Identification of the Methanococcus voltae S-layer structural gene.

We have established that the gene which we had previously identified as encoding the Methanococcus voltae P-type ATPase is, in fact, the structural gene for the M. voltae S-layer protein. This conclusion is based on a comparison of the N-terminal sequence of S-layer protein prepared by two independent methods with that derived from the nucleotide sequence of the cloned gene. This conclusion was further supported by immunocytochemical localization of the antigen directed against the antibodies used in the cloning experiments.     

Isolation of acetate auxotrophs of the methane-producing archaeon Methanococcus maripaludis by random insertional mutagenesis.

To learn more about autotrophic growth of methanococci, we isolated nine conditional mutants of Methanococcus maripaludis after transformation of the wild type with a random library in pMEB.2, a suicide plasmid bearing the puromycin-resistance cassette pac. These mutants grew poorly in mineral medium and required acetate or complex organic supplements such as yeast extract for normal growth. One mutant, JJ104, was a leaky acetate auxotroph. A plasmid, pWDK104, was recovered from this mutant by electroporation of a plasmid preparation into Escherichia coli. Transformation of wild-type M. maripaludis with pWDK104 produced JJ104-1, a mutant with the same phenotype as JJ104, thus establishing that insertion of pWDK104 into the genome was responsible for the phenotype. pWDK104 contained portions of the methanococcal genes encoding an ABC transporter closely related to MJ1367-MJ1368 of M. jannaschii. Because high levels of molybdate, tungstate, and selenite restored growth to wild-type levels, this transporter may be specific for these oxyanions. A second acetate auxotroph, JJ117, had an absolute growth requirement for either acetate or cobalamin, and wild-type growth was observed only in the presence of both. Cobinamide, 5', 6'-dimethylbenzimidazole, and 2-aminopropanol did not replace cobalamin. This phenotype was correlated with tandem insertions in the genome but not single insertions and appeared to have resulted from an indirect effect on cobamide metabolism. Plasmids rescued from other mutants contained portions of ORFs denoted in M. jannaschii as endoglucanase (MJ0555), transketolase (MJ0681), thiamine biosynthetic protein thiI (MJ0931), and several hypothetical proteins (MJ1031, MJ0835, and MJ0835.1).     

Cleavage of preflagellins by an aspartic acid signal peptidase is essential for flagellation in the archaeon Methanococcus voltae

The differences between archaeal and bacterial flagella are becoming more apparent as research on the archaeal structure progresses. One crucial difference is the presence of a leader peptide on archaeal preflagellins, which is removed from the flagellin prior to its incorporation into the flagellar filament. The enzyme responsible for the removal of the flagellin leader peptide was identified as FlaK. FlaK of Methanococcus voltae retains its preflagellin peptidase activity when expressed in Escherichia coli and used in an in vitro assay. Homologous recombination of an integration vector into the chromosomal copy of flaK resulted in a non-motile, non-flagellated phenotype. The flagellins of the mutant had larger molecular weights than their wild-type counterparts, as expected if they retained their 11- to 12-amino-acid leader peptide. Membranes of the flaK mutant were unable to process preflagellin in the in vitro assay. Site-directed mutagenesis demonstrated that two aspartic acid residues conserved with ones in type IV prepilin peptidases were necessary for proper recognition or processing of the preflagellin. As bacterial flagellins lack a leader peptide and a peptidase is not required for export and assembly, the requirement for FlaK further emphasizes the similarity archaeal flagella have with type IV pili, rather than with bacterial flagella.     

Ultrastructure and biochemistry of the cell wall of Methanococcus voltae.

The ultrastructure and chemical composition of the cell wall of the marine archaebacterium Methanococcus voltae were studied by negative-staining and freeze-etch electron microscopy and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. M. voltae possesses a single regularly structured (RS) protein layer external to the plasma membrane. Freeze-etch preparations of cells indicated that the protein subunits are hexagonally arranged with a center-to-center spacing of approximately 10 nm. The extracted RS protein had a molecular weight of 76,000. It was present on envelopes prepared by shearing in a French press, osmotic lysis, or sonication, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NaCl was not required for attachment of the RS protein to the underlying plasma membrane. The hexagonal array could be demonstrated by platinum shadowing and freeze-etching of envelopes, but negative staining in the abscence of NaCl failed to stabilize the array. The RS protein could be solubilized by urea, guanidine hydrochloride, dithiothreitol, and several detergents, including Nonidet P-40, Triton X-100, and Tween 20. However, the most specific release of the wall protein from envelopes occurred after a heat treatment in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at 50 to 60 degrees C.     

