and 6.8 g/L, respectively. A kinetic model with substrate inhibition was developed and can be used to simulate the methane formation from M. mazei strain S6 grown on acetate at 35 degrees C, pH 7.     

ATP-dependent H+ -pump activity in inverted vesicles of Methanosarcina mazei Gö1 and characterization of membrane ATPase.

ATP-dependent H+ -pump activity was found in inverted vesicles of Methanosarcina mazei Gö1 by using acridine orange as a fluorescent probe. The H+ -pump activity specifically required both Mg and sulfite ions, but azide, an inhibitor of F0F1-ATPase, did not inhibit the activity. The membranes prepared from M. mazei also had an Mg-ATPase activity, and at least the presence of vacuolar-type ATPase was detected.     

Purification and characterization of a beta-glucuronidase from Aspergillus niger.

A beta-glucuronidase from Pectinex Ultra SP-L, a commercial pectolytic enzyme preparation from Aspergillus niger, was purified 170-fold by ion-exchange chromatography and gel filtration. Apparent M(r) of the purified enzyme, estimated by denaturing gel electrophoresis and size-exclusion chromatography, were 68,000 and 71,000, respectively, indicating that the enzyme is a monomeric protein. It released uronic acids not only from p-nitrophenyl beta-glucosiduronic acid (PNP-GlcA) but also from acidic galactooligosaccharides carrying either beta-D-glucosyluronic or 4-O-methyl-beta-D-glucosyluronic residues at the nonreducing termini through beta-(1-->6)-glycosidic linkages. The enzyme exhibited a maximal activity toward these substrates at pH 3.0. A regioisomer, 3-O-beta-glucosyluronic acid-galactose, was unsusceptible to the enzyme. The enzyme did act on a polymer substrate, releasing uronic acid from the carbohydrate portion of a radish arabinogalactan-protein modified by treatment with fungal alpha-L-arabinofuranosidase. The enzyme produced acidic oligosaccharides by transglycosylation, catalyzing the transfer of uronic acid residues of PNP-GlcA and 6-O-beta-glucosyluronic acid-galactose to certain exogenous acceptor sugars such as Gal, N-acetylgalactosamine, Glc, and xylose.     

Purification and characterization of a catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1(T) exhibiting high catalase activity

Catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1(T), which was isolated from an environment exposed to H(2)O(2) and exhibited high catalase activity, was purified and characterized, and its localization in the cell was determined. Its molecular mass was 230 kDa, and the molecule consisted of four identical subunits. The enzyme, which was not apparently reduced by dithionite, showed a Soret peak at 406 nm in a resting state. The catalytic activity was 527,500 U. mg of protein(-1) under standard reaction conditions at 40 degrees C, 1.5 and 4.3 times faster, respectively, than those of the Micrococcus luteus and bovine catalases examined under the same reaction conditions, and showed a broad optimum pH range (pH 6 to 10). The catalase from strain S-1(T) is located not only in the cytoplasmic space but also in the periplasmic space. There is little difference in the activation energy for the activity between strain S-1(T) catalase and M. luteus and bovine liver catalases. The thermoinstability of the activity of the former catalase were significantly higher than those of the latter catalases. The thermoinstability suggests that the catalase from strain S-1(T) should be categorized as a psychrophilic enzyme. Although the catalase from strain S-1(T) is classified as a mammal type catalase, it exhibits the unique enzymatic properties of high intensity of enzymatic activity and thermoinstability. The results obtained suggest that these unique properties of the enzyme are in accordance with the environmental conditions under which the microorganism lives.     

Re-evaluation of the function of the F420 dehydrogenase in electron transport of Methanosarcina mazei

Methanosarcina mazei is a methanogenic archaeon that is able to thrive on various substrates and therefore contains a variety of redox-active proteins involved in both cytoplasmic and membrane-bound electron transport. The organism possesses a complex branched respiratory chain that has the ability to utilize different electron donors. In this study, two knockout mutants of the membrane-bound F(420) dehydrogenase (ΔfpoF and ΔfpoA-O) were constructed and analyzed. They exhibited severe growth deficiencies with trimethylamine, but not with acetate, as substrates. In cell lysates of the fpo mutants, the F(420):heterodisulfide oxidoreductase activity was strongly reduced, although soluble F(420) hydrogenase was still present. This led to the conclusion that the predominant part of cellular oxidation of the reduced form of F(420) (F(420)H(2)) in Ms. mazei is performed by F(420) dehydrogenase. Enzyme assays of cytoplasmic fractions revealed that ferredoxin (Fd):F(420) oxidoreductase activity was essentially absent in the ΔfpoF mutant. Subsequently, FpoF was produced in Escherichia coli and purified for further characterization. The purified FpoF protein catalyzed the Fd:F(420) oxidoreductase reaction with high specificity (the K(M) for reduced Fd was 0.5 μM) but with low velocity (V(max) = 225 mU·mg(-1)) and was present in the Ms. mazei cytoplasm in considerable amounts. Consequently, soluble FpoF might participate in electron carrier equilibrium and facilitate survival of the Ms. mazei Δech mutant that lacks the membrane-bound Fd-oxidizing Ech hydrogenase.     

