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1.  A Novel Inducible Protein Production System and Neomycin Resistance as Selection Marker for Methanosarcina mazei 
Archaea  2012;2012:973743.
Methanosarcina mazei is one of the model organisms for the methanogenic order Methanosarcinales whose metabolism has been studied in detail. However, the genetic toolbox is still limited. This study was aimed at widening the scope of utilizable methods in this group of organisms. (i) Proteins specific to methanogens are oftentimes difficult to produce in E. coli. However, a protein production system is not available for methanogens. Here we present an inducible system to produce Strep-tagged proteins in Ms. mazei. The promoter p1687, which directs the transcription of methyl transferases that demethylate methylamines, was cloned into plasmid pWM321 and its activity was determined by monitoring β-glucuronidase production. The promoter was inactive during growth on methanol but was rapidly activated when trimethylamine was added to the medium. The gene encoding the β-glucuronidase from E. coli was fused to a Strep-tag and was cloned downstream of the p1687 promoter. The protein was overproduced in Ms. mazei and was purified in an active form by affinity chromatography. (ii) Puromycin is currently the only antibiotic used as a selectable marker in Ms. mazei and its relatives. We established neomycin resistance as a second selectable marker by designing a plasmid that confers neomycin resistance in Ms. mazei.
doi:10.1155/2012/973743
PMCID: PMC3407599  PMID: 22851906
2.  Establishing a Markerless Genetic Exchange System for Methanosarcina mazei Strain Gö1 for Constructing Chromosomal Mutants of Small RNA Genes 
Archaea  2011;2011:439608.
A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gö1 using the hpt gene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of the hpt gene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP). The nonreplicating allelic exchange vector (pRS345) carrying the pac-resistance cassette for direct selection of chromosomal integration, and the hpt gene for counterselection was introduced into this strain. By a pop-in and ultimately pop-out event of the plasmid from the chromosome, allelic exchange is enabled. Using this system, we successfully generated a M. mazei deletion mutant of the gene encoding the regulatory non-coding RNA sRNA154. Characterizing M. mazeiΔsRNA154 under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA154 in regulation of nitrogen fixation by posttranscriptional regulation.
doi:10.1155/2011/439608
PMCID: PMC3177094  PMID: 21941461
3.  Stress Genes and Proteins in the Archaea 
The field covered in this review is new; the first sequence of a gene encoding the molecular chaperone Hsp70 and the first description of a chaperonin in the archaea were reported in 1991. These findings boosted research in other areas beyond the archaea that were directly relevant to bacteria and eukaryotes, for example, stress gene regulation, the structure-function relationship of the chaperonin complex, protein-based molecular phylogeny of organisms and eukaryotic-cell organelles, molecular biology and biochemistry of life in extreme environments, and stress tolerance at the cellular and molecular levels. In the last 8 years, archaeal stress genes and proteins belonging to the families Hsp70, Hsp60 (chaperonins), Hsp40(DnaJ), and small heat-shock proteins (sHsp) have been studied. The hsp70(dnaK), hsp40(dnaJ), and grpE genes (the chaperone machine) have been sequenced in seven, four, and two species, respectively, but their expression has been examined in detail only in the mesophilic methanogen Methanosarcina mazei S-6. The proteins possess markers typical of bacterial homologs but none of the signatures distinctive of eukaryotes. In contrast, gene expression and transcription initiation signals and factors are of the eucaryal type, which suggests a hybrid archaeal-bacterial complexion for the Hsp70 system. Another remarkable feature is that several archaeal species in different phylogenetic branches do not have the gene hsp70(dnaK), an evolutionary puzzle that raises the important question of what replaces the product of this gene, Hsp70(DnaK), in protein biogenesis and refolding and for stress resistance. Although archaea are prokaryotes like bacteria, their Hsp60 (chaperonin) family is of type (group) II, similar to that of the eukaryotic cytosol; however, unlike the latter, which has several different members, the archaeal chaperonin system usually includes only two (in some species one and in others possibly three) related subunits of ∼60 kDa. These form, in various combinations depending on the species, a large structure or chaperonin complex sometimes called the thermosome. This multimolecular assembly is similar to the bacterial chaperonin complex GroEL/S, but it is made of only the large, double-ring oligomers each with eight (or nine) subunits instead of seven as in the bacterial complex. Like Hsp70(DnaK), the archaeal chaperonin subunits are remarkable for their evolution, but for a different reason. Ubiquitous among archaea, the chaperonins show a pattern of recurrent gene duplication—hetero-oligomeric chaperonin complexes appear to have evolved several times independently. The stress response and stress tolerance in the archaea involve chaperones, chaperonins, other heat shock (stress) proteins including sHsp, thermoprotectants, the proteasome, as yet incompletely understood thermoresistant features of many molecules, and formation of multicellular structures. The latter structures include single- and mixed-species (bacterial-archaeal) types. Many questions remain unanswered, and the field offers extraordinary opportunities owing to the diversity, genetic makeup, and phylogenetic position of archaea and the variety of ecosystems they inhabit. Specific aspects that deserve investigation are elucidation of the mechanism of action of the chaperonin complex at different temperatures, identification of the partners and substitutes for the Hsp70 chaperone machine, analysis of protein folding and refolding in hyperthermophiles, and determination of the molecular mechanisms involved in stress gene regulation in archaeal species that thrive under widely different conditions (temperature, pH, osmolarity, and barometric pressure). These studies are now possible with uni- and multicellular archaeal models and are relevant to various areas of basic and applied research, including exploration and conquest of ecosystems inhospitable to humans and many mammals and plants.
