Light sensitivity of methanogenic archaebacteria.

Representatives of four families of methanogenic archaebacteria (archaea), Methanobacterium thermoautotrophicum delta H, Methanobacterium thermoautotrophicum Marburg, Methanosarcina acetivorans, Methanococcus voltae, and Methanomicrobium mobile, were found to be light sensitive. The facultative anaerobic eubacteria Escherichia coli and Salmonella typhimurium, however, were tolerant of light when grown anaerobically under identical light conditions. Interference filters were used to show that growth of the methanogens is inhibited by light in the blue end of the visible spectrum (370 to 430 nm).     

Organic osmolytes in methanogenic archaebacteria

Methanogenic archaebacteria have developed unique ways of dealing with osmotic stress. While several of them have transport systems capable of internalizing betaine, an osmolyte in many eubacteria, in general they have developed de novo synthesis of a novel series of beta-amino acids as compatible solutes. 13C-NMR spectroscopy has been the key tool in elucidating both the identity of these organic osmolytes and in investigating their dynamics.     

Distribution of compatible solutes in the halophilic methanogenic archaebacteria.

Accumulation of compatible solutes, by uptake or de novo synthesis, enables bacteria to reduce the difference between osmotic potentials of the cell cytoplasm and the extracellular environment. To examine this process in the halophilic and halotolerant methanogenic archaebacteria, 14 strains were tested for the accumulation of compatible solutes in response to growth in various extracellular concentrations of NaCl. In external NaCl concentrations of 0.7 to 3.4 M, the halophilic methanogens accumulated K+ ion and low-molecular-weight organic compounds. beta-Glutamate was detected in two halotolerant strains that grew below 1.5 M NaCl. Two unusual beta-amino acids, N epsilon-acetyl-beta-lysine and beta-glutamine (3-aminoglutaramic acid), as well as L-alpha-glutamate were compatible solutes among all of these strains. De novo synthesis of glycine betaine was also detected in several strains of moderately and extremely halophilic methanogens. The zwitterionic compounds (beta-glutamine, N epsilon-acetyl-beta-lysine, and glycine betaine) and potassium were the predominant compatible solutes among the moderately and extremely halophilic methanogens. This is the first report of beta-glutamine as a compatible solute and de novo biosynthesis of glycine betaine in the methanogenic archaebacteria.     

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.     

A novel biosynthesis of medium chain length alpha-ketodicarboxylic acids in methanogenic archaebacteria

Gas chromatographic-mass spectrometric analysis on the distribution of alpha-ketodicarboxylic acids in various bacteria determined that alpha-ketoglutarate and alpha-ketoadipate are widely distributed in all the bacteria examined, whereas alpha-ketopimelate and alpha-ketosuberate are found only in the methanogenic archaebacteria. Labeling experiments with stable isotopes indicated that each of these acids arises from alpha-ketoglutarate by repeated alpha-ketoacid chain elongation. The final product in this series of reactions, alpha-ketosuberate, serves in the methanogenic bacteria as the biosynthetic precursor to the 7-mercaptoheptanoic acid portion of 7-mercaptoheptanoylthreonine phosphate, a methanogenic coenzyme.     

N 5-Methyltetrahydromethanopterin: coenzyme M methyltransferase in methanogenic archaebacteria is a membrane protein

An assay is described that allows the direct measurement of the enzyme activity catalyzing the transfer of the methyl group from N ⁵-methyltetrahydromethanopterin (CH₃–H₄MPT) to coenzyme M (H–S–CoM) in methanogenic archaebacteria. With this method the topology, the partial purification, and the catalytic properties of the methyltransferase in methanol- and acetate-grown Methanosarcina barkeri and in H₂/CO₂-grown Methanobacterium thermoautotrophicum were studied. The enzyme activity was found to be associated almost completely with the membrane fraction and to require detergents for solubilization. The transferase activity in methanol-grown M. barkeri was studied in detail. The membrane fraction exhibited a specific activity of CH₃–S–CoM formation from CH₃–H₄MPT (apparent K _m=50 M) and H–S–CoM (apparent K _m=250 M) of approximately 0.6 mol·min^-1·mg protein^-1. For activity the presence of Ti(III) citrate (apparent K _m=15 M) and of ATP (apparent K _m=30 M) were required in catalytic amounts. Ti(III) could be substituted by reduced ferredoxin. ATP could not be substituted by AMP, CTP, GTP, S-adenosylmethionine, or by ATP analogues. The membrane fraction was methylated by CH₃–H₄MPT in the absence of H–S–CoM. This methylation was dependent on Ti(III) and ATP. The methylated membrane fraction catalyzed the methyltransfer from CH₃–H₄MPT to H–S–CoM in the absence of ATP and Ti(III). Demethylation in the presence of H–S–CoM also did not require Ti(III) or ATP. Based on these findings a mechanism for the methyltransfer reaction and for the activation of the enzyme is proposed.Abbreviations H₄MPT tetrahydromethanopterin - CH₃–H₄MPT N ⁵-methyl-H₄MPT - H–S–CoM 2-mercaptoethanesulfonate or coenzyme M - CH₃–S–CoM 2(methylthio)ethanesulfonate or methylcoenzyme M - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - DTT dithiothreitol - MOPS morpholinopropanesulfonate - CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate - 1 U = 1 mol/min     

