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1.
FEMS Microbiol Lett ; 367(17)2020 09 01.
Article in English | MEDLINE | ID: mdl-32840570

ABSTRACT

In microbial corrinoid-dependent methyltransferase systems, adventitious Co(I)-corrinoid oxidation halts catalysis and necessitates repair by ATP-dependent reductive activases. RamA, an activase with a C-terminal ferredoxin domain with two [4Fe-4S] clusters from methanogenic archaea, has been far less studied than the bacterial activases bearing an N-terminal ferredoxin domain with one [2Fe-2S] cluster. These differences suggest RamA might prove to have other distinctive characteristics. Here, we examine RamA kinetics and the stoichiometry of the corrinoid protein:RamA complex. Like bacterial activases, K+ stimulates RamA. Potassium stimulation had been questioned due to differences in the primary structure of bacterial and methanogen activases. Unlike one bacterial activase, ATP is not inhibitory allowing the first determination of apparent kinetic parameters for any corrinoid activase. Unlike bacterial activases, a single RamA monomer complexes a single corrinoid protein monomer. Alanine replacement of a RamA serine residue corresponding to the serine of one bacterial activase which ligates the corrinoid cobalt during complex formation led to only moderate changes in the kinetics of RamA. These results reveal new differences in the two types of corrinoid activases, and provide direct evidence for the proposal that corrinoid activases act as catalytic monomers, unlike other enzymes that couple ATP hydrolysis to difficult reductions.


Subject(s)
Archaeal Proteins/metabolism , Methanosarcina barkeri/enzymology , Tissue Plasminogen Activator/metabolism , Archaeal Proteins/genetics , Enzyme Activation/drug effects , Kinetics , Methanosarcina barkeri/drug effects , Potassium/pharmacology , Tissue Plasminogen Activator/genetics
2.
Colloids Surf B Biointerfaces ; 143: 518-525, 2016 Jul 01.
Article in English | MEDLINE | ID: mdl-27038917

ABSTRACT

The retention of selective biofilms of Methanosarcina species within anaerobic digesters could reduce start-up times and enhance the efficiency of the process in treating high-strength domestic sewage. The objective of the study was to examine the effect of the surface characteristics of six common polymer support materials on the initial adhesion of the model methanogen, Methanosarcina barkeri, and to assess the potential of these support materials as selective biofilm carriers. Results from both the initial adhesion tests and extended DLVO (xDLVO) model correlated with each other, with PVC (12% surface coverage/mm(2)), PTFE (6% surface coverage/mm(2)), and PP (6% surface coverage/mm(2)), shown to be the better performing support materials for initial adhesion, as well as subsequent biofilm formation by M. barkeri after 72h. Experimental results of these three support materials showed that the type of material strongly influenced the extent of adhesion from M. barkeri (p<0.0001), and the xDLVO model was able to explain the results in these environmental conditions. Therefore, DLVO physicochemical forces were found to be influential on the initial adhesion of M. barkeri. Scanning electron microscopy suggested that production of extracellular polymeric substances (EPS) from M. barkeri could facilitate further biofilm development. This study highlights the potential of using the xDLVO model to rapidly identify suitable materials for the selective adhesion of M. barkeri, which could be beneficial in both the start-up and long-term phases of anaerobic digestion.


Subject(s)
Bacterial Adhesion/drug effects , Biofilms/drug effects , Methanosarcina barkeri/drug effects , Polypropylenes/pharmacology , Polytetrafluoroethylene/pharmacology , Polyvinyl Chloride/pharmacology , Anaerobiosis/physiology , Biodegradation, Environmental , Biofilms/growth & development , Bioreactors , Methanosarcina barkeri/growth & development , Polypropylenes/chemistry , Polytetrafluoroethylene/chemistry , Polyvinyl Chloride/chemistry , Sewage/microbiology , Surface Properties
3.
Appl Environ Microbiol ; 78(21): 7596-602, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22904062

ABSTRACT

In natural environments methane is usually produced by aceticlastic and hydrogenotrophic methanogenic archaea. However, some methanogens can use C(1) compounds such as methanol as the substrate. To determine the contributions of individual substrates to methane production, the stable-isotope values of the substrates and the released methane are often used. Additional information can be obtained by using selective inhibitors (e.g., methyl fluoride, a selective inhibitor of acetoclastic methanogenesis). We studied stable carbon isotope fractionation during the conversion of methanol to methane in Methanosarcina acetivorans, Methanosarcina barkeri, and Methanolobus zinderi and generally found large fractionation factors (-83‰ to -72‰). We further tested whether methyl fluoride impairs methylotrophic methanogenesis. Our experiments showed that even though a slight inhibition occurred, the carbon isotope fractionation was not affected. Therefore, the production of isotopically light methane observed in the presence of methyl fluoride may be due to the strong fractionation by methylotrophic methanogens and not only by hydrogenotrophic methanogens as previously assumed.