PMCID: PMC98981  PMID: 10585970
4.  Disaggregation of Methanosarcina spp. and Growth as Single Cells at Elevated Osmolarity 
Applied and Environmental Microbiology  1993;59(11):3832-3839.
The effect of medium osmolarity on the morphology and growth of Methanosarcina barkeri, Methanosarcina thermophila, Methanosarcina mazei, Methanosarcina vacuolata, and Methanosarcina acetivorans was examined. Each strain was adapted for growth in NaCl concentrations ranging from 0.05 to 1.0 M. Methanosarcina spp. isolated from both marine and nonmarine sources exhibited similar growth characteristics at all NaCl concentrations tested, demonstrating that these species are capable of adapting to a similar range of medium osmolarities. Concomitant with the adaptation in 0.4 to 1.0 M NaCl, all strains disaggregated and grew as single cells rather than in the characteristic multicellular aggregates. Aggregated cells had a methanochondroitin outer layer, while disaggregated single cells lacked the outer layer but retained the protein S-layer adjacent to the cell membrane. Synthesis of glucuronic acid, a major component of methanochondroitin, was reduced 20-fold in the single-cell form of M. barkeri when compared with synthesis in aggregated cells. Strains with the methanochondroitin outer cell layer exhibited enhanced stability at low (<0.2 M NaCl) osmolarity and grew at higher temperatures. Disaggregated cells could be converted back to aggregated cells by gradually readapting cultures to lower NaCl (<0.2 M) and Mg2+ (<0.005 M) concentrations. Disaggregated Methanosarcina spp. could also be colonized and replica plated with greater than 95% recovery rates on solidified agar basal medium that contained 0.4 to 0.6 M NaCl and either trimethylamine, methanol, or acetate as the substrate. The ability to disaggregate and grow Methanosarcina spp. as viable, detergent-sensitive, single cells on agar medium makes these species amenable to mutant selection and screening for genetic studies and enables cells to be gently lysed for the isolation of intact genetic material.
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PMCID: PMC182538  PMID: 16349092
5.  Inhibition of Methanogenesis by Methyl Fluoride: Studies of Pure and Defined Mixed Cultures of Anaerobic Bacteria and Archaea 
Applied and Environmental Microbiology  1997;63(11):4552-4557.
Methyl fluoride (fluoromethane [CH(inf3)F]) has been used as a selective inhibitor of CH(inf4) oxidation by aerobic methanotrophic bacteria in studies of CH(inf4) emission from natural systems. In such studies, CH(inf3)F also diffuses into the anaerobic zones where CH(inf4) is produced. The effects of CH(inf3)F on pure and defined mixed cultures of anaerobic microorganisms were investigated. About 1 kPa of CH(inf3)F, similar to the amounts used in inhibition experiments, inhibited growth of and CH(inf4) production by pure cultures of aceticlastic methanogens (Methanosaeta spp. and Methanosarcina spp.) and by a methanogenic mixed culture of anaerobic microorganisms in which acetate was produced as an intermediate. With greater quantities of CH(inf3)F, hydrogenotrophic methanogens were also inhibited. At a partial pressure of CH(inf3)F of 1 kPa, homoacetogenic, sulfate-reducing, and fermentative bacteria and a methanogenic mixed culture of anaerobic microorganisms based on hydrogen syntrophy were not inhibited. The inhibition by CH(inf3)F of the growth and CH(inf4) production of Methanosarcina mazei growing on acetate was reversible. CH(inf3)F inhibited only acetate utilization by Methanosarcina barkeri, which is able to use acetate and hydrogen simultaneously, when both acetate and hydrogen were present. These findings suggest that the use of CH(inf3)F as a selective inhibitor of aerobic CH(inf4) oxidation in undefined systems must be interpreted with great care. However, by a careful choice of concentrations, CH(inf3)F may be useful for the rapid determination of the role of acetate as a CH(inf4) precursor.
