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PubMed1983

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.

Bacillus subtilis↗
PubMed1983

Growth and plating efficiency of methanococci on agar media.

Plating techniques for cultivation of methanogenic bacteria have been optimized for two members of the genus Methanococcus. Methanococcus maripaludis and Methanococcus voltae were cultivated on aerobically and anaerobically prepared agar media. Maintenance of O(2) levels below 5 mul/liter within an anaerobic glove box was necessary during plating and incubation for 90% recovery of plated cells. Under an atmosphere of H(2), CO(2), and H(2)S (79:20:1), 2 to 3 days of incubation at 37 degrees C were sufficient for the formation of visible colonies. The viability of plated cells was significantly affected by the growth phase of the culture, H(2)S concentration, and the volume of medium per plate. In addition, colony size of methanococci was affected by agar type, as well as by the concentrations of agar, H(2)S, and bicarbonate.

Journal Article↗
PubMed1987

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 DeltaH and Marburg were completely inhibited by 7 muM 1,2-dibromoethane and, to various degrees, by 51 to 1,084 muM 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 muM bromoethanesulfonic acid, whereas both Methanobacterium strains were only partly inhibited by 2,124 muM. Coenzyme M protected cells from bromoethanesulfonic acid but not from any of the other inhibitors.

Journal Article↗
PubMed1989

An archaebacterial promoter sequence assigned by RNA polymerase binding experiments.

To identify an archaebacterial promoter sequence, nuclease protection studies with the purified RNA polymerase of Methanococcus vannielii were performed. The enzyme binds specifically both at protein-encoding (hisA and methyl CoM reductase, component C) and tRNA-rRNA genes. The binding region of the RNA polymerase extends from 30 base pairs (bp) upstream (-30) to 20 bp downstream (+20) from the in vivo transcription start site. This finding indicates that the archaebacterial enzyme recognizes promoters without transacting transcription factors. The DNA segment protected from nuclease digestion by bound RNA polymerase contains an octanucleotide sequence centered at -25, which is conserved between the protein-encoding and the stable RNA genes. According to the specific binding of the enzyme to only DNA-fragments harbouring this motif, we propose the sequence TTTATATA as the major recognition signal of the Methanococcus RNA polymerase. Comparison of this motif with published archaebacterial DNA sequences revealed the presence of homologous sequences at the same location upstream of 36 genes. We therefore consider the overall consensus TTTATAATA as a general element of promoters in archaebacteria. In spite of the specific binding of the enzyme, most preparations of the Methanococcus vannielii RNA polymerase are unable to initiate transcription at the correct sites in vitro. Here we present first evidence for the possible existence of a transcription factor conferring the ability to the enzyme to initiate and terminate transcription specifically in vitro.

Archaea↗
PubMed1998

Immunoelectron microscopic studies indicate the existence of a cell shape preserving cytoskeleton in prokaryotes.

Immunoelectron microscopic studies of prokaryotes were performed with anti-actin antibodies directed against the C terminus of actin. Studies on ultrathin sections revealed high proportions of the overall label close to the cell periphery in Escherichia coli, Ralstonia eutropha, Thermoanaerobacterium thermosulfurigenes, T. thermosaccharolyticum, and Methanococcus jannaschii. Substantial label also in the cytoplasm was observed in Bacillus sp., Methanococcus voltae, and Methanobacterium thermoautotrophicum. Only very minor amounts of label were found in the nucleoid region of the cells. Whole-mount immunogold studies, combined with negative staining, revealed the existence of an intracellular network of fibrils which could be labeled by anti-actin antibodies. This network is assumed to be located below the cytoplasmic membrane all around the cytoplasm. It appears to have properties that would allow its function as a cytoskeleton-like structure preserving cell shape.

Actins↗
PubMed1999

Cysteinyl-tRNA formation: the last puzzle of aminoacyl-tRNA synthesis.

