Levels of water-soluble vitamins in methanogenic and non-methanogenic bacteria

The levels of seven water-soluble vitamins in Methanobacterium thermoautotrophicum, Methanococcus voltae, Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Bacteroides thetaiotaomicron were compared by using a vitamin-requiring Leuconostoc strain. Both methanogens contained levels of folic acid and pantothenic acid which were approximately two orders of magnitude lower than levels in the nonmethanogens. Methanobacterium thermoautotrophicum contained levels of thiamine, biotin, nicotinic acid, and pyridoxine which were approximately one order of magnitude lower than levels in the nonmethanogens. The thiamine level in Methanococcus voltae was approximately one order of magnitude lower than levels in the nonmethanogens. Only the levels of riboflavin (and nicotinic acid and pyridoxine in Methanococcus voltae) were approximately equal in the methanogens and nonmethanogens. Folic acid may have been present in extracts of methanogens merely as a precursor, by-product, or hydrolysis product of methanopterin.     

Ribose biosynthesis in methanogenic bacteria

NMR spectroscopy was used to determine the labeling patterns of the ribose moieties of ribonucleosides purified from Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii labeled with ¹³C-precursors. In most methanogens tested ribose was labeled in a manner consistent with the operation of the oxidative branch of the pentose phosphate pathway. In contrast, transaldolase and transketolase reactions typical of a partial nonoxidative pentose phosphate pathway are hypothesized to explain the different labeling patterns and enrichments of carbon atoms observed in the ribose moiety of Methanococcus voltae. The source of erythrose 4-phosphate needed for the transaldolase reaction proposed in Methanococcus voltae, and for biosynthesis of aromatic amino acids in methanogenic bacteria in general, was assessed. Phenylalanine carbon atom C-7 was labeled by [1-¹³C]pyruvate in Methanospirillum hungatei, Methanococcus voltae, and Methanococcus jannaschii, the only methanogens which incorporated sufficient label from pyruvate for testing. Reductive carboxylation of a triose precursor (derived from pyruvate) to synthesize erythrose 4-phosphate is consistent with the labeling patterns observed in phenylalanine and ribose.Abbreviation TCA Tricarboxylic acid Issued as NRCC Publication No. 37382     

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.     

Distribution of coenzyme F420 and properties of its hydrolytic fragments.

The ability of hydrolytic products of coenzyme F420 to substitute for F420 in the hydrogenase and nicotinamide adenine dinucleotide phosphate-liniked hydrogenase systems of Methanobacterium strain M.o.H. was kinetically determined. The nicotinamide adenine dinucleotide phosphate-linked hydrogenase system was employed to quantitate the levels of F420 in a number of methanogenic bacteria as well as in some nonmethanogens. Methanobacterium ruminantium and Methanosarcina barkeri contained low levels of F420, whereas other methanogens tested contained high levels (100 to 400 mg/kg of cells). F420 from six of the seven methanogens was tested by thin-layer electrophoresis and was found to be electrophoretically identical to that purified from Methanobacterium strain M.o.H. The only exception was M. barkeri, which contained a more electronegative derivative of F420. Acetobacterium woodii, Escherichia coli, and yeast extract contained no compounds able to substitute for F420 in the nicotinamide adenine dinucleotide phosphate-linked hydrogenase system.     

Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts

The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per μg of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per μg of vector DNA. However, a 380-fold improvement (3,417 transformants per μg of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.     

Enzyme-Linked Immunosorbent Assays for the Specific and Sensitive Quantification of Methanosarcina mazei and Methanobacterium bryantii