PMCID: PMC1389292  PMID: 16535736
6.  Cloning of the HSP70 gene from Halobacterium marismortui: relatedness of archaebacterial HSP70 to its eubacterial homologs and a model for the evolution of the HSP70 gene. 
Journal of Bacteriology  1992;174(14):4594-4605.
Heat shock induces the synthesis of a set of proteins in Halobacterium marismortui whose molecular sizes correspond to the known major heat shock proteins. By using the polymerase chain reaction and degenerate oligonucleotide primers for conserved regions of the 70-kDa heat shock protein (HSP70) family, we have successfully cloned and sequenced a gene fragment containing the entire coding sequence for HSP70 from H. marismortui. HSP70 from H. marismortui shows between 44 and 47% amino acid identity with various eukaryotic HSP70s and between 51 and 58% identity with its eubacterial and archaebacterial homologs. On the basis of a comparison of all available HSP70 sequences, we have identified a number of unique sequence signatures in this protein family that provide a clear distinction between eukaryotic organisms and prokaryotic organisms (archaebacteria and eubacteria). The archaebacterial (viz., H. marismortui and Methanosarcina mazei) HSP70s have been found to contain all of the signature sequences characteristic of eubacteria (particularly the gram-positive bacteria), which suggests a close evolutionary relationship between these groups. In addition, detailed analyses of HSP70 sequences that we have carried out have revealed a number of additional novel features of the HSP70 protein family. These include (i) the presence of an insertion of about 25 to 27 amino acids in the N-terminal quadrants of all known eukaryotic and prokaryotic HSP70s except those from archaebacteria and the gram-positive group of bacteria, (ii) significant sequence similarity in HSP70 regions comprising its first and second quadrants from organisms lacking the above insertion, (iii) highly significant similarity between a protein, MreB, of Escherichia coli and the N-terminal half of HSP70s, (iv) significant sequence similarity between the N-terminal quadrant of HSP70 (from gram-positive bacteria and archaebacteria) and the m-type thioredoxin of plant chloroplasts. To account for these and other observations, a model for the evolution of HSP70 proteins involving gene duplication is proposed. The model proposes that HSP70 from archaebacteria (H. marismortui and M. mazei) and the gram-positive group of bacteria constitutes the ancestral form of the protein and that all other HSP70s (viz., other eubacteria as well as eukaryotes) containing the insert have evolved from this ancient protein.
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PMCID: PMC206254  PMID: 1624448
7.  Characterization of GlnK1 from Methanosarcina mazei Strain Gö1: Complementation of an Escherichia coli glnK Mutant Strain by GlnK1 
Journal of Bacteriology  2002;184(4):1028-1040.
Trimeric PII-like signal proteins are known to be involved in bacterial regulation of ammonium assimilation and nitrogen fixation. We report here the first biochemical characterization of an archaeal GlnK protein from the diazotrophic methanogenic archaeon Methanosarcina mazei strain Gö1 and show that M. mazei GlnK1 is able to functionally complement an Escherichia coli glnK mutant for growth on arginine. This indicates that the archaeal GlnK protein substitutes for the regulatory function of E. coli GlnK. M. mazei GlnK1 is encoded in the glnK1-amtB1 operon, which is transcriptionally regulated by the availability of combined nitrogen and is only transcribed in the absence of ammonium. The deduced amino acid sequence of the archaeal glnK1 shows 44% identity to the E. coli GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure. M. mazei glnK1 was cloned and overexpressed in E. coli, and GlnK1 was purified to apparent homogeneity. A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer. GlnK1-specific antibodies were raised and used to confirm the in vivo trimeric form by Western analysis. In vivo ammonium upshift experiments and analysis of purified GlnK1 indicated significant differences compared to E. coli GlnK. First, GlnK1 from M. mazei is not covalently modified by uridylylation under nitrogen limitation. Second, heterotrimers between M. mazei GlnK1 and Klebsiella pneumoniae GlnK are not formed. Because M. mazei GlnK1 was able to complement growth of an E. coli glnK mutant with arginine as the sole nitrogen source, it is likely that uridylylation is not required for its regulatory function.
doi:10.1128/jb.184.4.1028-1040.2002
PMCID: PMC134814  PMID: 11807063
8.  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.
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PMCID: PMC373557  PMID: 16346753
9.  Functional organization of a single nif cluster in the mesophilic archaeon Methanosarcina mazei strain Gö1 
Archaea  2002;1(2):143-150.