A general method of isolating high molecular weight DNA from methanogenic archaea (archaebacteria).

High molecular weight DNA was readily isolated from all methanogens treated, as well as from thermophilic anaerobic eubacteria, by grinding cells frozen in liquid N2, prior to lysis with SDS. DNA can subsequently be purified by the usual phenol-chloroform extractions. The procedure yields DNA readily cut by restriction enzymes and suitable for oligonucleotide probing, as well as for mole percent G + C content determination by thermal denaturation. The method routinely yields DNA of high molecular weight and is an improvement over DNA isolation methods for many methanogens, which often involve an initial breakage of the cells in a French pressure cell.     

Vitamin contents of archaebacteria.

The levels of six water-soluble vitamins of seven archaebacterial species were determined and compared with the levels found in a eubacterium, Escherichia coli. Biotin, riboflavin, pantothenic acid, nicotinic acid, pyridoxine, and lipoic acid contents of Halobacterium volcanii, Methanobacterium thermoautotrophicum delta H, "Archaeoglobus fulgidus" VC-16, Thermococcus celer, Pyrodictium occultum, Thermoproteus tenax, and Sulfolobus solfataricus were measured by using bioassays. The archaebacteria examined were found to contain these vitamins at levels similar to or significantly below the levels found in in E. coli. Riboflavin was found at levels comparable to those in E. coli. Pyridoxine was as abundant among the archaebacteria of the methanogenhalophile branch as in E. coli. It was only one-half as abundant in the sulfur-metabolizing branch. "A. fulgidus," however, contained only 4% as much pyridoxine as E. coli. Nicotinic and pantothenic acids were approximately 10-fold less abundant (except for a 200-fold-lower nicotinic acid level in "A. fulgidus"). Nicotinic acid may be replaced by an 8-hydroxy-5-deazaflavin coenzyme (factor F420) in some archaebacteria (such as "A. fulgidus"). Compared with the level in E. coli, biotin was equally as abundant in Thermococcus celer and Methanobacterium thermoautotrophicum, about one-fourth less abundant in P. occultum and "A. fulgidus," and 25 to over 100 times less abundant in the others. The level of lipoic acid was up to 20 times lower in H. volcanii, Methanobacterium thermoautotrophicum, and Thermococcus celer. It was over two orders of magnitude lower among the remaining organisms. With the exception of "A. fulgidus," lipoic acid, pantothenic acid, and pyridoxine were more abundant in the members of the methanogen-halophile branch of the archaebacteria than in the sulfur-metabolizing branch.     

Transsulfuration in archaebacteria.

The transfer of sulfur from methionine to cysteine in the archaebacteria Sulfolobus acidocaldarius and Halobacterium marismortui was studied by feeding 34S-labeled methionine to cells and measuring the incorporation of 34S into protein-bound cellular cysteine and methionine by mass spectrometry. It was found that, as are eucaryotes, both of these archaebacteria were able to convert the sulfur of methionine to cysteine.     

Quinones from archaebacteria, II. Different types of quinones from sulphur-dependent archaebacteria

From the sulphur-dependent, anaerobically grown archaebacterium Sulfolobus ambivalens Caldariella quinone, CQ-6(12H) and the new Sulfolobus quinone SQ-6(12H), 6-(3,7,11,15,19,23-hexamethyltetracosyl)-5-methyl-benz[b]thioph en-4, 7-quinone have been isolated as main components. Lower homologues SQ-5-(10H), SQ-4(8H), SQ-3(6H), phylloquinone-like species CQ-6(10H), SQ-6(10H) and the menaquinone MK-6(12H) are present as minor components. The results are compared with those from Sulfolobus acidocaldarius. Thermococcus celer, Desulfurococcus mucosus and Desulfurococcus mobilis do not contain quinones in comparable amounts.     