With the exception of the methanogenic archaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum deltaH, all organisms surveyed contain orthologs of Escherichia coli cysteinyl-tRNA synthetase (CysRS). The characterization of CysRS-encoding (cysS) genes and the demonstration of their ability to complement an E. coli cysSts mutant reveal that Methanococcus maripaludis and Methanosarcina barkeri, two other methanogenic archaea, possess canonical CysRS proteins. A molecular phylogeny inferred from 40 CysRS sequences indicates that the CysRS of M. maripaludis and Methanosarcina spp. are specific relatives of the CysRS of Pyrococcus spp. and Chlamydia, respectively. This result suggests that the CysRS gene was acquired by lateral gene transfer in at least one euryarchaeotic lineage.

Amino Acid Sequence↗
PubMed2001

Heterologous expression of archaeal selenoprotein genes directed by the SECIS element located in the 3' non-translated region.

Previous in silico analysis of selenoprotein genes in Archaea revealed that the selenocysteine insertion (SECIS) motif necessary to recode UGA with selenocysteine was not adjacent to the UGA codon as is found in Bacteria. Rather, paralogous stem-loop structures are located in the 3' untranslated region (3' UTR), reminiscent of the situation in Eukarya. To assess the function of such putative SECIS elements, the Methanococcus jannaschii MJ0029 (fruA, which encodes the A subunit of the coenzyme F420-reducing hydrogenase) mRNA was mapped in vivo and probed enzymatically in vitro. It was shown that the SECIS element is indeed transcribed as part of the respective mRNA and that its secondary structure corresponds to that predicted by RNA folding programs. Its ability to direct selenocysteine insertion in vivo was demonstrated by the heterologous expression of MJ0029 in Methanococcus maripaludis, resulting in the synthesis of an additional selenoprotein, as analysed by 75Se labelling. The selective advantage of moving the SECIS element in the untranslated region may confer the ability to insert more than one selenocysteine into a single polypeptide. Evidence for this assumption was provided by the finding that the M. maripaludis genome contains an open reading frame with two in frame TGA codons, followed by a stem-loop structure in the 3' UTR of the mRNA that corresponds to the archaeal SECIS element.

3' Untranslated Regions↗
PubMed1997

Transcriptional regulation in Archaea: in vivo demonstration of a repressor binding site in a methanogen.

The status of the Archaea as one of the three primary Domains emphasizes the importance of understanding their molecular fundamentals. Basic transcription in the Archaea resembles eucaryal transcription. However, little is known about transcriptional regulation. We have taken an in vivo approach, using genetics to address transcriptional regulation in the methanogenic Archaeon Methanococcus maripaludis. We identified a repressor binding site that regulates nif (nitrogen fixation) gene expression. The repressor binding site was palindromic (an inverted repeat) and was located just after the transcription start site of nifH. Mutations that changed the sequence of the palindrome resulted in marked decreases in repression by ammonia, even when the palindromic nature of the site was retained. The same mutations greatly decreased binding to the site by components of cell extract. These results provide the first partial description of a transcriptional regulatory mechanism in the methanogenic Archaea. This work also illustrates the utility of genetic approaches in Methanococcus that have not been widely used in the methanogens: directed mutagenesis and reporter gene fusions with lacZ.

Base Sequence↗
PubMed2003

Microbial population dynamics in an anaerobic CSTR treating a chemical synthesis-based pharmaceutical wastewater.