The mesophilic methanogenic archaeon Methanosarcina mazei strain Gö1 is able to utilize molecular nitrogen (N2) as its sole nitrogen source. We have identified and characterized a single nitrogen fixation (nif) gene cluster in M. mazei Gö1 with an approximate length of 9 kbp. Sequence analysis revealed seven genes with sequence similarities to nifH, nifI1, nifI2, nifD, nifK, nifE and nifN, similar to other diazotrophic methanogens and certain bacteria such as Clostridium acetobutylicum, with the two glnB-like genes (nifI1 and nifI2) located between nifH and nifD. Phylogenetic analysis of deduced amino acid sequences for the nitrogenase structural genes of M. mazei Gö1 showed that they are most closely related to Methanosarcina barkeri nif2 genes, and also closely resemble those for the corresponding nif products of the gram-positive bacterium C. acetobutylicum. Northern blot analysis and reverse transcription PCR analysis demonstrated that the M. mazei nif genes constitute an operon transcribed only under nitrogen starvation as a single 8 kb transcript. Sequence analysis revealed a palindromic sequence at the transcriptional start site in front of the M. mazei nifH gene, which may have a function in transcriptional regulation of the nif operon.
PMCID: PMC2685556  PMID: 15803652
GlnB-like proteins; nif genes; nitrogen fixation; nitrogen regulation
10.  Identification of the Major Expressed S-Layer and Cell Surface-Layer-Related Proteins in the Model Methanogenic Archaea: Methanosarcina barkeri Fusaro and Methanosarcina acetivorans C2A 
Archaea  2012;2012:873589.
Many archaeal cell envelopes contain a protein coat or sheath composed of one or more surface exposed proteins. These surface layer (S-layer) proteins contribute structural integrity and protect the lipid membrane from environmental challenges. To explore the species diversity of these layers in the Methanosarcinaceae, the major S-layer protein in Methanosarcina barkeri strain Fusaro was identified using proteomics. The Mbar_A1758 gene product was present in multiple forms with apparent sizes of 130, 120, and 100 kDa, consistent with post-translational modifications including signal peptide excision and protein glycosylation. A protein with features related to the surface layer proteins found in Methanosarcina acetivorans C2A and Methanosarcina mazei Goel was identified in the M. barkeri genome. These data reveal a distinct conserved protein signature with features and implied cell surface architecture in the Methanosarcinaceae that is absent in other archaea. Paralogous gene expression patterns in two Methanosarcina species revealed abundant expression of a single S-layer paralog in each strain. Respective promoter elements were identified and shown to be conserved in mRNA coding and upstream untranslated regions. Prior M. acetivorans genome annotations assigned S-layer or surface layer associated roles of eighty genes: however, of 68 examined none was significantly expressed relative to the experimentally determined S-layer gene.
doi:10.1155/2012/873589
PMCID: PMC3361143  PMID: 22666082
11.  In silico description of cobalt and nickel assimilation systems in the genomes of methanogens 
Systems and Synthetic Biology  2011;5(3-4):105-114.
Methanogens are a diverse group of organisms that can live in a wide range of environments. Herein, cobalt and tungsten assimilation pathways have proposed to be established in the genomes of Methanococcus maripaludies C5 and Methanosarcina mazei Go1, respectively. All of the proteins involved in the proposed pathways were identified from public domain databases and then complied manually to reconstruct the pathways. The function of proteins with unknown function was assigned by a combined prediction approach. Totally, 17 proteins were identified to cobalt transport and assimilation processes whereas 7 proteins reported to tungsten assimilation system. Phylogenetic analysis of this study revealed that heavy metal transporter of methanogens could be evolved from closely related members in the different genera of methanogens. Nevertheless, genes encoding for metal resistance proteins could be originated from thermophilic and sulfur reducing bacteria. Many metalloenzymes in methanogens were very unique to the species of methanogens. It implied that these metal ions were utilized to produce the precursors for energy driven processes of methanogens. This study suggested that in combination of systems models and evolutionary inference can only correlate metabolic fluxes and physiological changes in methanogens. In silico models of this study will provide insights to design experiments for heavy metal assimilation processes of methanogens growing under heavy metal-rich environments and or in a laboratory condition.
doi:10.1007/s11693-011-9087-2
PMCID: PMC3234315  PMID: 23205154
Methanogens; Heavy metals assimilation; Metabolic behavior; Phylogeny; Metalloenzymes; Energetic metabolism
12.  A continuous fluorescence assay for the characterization of Nudix hydrolases 
Analytical Biochemistry  2013;437(2):178-184.