C15, C20, and C25 isoprenoid homologues in glycerol diether phospholipids of methanogenic archaebacteria

The glycerol diether phospholipids of 25 monocultures of methanogenic bacteria were isolated and degraded with hydriodic acid. The resulting alkyl iodides were converted to acetate esters and alcohols which were examined using capillary gas-liquid chromatography. The presence of C20 phytanol was observed in accordance with previous studies. Soft fragmentation by chemical ionization mass spectrometry combined with selected ion monitoring enabled the detection, for the first time, of C15 and C25 isoprenologues as components of the diether phospholipids in several strains.     

Use of the level of G+C n DNA for studying the molecular phylogeny o f methanogenic archaebacteria

A new method for theoretical analysis of the molecular phylogeny of bacteria, successfully applied earlier to nitrifying bacteria, was used to study the molecular phylogeny of methanogenic archaebacteria. The group studied included Methanococcus igneus, Methanococcus vannielii, Methanothermus fervidus, Methanolobus tindarius, Methanobacterium formicicum, Methanosarcina barkeri, Methanobacterium thermoformicicum, Methanoplanus limicola, Methanospirillum hungatei, and Methanobacterium thermoautotrophicum. Based on the hypothesis that direct linear regression always exists between evolutionary changes in the DNA G + C content and the primary structure of rRNA, the branching order of the phylogenetic tree of methanogenic archaebacteria was determined. For this tree, the values of the evolutionary distance between 16S rRNA primary structures Ei and the values of the G + C evolutionary distance P(i) exhibited a correlation coefficient 0.78. Thus, the DNA G + C content is not only an important taxonomic characteristics but also provides information helpful for the determination of the branching order of phylogenetic trees constructed based on 16S rRNA primary structures.     

Dihydrolipoamide dehydrogenase from halophilic archaebacteria.

Cadmium-binding proteins in the cytosol of testes from untreated rats were separated by Sephadex G-75 gel filtration. Three major testicular metal-binding proteins (TMBP), or groups of proteins, with relative elution volumes of approx. 1.0 (TMBP-1), 1.7 (TMBP-2) and 2.4 (TMBP-3) were separated. Elution of Zn-binding proteins exhibited a similar pattern. TMBP-3 has previously been thought to be metallothionein (MT), and hence this protein was further characterized and compared with hepatic MT isolated from Cd-treated rats. Estimation of Mr by gel filtration indicated a slight difference between MT (Mr 10000) and TMBP-3 (Mr 8000). Two major forms of MT (MT-I and MT-II) and TMBP-3 (TMBP-3 form I and TMBP-3 form II) were obtained after DEAE-Sephadex A-25 anion-exchange chromatography, with the corresponding subfractions being eluted at similar conductances. Non-denaturing polyacrylamide-gel electrophoresis on 7% acrylamide gels indicated that the subfractions of TMBP-3 had similar mobilities to those of the corresponding subfractions of MT. However, SDS (sodium dodecyl sulphate)/12% (w/v)-polyacrylamide-gel electrophoresis resulted in marked differences in migration of the two corresponding forms of MT and TMBP-3. Co-electrophoresis of MT-II and TMBP-3 form II by SDS/polyacrylamide-gel electrophoresis revealed two distinct proteins. Amino acid analysis indicated much lower content of cysteine in the testicular than in the hepatic proteins. TMBP-3 also contained significant amounts of tyrosine, phenylalanine and histidine, whereas MT did not. U.v.-spectral analysis of TMBP-3 showed a much lower A250/A280 ratio than for MT. Thus this major metal-binding protein in testes, which has been assumed to be MT is, in fact, a quite different protein.     

Pyrite oxidation by thermophilic archaebacteria.

Three species of thermophilic archaebacteria of the genera Sulfolobus (Sulfolobus acidocaldarius and S. solfataricus) and Acidianus (Acidianus brierleyi) were tested for their ability to oxidize pyrite and to grow autotrophically on pyrite, to explore their potential for use in coal desulfurization. Only A. brierleyi was able to oxidize and grow autotrophically on pyrite. Jarosite was formed during the pyrite oxidation, resulting in the precipitation of sulfate and iron. The medium composition affected the extent of jarosite formation.