Three microtitration plate enzyme-linked immunosorbent assays (ELISAs) have been developed: a competitive ELISA and a two-site (or indirect sandwich) ELISA for Methanosarcina mazei S6 and a two-site ELISA for Methanobacterium bryantii FR-2. The assays were sensitive, with limits of cell protein detection of 3 ng ml⁻¹, 5 ng ml⁻¹, and 50 ng ml⁻¹, respectively, and showed good precision. The M. mazei assays used monoclonal antibodies and were entirely species specific, showing no cross-reaction with methanogens of other genera or with other species of the same genus. The Methanobacterium bryantii assay, which used two polyclonal antisera, showed only a slight cross-reaction with one other Methanobacterium species but no cross-reaction with methanogens of other genera. The use of the ELISAs for quantitative analysis of mixed cultures and of sewage sludge samples was investigated. Sludge diluted at 1:10³ or more caused no significant interference in any of the three ELISAs. Various cultures of bacteria, methanogens, and nonmethanogens at a protein concentration of 50 μg ml⁻¹ showed no significant interference in the M. mazei competitive assay and the Methanobacterium bryantii two-site assay, although they did cause falsely low results in the M. mazei two-site assay.     

Defective formation and/or utilization of carbon monoxide in H2/CO2 fermenting methanogens dependent on acetate as carbon source

Autotrophic methanogens reduce CO₂ to CO and assimilate CO in a carbonylation reaction. Heterotrophic species were found not to form CO and/or to incorporate CO into cell matiral. The absence of CO formation correlated with the absence of carbon monoxide dehydrogenase activity. The heterotrophic Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanococcus voltae and Methanospirillum hungatei (strain GP 1) were investigated.     

Production of Ethane, Ethylene, and Acetylene from Halogenated Hydrocarbons by Methanogenic Bacteria

Several methanogenic bacteria were shown to produce ethane, ethylene, and acetylene when exposed to the halogenated hydrocarbons bromoethane, dibromo- or dichloroethane, and 1,2-dibromoethylene, respectively. They also produced ethylene when exposed to the coenzyme M analog and specific methanogenic inhibitor bromoethanesulfonic acid. The production of these gases from halogenated hydrocarbons has a variety of implications concerning microbial ecology, agriculture, and toxic waste treatment. All halogenated aliphatic compounds tested were inhibitory to methanogens. Methanococcus thermolithotrophicus, Methanococcus deltae, and Methanobacterium thermoautotrophicum ΔH and Marburg were completely inhibited by 7 μM 1,2-dibromoethane and, to various degrees, by 51 to 1,084 μM 1,2-dichloroethane, 1,2-dibromoethylene, 1,2-dichloroethylene, and trichloroethylene. In general, the brominated compounds were more inhibitory. The two Methanococcus species were fully inhibited by 1 μM bromoethanesulfonic acid, whereas both Methanobacterium strains were only partly inhibited by 2,124 μM. Coenzyme M protected cells from bromoethanesulfonic acid but not from any of the other inhibitors.     

Purine and pyrimidine biosynthesis in methanogenic bacteria

Following long-term labeling with [1-¹³C]acetate, [2-¹³C]acetate, ¹³CO₂, H¹³COOH, or ¹³CH₃OH, NMR spectroscopy was used to determine the labeling patterns of the purified ribonucleosides of Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii. Major differences were observed among the methanogens studied, specifically at carbon positions 2 and 8 of the purines, positions at which one-carbon carriers are involved during synthesis. In Methanospirillum hungatei and Methanosarcina barkeri, the labcl at both positions came from carbon atom C-2 of acetate, as predicted from known eubacterial pathways, whereas in Methanococcus voltae and Methanobacterium bryantii both originated from CO₂. In Methanosphaera stadtmanae grown in the presence of formate, the C-2 of purines originated exclusively from formate and the C-8 was labeled by the C-2 of acetate. When grown in media devoid of formate, the C-2 of the purine ring originated mainly from the C-2 of acetate and in part from CH₃OH. In Methanobrevibacter smithii grown in the presence of formate, C-2 and C-8 of purines were derived from CO₂ and/or formate. The labeling patterns obtained for pyrimidines are consistent with the biosynthetic pathways common to eubacteria and eucaryotes.Abbreviations CODH Carbon monoxide dehydrogenase - FH₄ tetrahydrofolate - H₄MPT tetrahydromethanopterin Issued as NRCC Publication No. 37383     

Diversity and Population Dynamics of Methanogenic Bacteria in a Granular Consortium