Effects of a chemical synthesis based pharmaceutical wastewater on performance of an anaerobic completely stirred tank reactor (CSTR), activity of acetoclastic methanogens and microbial composition were evaluated under various influent compositions. Initially, the CSTR was fed with glucose up to an organic loading rate (OLR) of 6 kg COD/m3 x d corresponding to an F/M ratio of 0.43 with a hydraulic retention time (HRT) of 2.5 days. A COD removal efficiency of 92% and a methane yield of 0.32 m3 CH4/kg COD(removed) were achieved whilst specific methanogenic activity (SMA) was found to be 336mL CH4/gTVS x d. After the CSTR was fed with pre-aerated wastewater diluted by glucose in different dilution ratios of 10% (w/v), 30% (w/v), 70% (w/v), and 100% (w/v) pre-aerated wastewater, gradual decreases in COD removal efficiency to 71%, methane yield to 0.28 m3CH4/kg COD(removed) and SMA to 166 mL CH4/gTVS d occurred whilst volatile fatty acid concentration reached to 1474 mg/L. After the raw wastewater diluted with the pre-aerated wastewater was fed into the CSTR in increasing ratios of 10% (w/v), 30% (w/v), and 60% (w/v), there was a proportional deterioration in performance in terms of COD removal efficiency, methane yield and acetoclastic methanogenic activity. Epifluorescence microscopy of the seed sludge revealed that Methanococcus-like species, short, and medium rods were found to be equally dominant. The short and medium rod species remained equally dominant groups in the CSTR throughout the feeding regime whilst Methanococcus-like species and long rods were found to be in insignificant numbers at the end of the study. Changes in archael diversity were determined using molecular analyses such as polymerase chain reaction (PCR), and denaturent gradient gel electrophoresis (DGGE). Results showed that overall archeal diversity did not change much whereas changes in composition of eubacterial population occurred.

Bioreactors↗
PubMed1999

Control of ribosomal protein L1 synthesis in mesophilic and thermophilic archaea.

The mechanisms for the control of ribosomal protein synthesis have been characterized in detail in Eukarya and in Bacteria. In Archaea, only the regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10, and MvaL12) of the mesophilic Methanococcus vannielii has been extensively investigated. As in Bacteria, regulation takes place at the level of translation. The regulator protein MvaL1 binds preferentially to its binding site on the 23S rRNA, and, when in excess, binds to the regulatory target site on its mRNA and thus inhibits translation of all three cistrons of the operon. The regulatory binding site on the mRNA, a structural mimic of the respective binding site on the 23S rRNA, is located within the structural gene about 30 nucleotides downstream of the ATG start codon. MvaL1 blocks a step before or at the formation of the first peptide bond of MvaL1. Here we demonstrate that a similar regulatory mechanism exists in the thermophilic M. thermolithotrophicus and M. jannaschii. The L1 gene is cotranscribed together with the L10 and L11 gene, in all genera of the Euryarchaeota branch of the Archaea studied so far. A potential regulatory L1 binding site located within the structural gene, as in Methanococcus, was found in Methanobacterium thermoautotrophicum and in Pyrococcus horikoshii. In contrast, in Archaeoglobus fulgidus a typical L1 binding site is located in the untranslated leader of the L1 gene as described for the halophilic Archaea. In Sulfolobus, a member of the Crenarchaeota, the L1 gene is part of a long transcript (encoding SecE, NusG, L11, L1, L10, L12). A previously suggested regulatory L1 target site located within the L11 structural gene could not be confirmed as an L1 binding site.

Amino Acid Sequence↗
PubMed1997

A euryarchaeal lysyl-tRNA synthetase: resemblance to class I synthetases.

The sequencing of euryarchaeal genomes has suggested that the essential protein lysyl-transfer RNA (tRNA) synthetase (LysRS) is absent from such organisms. However, a single 62-kilodalton protein with canonical LysRS activity was purified from Methanococcus maripaludis, and the gene that encodes this protein was cloned. The predicted amino acid sequence of M. maripaludis LysRS is similar to open reading frames of unassigned function in both Methanobacterium thermoautotrophicum and Methanococcus jannaschii but is unrelated to canonical LysRS proteins reported in eubacteria, eukaryotes, and the crenarchaeote Sulfolobus solfataricus. The presence of amino acid motifs characteristic of the Rossmann dinucleotide-binding domain identifies M. maripaludis LysRS as a class I aminoacyl-tRNA synthetase, in contrast to the known examples of this enzyme, which are class II synthetases. These data question the concept that the classification of aminoacyl-tRNA synthetases does not vary throughout living systems.

Acylation↗
PubMed2003

Lysine-2,3-aminomutase and beta-lysine acetyltransferase genes of methanogenic archaea are salt induced and are essential for the biosynthesis of Nepsilon-acetyl-beta-lysine and growth at high salinity.