The common substrate structure for the functionally diverse Nudix protein superfamily is nucleotide-diphosphate-X, where X is a large variety of leaving groups. The substrate specificity is known for less than 1% of the 29,400 known members. Most activities result in the release of an inorganic phosphate ion or of a product bearing a terminal phosphate moiety. Reactions have typically been monitored by a modification of the discontinuous Fiske–SubbaRow assay, which is relatively insensitive and slow. We report here the development of a continuous fluorescence assay that enables the rapid and accurate determination of substrate specificities in a 96-well format. We used this novel assay to confirm the reported substrate characterizations of MutT and NudD of Escherichia coli and to characterize DR_1025 of Deinococcus radiodurans and MM_0920 of Methanosarcina mazei. Novel findings enabled by the new assay include the following. First, in addition to the well-characterized hydrolysis of 8-oxo-dGTP at the α–β position, MutT cleaves at the β–γ phosphate bond at a rate of 3% of that recorded for hydrolysis at the α–β position. Second, MutT also catalyzes the hydrolysis of 5-methyl-dCTP. Third, 8-oxo-dGTP was observed to be the best substrate for DR_1025 of the 41 compounds screened.
doi:10.1016/j.ab.2013.02.023
PMCID: PMC3744803  PMID: 23481913
Nudix; Continuous assay; Fluorescence; Substrate screening; Kinetics
13.  Application of a Novel Microtitre Plate-Based Assay for the Discovery of New Inhibitors of DNA Gyrase and DNA Topoisomerase VI 
PLoS ONE  2013;8(2):e58010.
DNA topoisomerases are highly exploited targets for antimicrobial drugs. The spread of antibiotic resistance represents a significant threat to public health and necessitates the discovery of inhibitors that target topoisomerases in novel ways. However, the traditional assays for topoisomerase activity are not suitable for the high-throughput approaches necessary for drug discovery. In this study we validate a novel assay for screening topoisomerase inhibitors. A library of 960 compounds was screened against Escherichia coli DNA gyrase and archaeal Methanosarcina mazei DNA topoisomerase VI. Several novel inhibitors were identified for both enzymes, and subsequently characterised in vitro and in vivo. Inhibitors from the M. mazei topoisomerase VI screen were tested for their ability to inhibit Arabidopsis topoisomerase VI in planta. The data from this work present new options for antibiotic drug discovery and provide insight into the mechanism of topoisomerase VI.
doi:10.1371/journal.pone.0058010
PMCID: PMC3582512  PMID: 23469129
14.  Two CRISPR-Cas systems inMethanosarcina mazeistrain Gö1 display common processing features despite belonging to different types I and III 
RNA Biology  2013;10(5):779-791.
The clustered regularly interspaced short palindromic repeats (CRISPR) system represents a highly adaptive and heritable defense system against foreign nucleic acids in bacteria and archaea. We analyzed the two CRISPR-Cas systems in Methanosarcina mazei strain Gö1. Although belonging to different subtypes (I-B and III-B), the leaders and repeats of both loci are nearly identical. Also, despite many point mutations in each array, a common hairpin motif was identified in the repeats by a bioinformatics analysis and in vitro structural probing. The expression and maturation of CRISPR-derived RNAs (crRNAs) were studied in vitro and in vivo. Both respective potential Cas6b-type endonucleases were purified and their activity tested in vitro. Each protein showed significant activity and could cleave both repeats at the same processing site. Cas6b of subtype III-B, however, was significantly more efficient in its cleavage activity compared with Cas6b of subtype I-B. Northern blot and differential RNAseq analyses were performed to investigate in vivo transcription and maturation of crRNAs, revealing generally very low expression of both systems, whereas significant induction at high NaCl concentrations was observed. crRNAs derived proximal to the leader were generally more abundant than distal ones and in vivo processing sites were clarified for both loci, confirming the previously well-established 8 nt 5′ repeat tags. The 3′-ends were more diverse, but generally ended in a prefix of the following repeat sequence (3′-tag). The analysis further revealed a 5′-hydroxy and 3′-phosphate termini architecture of small crRNAs specific for cleavage products of Cas6 endonucleases from type I-E and I-F and type III-B.
doi:10.4161/rna.23928
PMCID: PMC3737336  PMID: 23619576
methanoarchaea; CRISPR-Cas system; immunity of prokaryotes; regulatory RNA; phages; Methanosarcina mazei
15.  Discovery and Characterization of the First Archaeal Dihydromethanopterin Reductase, an Iron-Sulfur Flavoprotein from Methanosarcina mazei 
Journal of Bacteriology  2014;196(2):203-209.