Upflow anaerobic sludge blanket bioreactor granules were used as an experimental model microbial consortium to study the dynamics and distribution of methanogens. Immunologic methods revealed a considerable diversity of methanogens that was greater in mesophilic granules than in the same granules 4 months after a temperature shift from 38 to 55°C. During this period, the sizes of the methanogenic subpopulations changed with distinctive profiles after the initial reduction caused by the shift. Methanogens antigenically related to Methanobrevibacter smithii PS and ALI, Methanobacterium hungatei JF1, and Methanosarcina thermophila TM1 increased rapidly, reached a short plateau, and then fell to lower concentrations that persisted for the duration of the experiment. A methanogen related to Methanogenium cariaci JR1 followed a similar profile at the beginning, but it soon diminished below detection levels. Methanothrix rods weakly related to the strain Opfikon increased rapidly, reaching a high-level, long-lasting plateau. Two methanogens related to Methanobrevibacter arboriphilus AZ and Methanobacterium thermoautotrophicum ΔH emerged from very low levels before the temperature shift and multiplied to attain their highest numbers 4 months after the shift. Histochemistry and immunohistochemistry revealed thick layers, globular clusters, and lawns of variable density which were distinctive of the methanogens related to M. thermoautotrophicum ΔH, M. thermophila TM1, and M. arboriphilus AZ and M. soehngenii Opfikon, respectively, in thin sections of granules grown at 55°C for 4 months. Mesophilic granules showed a different pattern of methanogenic subpopulations.     

AglC and AglK are involved in biosynthesis and attachment of diacetylated glucuronic acid to the N-glycan in Methanococcus voltae

Recent advances in the field of prokaryotic N-glycosylation have established a foundation for the pathways and proteins involved in this important posttranslational protein modification process. To continue the study of the Methanococcus voltae N-glycosylation pathway, characteristics of known eukaryotic, bacterial, and archaeal proteins involved in the N-glycosylation process were examined and used to select candidate M. voltae genes for investigation as potential glycosyl transferase and flippase components. The targeted genes were knocked out via linear gene replacement, and the resulting effects on N-glycan assembly were identified through flagellin and surface (S) layer protein glycosylation defects. This study reports the finding that deletion of two putative M. voltae glycosyl transferase genes, designated aglC (for archaeal glycosylation) and aglK, interfered with proper N-glycosylation. This resulted in flagellin and S-layer proteins with significantly reduced apparent molecular masses, loss of flagellar assembly, and absence of glycan attachment. Given previous knowledge of both the N-glycosylation pathway in M. voltae and the general characteristics of N-glycosylation components, it appears that AglC and AglK are involved in the biosynthesis or transfer of diacetylated glucuronic acid within the glycan structure. In addition, a knockout of the putative flippase candidate gene (Mv891) had no effect on N-glycosylation but did result in the production of giant cells with diameters three to four times that of wild-type cells.     

Role of Amino Acids and Vitamins in Nutrition of Mesophilic Methanococcus spp

In this study we found that autotrophic methanococci similar to Methanococcus maripaludis obtained up to 57% of their cellular carbon from exogenous amino acids. About 85% of the incorporation was into protein. Primarily nonpolar and basic amino acids and glycine were incorporated; only small amounts of acidic and some polar amino acids were taken up. An additional 10% of the incorporation was into the nucleic acid fraction. Because little ¹⁴CO₂ was formed from the ¹⁴C-amino acids, little metabolism of the amino acids occurred. Therefore the growth stimulation by amino acids was probably due to the sparing of anabolic energy requirements. Of the amino acids incorporated, only alanine was also a sole nitrogen source for these methanococci. In contrast, Methanococcus vannielii and “Methanococcus aeolicus” are autotrophic methanococci which did not incorporate amino acids and did not utilize alanine as a sole nitrogen source. Although glutamine served as a sole nitrogen source for the autotrophic methanococci and Methanococcus voltae, a heterotrophic methanococcus, growth was due to chemical deamination in the medium. M. voltae requires leucine and isoleucine for growth. However, these amino acids were not significant nitrogen sources, and alanine was not a sole nitrogen source for the growth of M. voltae. The branched-chain amino acids were not extensively metabolized by M. voltae. Pantoyl lactone and pantoic acid were readily incorporated by M. voltae. The intact vitamin pantothenate was neither stimulatory to growth nor incorporated. In conclusion, although amino acids and vitamins are nutritionally important to both autotrophic and heterotrophic methanococci, generally they are not subject to extensive catabolism.     