The compatible solute N(epsilon)-acetyl-beta-lysine is unique to methanogenic archaea and is produced under salt stress only. However, the molecular basis for the salt-dependent regulation of N(epsilon)-acetyl-beta-lysine formation is unknown. Genes potentially encoding lysine-2,3-aminomutase (ablA) and beta-lysine acetyltransferase (ablB), which are assumed to catalyze N(epsilon)-acetyl-beta-lysine formation from alpha-lysine, were identified on the chromosomes of the methanogenic archaea Methanosarcina mazei Gö1, Methanosarcina acetivorans, Methanosarcina barkeri, Methanococcus jannaschii, and Methanococcus maripaludis. The order of the two genes was identical in the five organisms, and the deduced proteins were very similar, indicating a high degree of conservation of structure and function. Northern blot analysis revealed that the two genes are organized in an operon (termed the abl operon) in M. mazei Gö1. Expression of the abl operon was strictly salt dependent. The abl operon was deleted in the genetically tractable M. maripaludis. Delta(abl) mutants of M. maripaludis no longer produced N(epsilon)-acetyl-beta-lysine and were incapable of growth at high salt concentrations, indicating that the abl operon is essential for N(epsilon)-acetyl-beta-lysine synthesis. These experiments revealed the first genes involved in the biosynthesis of compatible solutes in methanogens.

Acetyltransferases↗
PubMed1994

Fibrillarin-like proteins occur in the domain Archaea.

Fibrillarin is found in the nucleolus of Eucarya and associated with small nucleolar RNAs. It is involved in the processing of precursor rRNA. Two genes, encoding fibrillarin-like proteins from Methanococcus voltae and Methanococcus vannielii, have been isolated. The genes were named flpA (fibrillarin-like protein).

Amino Acid Sequence↗
PubMed1998

Sequence divergence of seryl-tRNA synthetases in archaea.

The genomic sequences of Methanococcus jannaschii and Methanobacterium thermoautotrophicum contain a structurally uncommon seryl-tRNA synthetase (SerRS) sequence and lack an open reading frame (ORF) for the canonical cysteinyl-tRNA synthetase (CysRS). Therefore, it is not clear if Cys-tRNACys is formed by direct aminoacylation or by a transformation of serine misacylated to tRNACys. To address this question, we prepared SerRS from two methanogenic archaea and measured the enzymatic properties of these proteins. SerRS was purified from M. thermoautotrophicum; its N-terminal peptide sequence matched the sequence deduced from the relevant ORF in the genomic data of M. thermoautotrophicum and M. jannaschii. In addition, SerRS was expressed from a cloned Methanococcus maripaludis serS gene. The two enzymes charged serine to their homologous tRNAs and also accepted Escherichia coli tRNA as substrate for aminoacylation. Gel shift experiments showed that M. thermoautotrophicum SerRS did not mischarge tRNACys with serine. This indicates that Cys-tRNACys is formed by direct acylation in these organisms.

Amino Acid Sequence↗
PubMed2001

Events during initiation of archaeal transcription: open complex formation and DNA-protein interactions.

Transcription in Archaea is initiated by association of a TATA box binding protein (TBP) with a TATA box. This interaction is stabilized by the binding of the transcription factor IIB (TFIIB) orthologue TFB. We show here that the RNA polymerase of the archaeon Methanococcus, in contrast to polymerase II, does not require hydrolysis of the beta-gamma bond of ATP for initiation of transcription and open complex formation on linearized DNA. Permanganate probing revealed that the archaeal open complex spanned at least the DNA region from -11 to -1 at a tRNA(Val) promoter. The Methanococcus TBP-TFB promoter complex protected the DNA region from -40 to -14 on the noncoding DNA strand and the DNA segment from -36 to -17 on the coding DNA strand from DNase I digestion. This DNase I footprint was extended only to the downstream end by the addition of the RNA polymerase to position +17 on the noncoding strand and to position +13 on the coding DNA strand.