The microbial production of methane by methanogenic archaea is dependent on the synthesis of the pterin-containing cofactor tetrahydromethanopterin (H4MPT). The enzyme catalyzing the last step of H4MPT biosynthesis (dihydromethanopterin reductase) has not previously been identified in methane-producing microorganisms. Previous complementation studies with the methylotrophic bacterium Methylobacterium extorquens have indicated that an uncharacterized archaeal-flavoprotein-like flavoprotein (AfpA) from Methylobacillus flagellatus or Burkholderia xenovorans can replace the activity of a phylogenetically unrelated bacterial dihydromethanopterin reductase (DmrA). We propose that MM1854, a homolog of AfpA from Methanosarcina mazei, catalyzes the last step of H4MPT biosynthesis in methane-producing microorganisms. To test this hypothesis, a six-histidine (His6)-tagged version of MM1854 was produced. Bioinformatic analysis revealed the presence of one flavin mononucleotide (FMN)-binding site and two iron-sulfur cluster sites, consistent with an oxidoreductase enzyme. Purified His6-MM1854 occurred as a homodimer of 29-kDa subunits, and the UV-visible spectrum of the purified protein showed absorbance peaks at 380 and 460 nm, characteristic of oxidized FMN. NAD(P)H was incapable of directly reducing the flavin cofactor, but dithionite eliminated the FMN peaks, indicating successful electron transfer to MM1854. An electron transfer system of NADPH, spinach NADPH-ferredoxin oxidoreductase, and ferredoxin could also reduce the FMN peaks. A newly developed assay indicated that dithiothreitol-reduced MM1854 could transfer electrons to dihydromethanopterin. This assay was also effective with a heat-stable DmrX analog from Methanocaldococcus jannaschii (MJ0208). These results provide the first biochemical evidence that MM1854 and MJ0208 function as archaeal dihydromethanopterin reductases (DmrX) and that ferredoxin may serve as an electron donor.
doi:10.1128/JB.00457-13
PMCID: PMC3911254  PMID: 23995635
16.  Identification of the gene for disaggregatase from Methanosarcina mazei  
Archaea  2008;2(3):185-191.
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-6T, 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.
PMCID: PMC2685598  PMID: 19054745
methanochondroitin; morphological change
17.  Transcriptional Profiling of Methyltransferase Genes during Growth of Methanosarcina mazei on Trimethylamine▿ †  
Journal of Bacteriology  2009;191(16):5108-5115.
The genomic expression patterns of Methanosarcina mazei growing with trimethylamine were measured in comparison to those of cells grown with methanol. We identified a total of 72 genes with either an increased level (49 genes) or a decreased level (23 genes) of mRNA during growth on trimethylamine with methanol-grown cells as the control. Major differences in transcript levels were observed for the mta, mtb, mtt, and mtm genes, which encode enzymes involved in methane formation from methanol and trimethylamine, respectively. Other differences in mRNA abundance were found for genes encoding enzymes involved in isopentenyl pyrophosphate synthesis and in the formation of aromatic amino acids, as well as a number of proteins with unknown functions. The results were verified by in-depth analysis of methyltransferase genes using specific primers for real-time quantitative reverse transcription-PCR (RT-PCR). The monitored transcript levels of genes encoding corrinoid proteins involved in methyl group transfer from methylated C1 compounds (mtaC, mtbC, mttC, and mtmC) indicated increased amounts of mRNA from the mtaBC1, mtaBC2, and mtaBC3 operons in methanol-grown cells, whereas mRNA of the mtb1-mtt1 operon was found in high concentrations during trimethylamine consumption. The genes of the mtb1-mtt1 operon encode methyltransferases that are responsible for sequential demethylation of trimethylamine. The analysis of product formation of trimethylamine-grown cells at different optical densities revealed that large amounts of dimethylamine and monomethylamine were excreted into the medium. The intermediate compounds were consumed only in the very late exponential growth phase. RT-PCR analysis of key genes involved in methanogenesis led to the conclusion that M. mazei is able to adapt to changing trimethylamine concentrations and the consumption of intermediate compounds. Hence, we assume that the organism possesses a regulatory network for optimal substrate utilization.
doi:10.1128/JB.00420-09
PMCID: PMC2725588  PMID: 19525341
18.  Connection between Multimetal(loid) Methylation in Methanoarchaea and Central Intermediates of Methanogenesis▿† 
Applied and Environmental Microbiology  2011;77(24):8669-8675.
In spite of the significant impact of biomethylation on the mobility and toxicity of metals and metalloids in the environment, little is known about the biological formation of these methylated metal(loid) compounds. While element-specific methyltransferases have been isolated for arsenic, the striking versatility of methanoarchaea to methylate numerous metal(loid)s, including rare elements like bismuth, is still not understood. Here, we demonstrate that the same metal(loid)s (arsenic, selenium, antimony, tellurium, and bismuth) that are methylated by Methanosarcina mazei in vivo are also methylated by in vitro assays with purified recombinant MtaA, a methyltransferase catalyzing the methyl transfer from methylcobalamin [CH3Cob(III)] to 2-mercaptoethanesulfonic acid (CoM) in methylotrophic methanogenesis. Detailed studies revealed that cob(I)alamin [Cob(I)], formed by MtaA-catalyzed demethylation of CH3Cob(III), is the causative agent for the multimetal(loid) methylation observed. Moreover, Cob(I) is also capable of metal(loid) hydride generation. Global transcriptome profiling of M. mazei cultures exposed to bismuth did not reveal induced methyltransferase systems but upregulated regeneration of methanogenic cofactors in the presence of bismuth. Thus, we conclude that the multimetal(loid) methylation in vivo is attributed to side reactions of CH3Cob(III) with reduced cofactors formed in methanogenesis. The close connection between metal(loid) methylation and methanogenesis explains the general capability of methanoarchaea to methylate metal(loid)s.