Extracellular hydroxyproline-rich glycoprotein of suspension-cultured tobacco cells

Hydroxyproline-rich glycoprotein was isolated from the used media of tobacco XD-6 cells cultured in suspension and purified by repeated ion exchange- and gel filtration-chromatography. The preparation was judged to be homogeneous from ultracentrifugation and isoelectric focusing. The dry wt of the glycoprotein was composed of 94% polysaccharide and 6% protein. Hydroxyproline accounted for 23% of the amino acids in the protein moiety. The polysaccharide moiety consisted of 44% galactose, 30% arabinose, 5% rhamnose, and 21% uronic acid. About 10% of the uronic acid residues were present as methyl esters.     

The evaluation of protective effect of lycopene against genotoxic influence of X-irradiation in human blood lymphocytes

Many studies suggest that exogenous antioxidants may protect cells against DNA damage caused with ionizing radiation. One of the most powerful antioxidants is lycopene (LYC), a carotenoid derived from tomatoes. The aim of this study was to investigate, using the comet assay, whether LYC can act as protectors/modifiers and prevent DNA damage induced in human blood lymphocytes, as well as to mitigate the effects of radiation exposure. In this project, LYC, dissolved in DMSO at a concentration of 10, 20 or 40 μM/ml of cell suspension, was added to the isolated lymphocytes from human blood at appropriate intervals before or after the X-irradiation at doses of 0.5, 1 and 2 Gy. Cell viability in all groups was maintained at above 70%. The results showed the decrease of DNA damage in cells treated with various concentrations of LYC directly and 1 h before exposure to X-rays compared to the control group exposed to irradiation alone. Contrary results were observed in cells exposed to LYC immediately after exposure to ionizing radiation. The studies confirmed the protective effect of LYC against DNA damage induced by ionizing radiation, but after irradiation the carotenoid did not stimulate of DNA repair and cannot act as modifier. However, supplementation with LYC, especially at lower doses, may be useful in protection from radiation-induced oxidative damage.     

Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei.

Coenzyme F420 has been assayed by high-performance liquid chromatography with fluorimetric detection; this permits quantification of individual coenzyme F420 analogs whilst avoiding the inclusion of interfering material. The total intracellular coenzyme F420 content of Methanosarcina barkeri MS cultivated on methanol and on H2-CO2 and of Methanosarcina mazei S-6 cultured on methanol remained relatively constant during batch growth. The most abundant analogs in M. barkeri were coenzymes F420-2 and F420-4, whilst in M. mazei coenzymes F420-2 and F420-3 predominated. Significant changes in the relative proportions of the coenzyme F420 analogs were noted during batch growth, with coenzymes F420-2 and F420-4 showing opposite responses to each other and the same being also true for coenzymes F420-3 and F420-5. This suggests that an enzyme responsible for transferring pairs of glutamic acid residues may be active. The degradation fragment FO was also detected in cells in late exponential and stationary phase. Coenzyme F420 analogs were present in the culture supernatant of both methanogens, in similar proportions to that in the cells, except for FO which was principally located in the supernatant.     

Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei

Coenzyme F420 has been assayed by high-performance liquid chromatography with fluorimetric detection; this permits quantification of individual coenzyme F420 analogs whilst avoiding the inclusion of interfering material. The total intracellular coenzyme F420 content of Methanosarcina barkeri MS cultivated on methanol and on H2-CO2 and of Methanosarcina mazei S-6 cultured on methanol remained relatively constant during batch growth. The most abundant analogs in M. barkeri were coenzymes F420-2 and F420-4, whilst in M. mazei coenzymes F420-2 and F420-3 predominated. Significant changes in the relative proportions of the coenzyme F420 analogs were noted during batch growth, with coenzymes F420-2 and F420-4 showing opposite responses to each other and the same being also true for coenzymes F420-3 and F420-5. This suggests that an enzyme responsible for transferring pairs of glutamic acid residues may be active. The degradation fragment FO was also detected in cells in late exponential and stationary phase. Coenzyme F420 analogs were present in the culture supernatant of both methanogens, in similar proportions to that in the cells, except for FO which was principally located in the supernatant.     

General methods for the synthesis of glycopyranosyluronic acid azides

Per-O-acetylated D-glycopyranoses derived from both mono- and disaccharides were first converted to glycosyl iodides and subsequently reacted with an azide source to achieve the stereoselective synthesis of beta-D-glycosyl azides after deacetylation. Low-temperature (4 degrees C) TEMPO oxidation of the monosaccharides provided the corresponding uronic acids, which were purified as the free acids. Oxidation of the lactosyl- and cellobiosyl azides resulted in diacid formation. However, 4',6'-O-benzylidene protection enabled selective oxidation of the C-6 hydroxyl. 2-Acetamido-2-deoxy-D-glycopyranosyl azides were also prepared and converted to uronic acids completing the library synthesis.     

Structural basis of the interspecies interaction between the chaperone DnaK(Hsp70) and the co-chaperone GrpE of archaea and bacteria

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.