Subject(s)
Methane/biosynthesis , Methanol/metabolism , Methanosarcina barkeri/metabolism , Carbon Isotopes , Hydrocarbons, Fluorinated/pharmacology , Methanosarcina barkeri/drug effects
4.
Antimicrob Agents Chemother ; 54(1): 267-72, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19822707

ABSTRACT

The recently discovered antibacterial compound alaremycin, produced by Streptomyces sp. A012304, structurally closely resembles 5-aminolevulinic acid, the substrate of porphobilinogen synthase. During the initial steps of heme biosynthesis, two molecules of 5-aminolevulinic acid are asymmetrically condensed to porphobilinogen. Alaremycin was found to efficiently inhibit the growth of both Gram-negative and Gram-positive bacteria. Using the newly created heme-permeable strain Escherichia coli CSA1, we are able to uncouple heme biosynthesis from bacterial growth and demonstrate that alaremycin targets the heme biosynthetic pathway. Further studies focused on the activity of alaremycin against the opportunistic pathogenic bacterium Pseudomonas aeruginosa. The MIC of alaremycin was determined to be 12 mM. Alaremycin was identified as a direct inhibitor of recombinant purified P. aeruginosa porphobilinogen synthase and had a K(i) of 1.33 mM. To understand the molecular basis of alaremycin's antibiotic activity at the atomic level, the P. aeruginosa porphobilinogen synthase was cocrystallized with the alaremycin. At 1.75-A resolution, the crystal structure reveals that the antibiotic efficiently blocks the active site of porphobilinogen synthase. The antibiotic binds as a reduced derivative of 5-acetamido-4-oxo-5-hexenoic acid. The corresponding methyl group is, however, not coordinated by any amino acid residues of the active site, excluding its functional relevance for alaremycin inhibition. Alaremycin is covalently bound by the catalytically important active-site lysine residue 260 and is tightly coordinated by several active-site amino acids. Our data provide a solid structural basis to further improve the activity of alaremycin for rational drug design. Potential approaches are discussed.


Subject(s)
Aminocaproates/pharmacology , Anti-Bacterial Agents/pharmacology , Heme/biosynthesis , Porphobilinogen Synthase/antagonists & inhibitors , Porphobilinogen Synthase/chemistry , Pseudomonas aeruginosa/metabolism , Bacteria/drug effects , Bacterial Proteins/biosynthesis , Crystallization , Drug Resistance, Bacterial/genetics , Genetic Vectors , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Kinetics , Magnesium/pharmacology , Methanosarcina barkeri/drug effects , Methanosarcina barkeri/genetics , Methanosarcina barkeri/metabolism , Microbial Sensitivity Tests , Models, Molecular , Protein Conformation , Zinc/pharmacology
5.
Mikrobiologiia ; 78(1): 5-11, 2009.
Article in Russian | MEDLINE | ID: mdl-19334592

ABSTRACT

Methanosarcina barkeri is capable of synthesizing large amounts of corrinoids, compounds of the vitamin B12 group, although not cobalamin. In the present work, exogenous cobalamin was demonstrated to upregulate DNA synthesis in M. harkeri cell suspensions incubated under air. The effect is similar to the one in Propionibacterium freudenreichii cells, though less pronounced. The growth of the archaeon under anaerobic conditions was shown to be suppressed by cobalamin and 5,6-dimethylbenzimidazole. The data obtained suggest the presence of a corrinoid-dependent ribonucleotide reductase in the archaeon cells which provides for deoxyribose precursors for DNA biosynthesis independently of the presence of molecular oxygen in the medium. Growth suppression under anoxic conditions by cobalamin and 5,6-dimethylbenzimidazole may be due to a decrease in the concentration of factor III, a polyfunctional corrinoid dominating in M. barkeri cells.


Subject(s)
DNA Replication/drug effects , DNA, Archaeal/biosynthesis , Methanosarcina barkeri/drug effects , Methanosarcina barkeri/metabolism , Vitamin B 12/pharmacology , Aerobiosis , Anaerobiosis , DNA, Archaeal/genetics , Methanosarcina barkeri/genetics , Propionibacterium/growth & development , Propionibacterium/metabolism , Vitamin B 12/metabolism
6.
Appl Environ Microbiol ; 72(10): 6819-21, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17021235

ABSTRACT

Octamethylcyclotetrasiloxane (OMCTS), a common constituent of household products, triggers the transformation of bismuth to the volatile toxic derivative trimethylbismuth by Methanosarcina barkeri, which is a representative member of the sewage sludge microflora. Comparative studies with the ionophores monensin and lasalocid, which induce effects similar to those observed for OMCTS, indicated that the stimulation of bismuth methylation is not specific for the siloxane and suggested that the stimulation observed is mainly due to facilitated membrane permeation of the metal ion.


Subject(s)
Bismuth/metabolism , Methanosarcina barkeri/drug effects , Methylation/drug effects , Siloxanes/pharmacology , Methanosarcina barkeri/metabolism
7.
Mikrobiologiia ; 73(1): 25-30, 2004.
Article in Russian | MEDLINE | ID: mdl-15074036

ABSTRACT

The proteins of parasporal inclusions from three Bacillus thuringiensis subspecies (kurstaki, amagiensis, and monterrey) inhibited growth of methanogenic archaea of two species belonging to two genera, Methanobrevibacter arboriphilus and Methanosarcina barkeri. The minimal inhibitory concentrations of these proteins were 20 to 50 micrograms/ml. Lysozyme exhibited similar bactericidal effect on archaea. The perspective of comparative studies on the effect of polyfunctional proteins on bacteria and archaea is discussed.