doi:10.1128/AEM.06406-11
PMCID: PMC3233109  PMID: 22003009
19.  Identification and analysis of proton-translocating pyrophosphatases in the methanogenic archaeon Methanosarcina mazei  
Archaea  2001;1(1):1-7.
Analysis of genome sequence data from the methanogenic archaeon Methanosarcina mazei Gö1 revealed the existence of two open reading frames encoding proton-translocating pyrophosphatases (PPases). These open reading frames are linked by a 750-bp intergenic region containing TC-rich stretches and are transcribed in opposite directions. The corresponding polypeptides are referred to as Mvp1 and Mvp2 and consist of 671 and 676 amino acids, respectively. Both enzymes represent extremely hydrophobic, integral membrane proteins with 15 predicted transmembrane segments and an overall amino acid sequence similarity of 50.1%. Multiple sequence alignments revealed that Mvp1 is closely related to eukaryotic PPases, whereas Mvp2 shows highest homologies to bacterial PPases. Northern blot experiments with RNA from methanol-grown cells harvested in the mid-log growth phase indicated that only Mvp2 was produced under these conditions. Analysis of washed membranes showed that Mvp2 had a specific activity of 0.34 U mg (protein)–1. Proton translocation experiments with inverted membrane vesicles prepared from methanol-grown cells showed that hydrolysis of 1 mol of pyrophosphate was coupled to the translocation of about 1 mol of protons across the cytoplasmic membrane. Appropriate conditions for mvp1 expression could not be determined yet. The pyrophosphatases of M. mazei Gö1 represent the first examples of this enzyme class in methanogenic archaea and may be part of their energy-conserving system. Abbreviations: DCCD, N,N′-dicyclohexylcarbodiimide; PPase, inorganic pyrophosphatase; PPi, inorganic pyrophosphate; Δp, proton motive force.
PMCID: PMC2685546  PMID: 15803653
energy conservation; inorganic pyrophosphate; methanogenesis; proton pump; pyrophosphatase
20.  Generation of Dominant Selectable Markers for Resistance to Pseudomonic Acid by Cloning and Mutagenesis of the ileS Gene from the Archaeon Methanosarcina barkeri Fusaro 
Journal of Bacteriology  2000;182(9):2611-2618.
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 (PAr) 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 PAr 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 PAr 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 PAr clones. The MIC of pseudomonic acid for M. acetivorans transformed with a plasmid carrying this single mutation is 70 μg/ml of medium (for the wild type, the MIC is 12 μg/ml). The highest MICs (560 μg/ml) were observed with two triple mutants, A440V/A482T/G594E and A440V/G593D/G594E. Plasmid shuttle vectors and insertion cassettes that encode PAr 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.
PMCID: PMC111328  PMID: 10762266
21.  Identification of a Salt-Induced Primary Transporter for Glycine Betaine in the Methanogen Methanosarcina mazei Gö1 
The salt adaptation of the methanogenic archaeon Methanosarcina mazei Gö1 was studied at the physiological and molecular levels. The freshwater organism M. mazei Gö1 was able to adapt to salt concentrations up to 1 M, and the addition of the compatible solute glycine betaine to the growth medium facilitated adaptation to higher salt concentrations. Transport studies with cell suspensions revealed a salt-induced glycine betaine uptake activity in M. mazei Gö1, and inhibitor studies argue for a primary transport device. Analysis of the genome of M. mazei Gö1 identified a homolog of known primary glycine betaine transporters. This gene cluster was designated Ota (osmoprotectant transporter A). Its sequence and gene organization are very similar to those of the glycine betaine transporter OpuA of Bacillus subtilis. Northern blot analysis of otaC revealed a salt-dependent transcription of this gene. Ota is the first identified salt-induced transporter for compatible solutes in Archaea.
doi:10.1128/AEM.68.5.2133-2139.2002
PMCID: PMC127567  PMID: 11976081
22.  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.
PMCID: PMC202587  PMID: 16347594
23.  Carbon-dependent control of electron transfer and central carbon pathway genes for methane biosynthesis in the Archaean, Methanosarcina acetivorans strain C2A 
BMC Microbiology  2010;10:62.