Adenosine Triphosphate↗
PubMed1997

A structural census of genomes: comparing bacterial, eukaryotic, and archaeal genomes in terms of protein structure.

Representative genomes from each of the three kingdoms of life are compared in terms of protein structure, in particular, those of Haemophilus influenzae (a bacteria), Methanococcus jannaschii (an archaeon), and yeast (a eukaryote). The comparison is in the form of a census (or comprehensive accounting) of the relative occurrence of secondary and tertiary structures in the genomes, which particular emphasis on patterns of supersecondary structure. Comparison of secondary structure shows that the three genomes have nearly the same overall secondary-structure content, although they differ markedly in amino acid composition. Comparison of super-secondary structure, using a novel "frequent-words" approach, shows that yeast has a preponderance of consecutive strands (e.g. beta-beta-beta patterns), Haemophilus, consecutive helices (alpha-alpha-alpha), and Methanococcus, alternating helix-strand structures (beta-alpha-beta). Yeast also has significantly more helical membrane proteins than the other two genomes, with most of the differences concentrated in proteins containing two transmembrane segments. Comparison of tertiary structure (by sequence matching and domain-level clustering) highlights the substantial duplication in each genome (approximately 30% to 50%), with the degree of duplication following similar patterns in all three. Many sequence families are shared among the genomes, with the degree of overlap between any two genomes being roughly similar. In total, the three genomes contain 148 of the approximately 300 known protein folds. Forty-five of these 148 that are present in all three genomes are especially enriched in mixed super-secondary structures (alpha/beta). Moreover, the five most common of these 45 (the "top-5") have a remarkably similar super-secondary structure architecture, containing a central sheet of parallel strands with helices packed onto at least one face and beta-alpha-beta connections between adjacent strands. These most basic molecular parts, which, presumably, were present in the last common ancestor to the three Kingdoms, include the TIM-barrel, Rossmann, flavodoxin, thiamin-binding, and P-loop-hydrolase folds.

Archaea↗
PubMed2001

Archaeal RecA homologues: different response to DNA-damaging agents in mesophilic and thermophilic Archaea.

Two archaeal proteins, RadA and RadB, share similarity with the RecA/Rad51 family of recombinases, with RadA being the functional homologue. We have studied and compared the RadA and RadB proteins of mesophilic and thermophilic Archaea. In growing cells, RadA levels are similar in mesophilic Methanococcus species and the hyperthermophile Methanococcus jannaschii. Treatment of cells with mutagenic agents (methylmethane sulfonate or UV light) increased the expression of RadA (as evidenced by higher levels of both mRNA and protein) in all organisms tested, but the increase was greater in the mesophiles than in the thermophiles M. jannaschii and Sulfolobus solfataricus. Recombinantly expressed RadA proteins from the mesophile M. voltae and the thermophile M. jannaschii were similar in their ATPase- and DNA-binding activities. All the data are consistent with proposals that RadA plays the same role as eukaryotic Rad51. Surprisingly, the data also suggested that the thermophiles do not need more RadA protein or activity than the mesophiles. On the other hand, RadB is not coregulated with RadA, and its role remains unclear. Neither RadA nor RadB from a mesophile or from a thermophile rescued the UV-sensitive phenotype of an Escherichia coli recA- host.

Archaea↗
PubMed1991

Cloning and sequencing of the fus-gene encoding elongation factor 2 in the archaebacterium Thermoplasma acidophilum.

We have cloned a 1.6-kb region of chromosomal DNA from Thermoplasma acidophilum into Escherichia coli using as a probe part of the Methanococcus vannielii fus-gene. The sequence of the clone was highly homologous to part of the corresponding Methanococcus vannielii gene. By chromosome walking, a 4.7-kb EcoRI fragment containing the complete gene was isolated. Nucleotide sequencing revealed an open reading frame of 2196 nucleotides. The deduced amino acid sequence contains the known peptide sequence around the ADP-ribosylation site of T. acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned. The amino acid sequence was compared to that of hamster and E. coli, as well as to known archaebacterial EF-2 sequences.

Amino Acid Sequence↗