Relatedness of the flagellins from methanogens

Purified flagellar filaments isolated from six methanogens were composed of multiple flagellins. Two flagellins were present in Methanococcus deltae (M _r =34000 and 32000), Methanoculleus marisnigri (M _r =31000 and 25500) and Methanococcus jannaschii (M _r =31000 and 27500), three in Methanothermus fervidus (M _r =34000, 25000 and 24000) and four or more in both Methanococcus vanniellii and Methanococcus maripaludis (M _r ranging from 27500 to 32000). The flagellins of M. fervidus and M. deltae reacted positively with glycoprotein-specific stains. The flagellins of M. deltae, M. maripaludis and M. vannielii were closely related to those of M. voltae based on cross-reactivity with antisera raised against M. voltae flagellins and homology with flagellin-specific oligonucleotide probes to the N-terminus and leader peptide of M. voltae flagellins. Similarities appear to exist among the flagellins of M. fervidus, M. marisnigri and Halobacterium halobium based on cross-reactivity with antisera produced against the flagella of Methanospirillum hungatei JF1. The N-termini of the flagellins from the mesophilic Methanococcus spp. and M. marisnigri show homology with the N-termini of other archaebacterial flagellins. These N-termini may undergo a modification involving removal of a leader peptide.     

Role of 2,4-dichlorophenoxyacetic acid and of myoinositol in the formation of the callus on excised roots ofSolanum laciniatum AIT

Exeised roots ofSolanum laciniatum Ait. grown in vitro in a liquid medium will form the typical rich white callus with a high water content. Its formation is made possible by the presence of 2,4-dichlorophenoxyacetic acid and niyo-inositol in the nutrient medium. Choline, ascorbic acid, riboflavin, calcium pantothenate and biotin are inactive. A mixture of thiamine, pyridoxine and nicotinic acid will induce only slight proliferation.     

Dienoic acid contents of rats with linoleic acid and pyridoxine deficiencies

Low dienoic acid content of the tissues of rats has been produced in two ways: (a) withdrawal of essential fatty acid from the diet, and (b) exclusion of pyridoxine or thiamine from the diet or severe restriction of food (caloric deficiency).By the first method dienoic acid content of the tissues fell to lower levels, and typical symptoms of the deficiency were produced. By the second procedure, the fall was more rapid in the acute vitamin deficiencies but no symptoms characteristic of linoleic acid deficiency occurred. A similar but less rapid course was followed in caloric restriction.Since no essential difference in dienoic acid content of the tissues of rats was found under these several dietary conditions, it was concluded that pyridoxine under the conditions of these experiments does not exert a special supplementary action in the production or conservation of polyenoic fatty acids.     

Withania somnifera (Ashwagandha): Regeneration through Meristem Culture

High frequency of callus induction was achieved from shoot tip explants (80%) of Withania somnifera (Hindi name-Ashwagandha) as compared to leaf explants (70%), on MS medium supplemented with IAA (56μM) and Kinetin (56μM). When hormone-free MS medium was fortified with vitamins [thiamine. HCI (3μM), nicotinic acid (40.7μM) and pyridoxine. HCI (24.3μM)], it was found suitable for complete plant regeneration through meristem culture.     

Immunocytochemical localization of methyl-coenzyme M reductase in Methanobacterium thermoautotrophicum

Cells of Methanobacterium thermoautotrophicum were fixed with glutaraldehyde, sectioned and labeled with antibodies against the subunit of component C (=methyl-CoM reductase) of methyl-CoM reductase system and with colloidal gold-labeled protein A. It was found that the gold particles were located predominantly in the vicinity of the cytoplasmic membrane, when the cells were grown under conditions where methyl-CoM reductase was not overproduced. This finding confirms the recent data obtained with Methanococcus voltae showing via the same immunocytochemical localization technique that in this organism methyl-CoM reductase is membrane associated.     