Background
The archaeon, Methanosarcina acetivorans strain C2A forms methane, a potent greenhouse gas, from a variety of one-carbon substrates and acetate. Whereas the biochemical pathways leading to methane formation are well understood, little is known about the expression of the many of the genes that encode proteins needed for carbon flow, electron transfer and/or energy conservation. Quantitative transcript analysis was performed on twenty gene clusters encompassing over one hundred genes in M. acetivorans that encode enzymes/proteins with known or potential roles in substrate conversion to methane.
Results
The expression of many seemingly "redundant" genes/gene clusters establish substrate dependent control of approximately seventy genes for methane production by the pathways for methanol and acetate utilization. These include genes for soluble-type and membrane-type heterodisulfide reductases (hdr), hydrogenases including genes for a vht-type F420 non-reducing hydrogenase, molybdenum-type (fmd) as well as tungsten-type (fwd) formylmethanofuran dehydrogenases, genes for rnf and mrp-type electron transfer complexes, for acetate uptake, plus multiple genes for aha- and atp-type ATP synthesis complexes. Analysis of promoters for seven gene clusters reveal UTR leaders of 51-137 nucleotides in length, raising the possibility of both transcriptional and translational levels of control.
Conclusions
The above findings establish the differential and coordinated expression of two major gene families in M. acetivorans in response to carbon/energy supply. Furthermore, the quantitative mRNA measurements demonstrate the dynamic range for modulating transcript abundance. Since many of these gene clusters in M. acetivorans are also present in other Methanosarcina species including M. mazei, and in M. barkeri, these findings provide a basis for predicting related control in these environmentally significant methanogens.
doi:10.1186/1471-2180-10-62
PMCID: PMC2838876  PMID: 20178638
24.  Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage [published erratum appears in J Cell Biol 1995 Feb;128(4):following 713] 
The Journal of Cell Biology  1994;127(6):1755-1766.
The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP- 2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)- positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24-48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84% of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-beta 1 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF-beta 1 in C2C12 cells. TGF-beta 1 potentiated the inhibitory effect of BMP-2 on myotube formation, whereas TGF-beta 1 reduced ALP activity and osteocalcin production induced by BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.
PMCID: PMC2120318  PMID: 7798324
25.  CD2 regulates responsiveness of activated T cells to interleukin 12 [published erratum appears in J Exp Med 1995 Oct 1;182(4):1175] 
Interleukin (IL) 12 is a 70-kD heterodimeric cytokine produced by antigen-presenting cells (APCs) such as macrophages in response to infectious pathogens and interferon (IFN) gamma. The varied immunomodulatory effects of IL-12 include the stimulation of proliferation and IFN-gamma production by T cells, and it also has a central role in the development of the T helper cell type 1 immune phenotype. We undertook the production of antibodies capable of modulating the response of T cells to IL-12, and in the process we discovered two antibodies that inhibited the ability of IL-12 to stimulate T cell proliferation. In this report, we demonstrate that these anti-bodies recognize CD2, and we show how antibodies directed toward either the adhesion domain of CD2 or its ligand, CD58, specifically inhibit IL-12 induced proliferation and IFN-gamma production by phytohemagglutinin-activated T cells, leaving the response to IL-12 unaffected. A three-to fourfold reduction in proliferation and IFN-gamma production was observed at IL-12 concentrations as high as 1 nM, with complete inhibition occurring at < or = 1 pM. This novel effect is not directly mediated at the level of the IL-12 receptor, as shown by the inability of these antibodies to block IL-12 binding to activated T cells. Furthermore, by using activating pairs of CD2 antibodies, we show that CD2 stimulation strongly synergizes with IL-12, even at 0.1 pM, in inducing both T cell proliferation and IFN-gamma production. Cytolytic T lymphocyte- associated antigen 4-immunoglobulin-mediated inhibition of the B7/CD28 interaction did not affect the T cell response to either IL-12 or IL-2, but the removal of APCs selectively diminished the proliferative response to IL-12. Based on this data, we hypothesize that CD2 has a central role in an IL-12/IFN-gamma positive feedback loop between T cell and APC, providing the key functional link via a CD2/CD58 interaction that controls T cell responsiveness to IL-12. This model provides a basis for future investigations aimed at defining the signaling mechanisms that mediate this cytokine-specific regulatory effect of CD2, and it offers insight into how a cytokine receptor and distinct adhesion molecule can interact to modulate responsiveness to that cytokine. In addition, it underscores the possibility that the clinical potential of an immunomodulatory drug like IL-12 may be governed by the presence or absence of specific costimulation.
PMCID: PMC2192171  PMID: 7544396

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