Subject(s)
Archaea/drug effects , Bacillus thuringiensis/metabolism , Bacterial Proteins/pharmacology , Archaea/growth & development , Bacillus thuringiensis/chemistry , Bacterial Proteins/isolation & purification , Inclusion Bodies/metabolism , Methanosarcina barkeri/drug effects , Microbial Sensitivity Tests , Species Specificity , Spores, Bacterial/metabolism
8.
Indian J Exp Biol ; 41(8): 915-7, 2003 Aug.
Article in English | MEDLINE | ID: mdl-15248497

ABSTRACT

Methane emission was inhibited by aluminium ion in paddy fields. Addition of Al3+ (20 mM) to the culture medium containing cells of pure Methanosarcina barkeri, inhibited methanogenesis. Methanogenic co-factor, F-420, was isolated and purified from Methanosarcina barkeri MS. Spectrophotometric and spectrofluorometric analysis of interaction between co-factor, F-420, and Al3+ revealed that they formed a complex compound that might have blocked methanogenesis.


Subject(s)
Aluminum Compounds/pharmacology , Methane/metabolism , Methanosarcina barkeri/drug effects , Oxidoreductases/metabolism , Aluminum Compounds/chemistry , Cations , Drug Interactions , Electron Transport , Methanosarcina barkeri/enzymology , Methanosarcina barkeri/growth & development , Oxidoreductases/chemistry , Spectrometry, Fluorescence
9.
J Bacteriol ; 182(9): 2611-8, 2000 May.
Article in English | MEDLINE | ID: mdl-10762266

ABSTRACT

Currently, only one selectable marker is available for genetic studies in the archaeal genus Methanosarcina. Here we report the generation of selectable markers that encode resistance to pseudomonic acid (PA(r)) in Methanosarcina species by mutagenesis of the isoleucyl-tRNA synthetase gene (ileS) from Methanosarcina barkeri Fusaro. The M. barkeri ileS gene was obtained by screening of a genomic library for hybridization to a PCR fragment. The complete 3,787-bp DNA sequence surrounding and including the ileS gene was determined. As expected, M. barkeri IleS is phylogenetically related to other archaeal IleS proteins. The ileS gene was cloned into a Methanosarcina-Escherichia coli shuttle vector and mutagenized with hydroxylamine. Nine independent PA(r) clones were isolated after transformation of Methanosarcina acetivorans C2A with the mutagenized plasmids. Seven of these clones carry multiple changes from the wild-type sequence. Most mutations that confer PA(r) were shown to alter amino acid residues near the KMSKS consensus sequence of class I aminoacyl-tRNA synthetases. One particular mutation (G594E) was present in all but one of the PA(r) clones. The MIC of pseudomonic acid for M. acetivorans transformed with a plasmid carrying this single mutation is 70 microgram/ml of medium (for the wild type, the MIC is 12 microgram/ml). The highest MICs (560 microgram/ml) were observed with two triple mutants, A440V/A482T/G594E and A440V/G593D/G594E. Plasmid shuttle vectors and insertion cassettes that encode PA(r) based on the mutant ileS alleles are described. Finally, the implications of the specific mutations we isolated with respect to binding of pseudomonic acid by IleS are discussed.


Subject(s)
Anti-Bacterial Agents/pharmacology , Isoleucine-tRNA Ligase/genetics , Methanosarcina barkeri/drug effects , Methanosarcina barkeri/enzymology , Mupirocin/pharmacology , Base Sequence , Biomarkers , Chromosomes, Archaeal , Cloning, Molecular , DNA, Archaeal , Drug Resistance, Microbial , Escherichia coli/genetics , Genes, Archaeal , Genetic Vectors/genetics , Isoleucine-tRNA Ligase/chemistry , Isoleucine-tRNA Ligase/classification , Methanosarcina barkeri/genetics , Molecular Sequence Data , Mutagenesis , Protein Conformation , Recombination, Genetic , Sequence Analysis, DNA
10.
FEMS Microbiol Lett ; 69(2): 201-4, 1992 Jan 01.
Article in English | MEDLINE | ID: mdl-1537550

ABSTRACT

Methanobacterium barkeri was found to catalyze the reductive dehalogenation of trichlorofluoromethane (CFC-11), also known as FREON 11. Products detected were CHFCl2, CH2FCl, CO and fluoride.


Subject(s)
Chlorofluorocarbons, Methane/metabolism , Methanosarcina barkeri/metabolism , Biodegradation, Environmental , Carbon Monoxide/metabolism , Chlorofluorocarbons, Methane/pharmacology , Kinetics , Methanosarcina barkeri/drug effects , Methanosarcina barkeri/growth & development , Oxidation-Reduction
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