维生素在丙酮酸过量合成中的重要作用

研究了烟酸、硫胺素、吡哆醇、生物素和核黄素对一株光滑球拟酵母(\%Torulopsis glabrata\%) WSH\|IP303以葡萄糖为碳源、以氯化铵为唯一氮源生产丙酮酸的影响。利用正交试验方法,确证了硫胺素是影响WSH\|IP303生产丙酮酸的最重要因素。在硫胺素浓度一定(0.01～0.015mg/L)的前提下,提高烟酸浓度有助于加快耗糖速度。当烟酸、硫胺素、吡哆醇、生物素和核黄素的浓度分别为8、0.015、0.4、0.04和01mg/L时,摇瓶发酵48h,丙酮酸产量和产率可分别达到52.4g/L和0525g/g。采用优化的维生素组合方式,进行2.5L罐分批发酵,在初糖浓度120g/L的条件下发酵57.5h,丙酮酸产量和产率分别达到69.4g/L和0593g/g,分别比摇瓶培养的最好结果提高了32.%和13%。     

Cloning and characterization of archaeal type I signal peptidase from Methanococcus voltae

Archaeal protein trafficking is a poorly characterized process. While putative type I signal peptidase genes have been identified in sequenced genomes for many archaea, no biochemical data have been presented to confirm that the gene product possesses signal peptidase activity. In this study, the putative type I signal peptidase gene in Methanococcus voltae was cloned and overexpressed in Escherichia coli, the membranes of which were used as the enzyme source in an in vitro peptidase assay. A truncated, His-tagged form of the M. voltae S-layer protein was generated for use as the substrate to monitor the signal peptidase activity. With M. voltae membranes as the enzyme source, signal peptidase activity in vitro was optimal between 30 and 40°C; it was dependent on a low concentration of KCl or NaCl but was effective over a broad concentration range up to 1 M. Processing of the M. voltae S-layer protein at the predicted cleavage site (confirmed by N-terminal sequencing) was demonstrated with the overexpressed archaeal gene product. Although E. coli signal peptidase was able to correctly process the signal peptide during overexpression of the M. voltae S-layer protein in vivo, the contribution of the E. coli signal peptidase to cleavage of the substrate in the in vitro assay was minimal since E. coli membranes alone did not show significant activity towards the S-layer substrate in in vitro assays. In addition, when the peptidase assays were performed in 1 M NaCl (a previously reported inhibitory condition for E. coli signal peptidase I), efficient processing of the substrate was observed only when the E. coli membranes contained overexpressed M. voltae signal peptidase. This is the first proof of expressed type I signal peptidase activity from a specific archaeal gene product.     

Efficient pyruvate production by a multi-vitamin auxotroph of Torulopsis glabrata: key role and optimization of vitamin levels

A multi-vitamin auxotroph, Torulopsis glabrata strain WSH-IP303, which can use ammonium chloride as a sole nitrogen source for pyruvate production, was selected. To optimize pyruvate yield and productivity, a simple but useful, orthogonal design method, was used to investigate the relationship between thiamine, nicotinic acid, pyridoxine, biotin, and riboflavin. Thiamine was confirmed to be the most important factor affecting pyruvate production. When the concentration of thiamine was 0.01 mg/l or 0.015 mg/l, glucose consumption was improved by increasing the nicotinic acid concentration. When the concentrations of nicotinic acid, thiamine, pyridoxine, biotin, and riboflavin were 8.0, 0.015, 0.4, 0.04, and 0.1 mg/l, respectively, pyruvate concentration and yield reached 52 g/l and 0.52 g/g, respectively, in a 48-h flask culture. By employing a combination of the optimum vitamin concentrations, a batch culture was conducted in a 2.5-l fermentor with an initial glucose concentration of 112 g/l; and the pyruvate concentration reached 69 g/l after 56 h (yielding 0.62 g/g).