A lysR-type regulator is involved in the negative regulation of genes encoding selenium-free hydrogenases in the archaeon Methanococcus voltae

The archaeon Methanococcus voltae encodes two pairs of NiFe-hydrogenase isoenzymes. One hydrogenase of each pair contains selenium in the active site, whereas the other one is selenium-free. The gene groups for the selenium-free hydrogenases, called vhc and frc, are linked by a common intergenic region. They are only transcribed under selenium limitation. A protein binding to a negative regulatory element involved in the regulation of the two operons was purified by DNA-affinity chromatography. Through the identification of the corresponding gene the protein was found to be a LysR-type regulator. It was named HrsM (hydrogenase gene regulator, selenium dependent in M. voltae). hrsM knockout mutants constitutively transcribed the vhc and frc operons in the presence of selenium. A putative HrsM binding site was also detected in the intergenic region in front of the hrsM gene. Northern blot analysis indicated that the hrsM gene might be autoregulated.     

Identification and characterisation of structural maintenance of chromosome 1 (smc1) mutants of Coprinopsis cinerea

A Coprinopsis cinerea homokaryotic fruiting strain was mutagenised, identifying a mutant that exhibited a hyphal growth temperature sensitive defect and hyphal knot development defect at an early fruiting stage, even at the hyphal growth permissive temperature. Microscopic observation suggested that the mutant nuclei exhibited defects in the metaphase to anaphase transition at the restrictive temperature. The gene in which the mutation occurred was cloned, sequenced and determined to be homologous to smc1. Sequence analyses of the mutant revealed deletion of 28 base pairs in the 19th intron of the Cc.smc1 gene, resulting in complete failure of splicing of that intron and in insertion of 14 amino acids in the C-terminal region of the Cc.Smc1 protein. We isolated eight hyphal growth revertants and identified four intragenic suppressors. All were the result of amino acid substitutions in the C-terminal region. Three of the suppressors caused reversion of the arrest in an early fruiting stage. One of the suppressors exhibited cold sensitivity and failed to suppress the fruiting defect, suggesting that flexibility of a lobe in the C-terminal region is important for proper function of Cc.Smc1.     

Distribution of the Escherichia coli structural maintenance of chromosomes (SMC)-like protein MukB in the cell

Fluorescent polyclonal antibodies specific for MukB have been used to study its localization in Escherichia coli. In wild-type cells, the MukB protein appeared as a limited number of oblong shapes embracing the nucleoid. MukB remained associated with the nucleoid in the absence of DNA replication. The centre of gravity of the dispersed MukB signal initially localized near mid-cell, but moved to approximately quarter positions well before the termination of DNA replication and its subsequent reinitiation. Because MukB had been reported to bind to FtsZ and to its eukaryotic homologue tubulin in vitro, cells were co-labelled with MukB- and FtsZ-specific fluorophores. No co-localization of MukB with polymerized FtsZ (the FtsZ ring) was observed at any time during the cell cycle. A possible role for MukB in preventing premature FtsZ polymerization and in DNA folding that might assist DNA segregation is discussed.     

SMC (structural maintenance of chromosomes) structural protein family and their role in chromatin reorganization

Structural chromatin proteins of the SMC (Structural Maintenance of Chromosomes) family play an important role in structural DNA reorganization in pro- and eukaryotes. Eukaryotic SMC proteins are the core components of the cohesin and condensin complexes. The cohesin complex is responsible for sister chromatid and homolog cohesion in mitosis and meiosis. The condensin complex uses ATP energy to induce positive coiled-coils in DNA, which results in compaction of the latter and formation of mitotic chromosome scaffold. In addition, the SMC proteins constitute recombination and recombination repair complexes. In hermaphrodites of nematode Caenorhabditis elegans, the SMC protein-containing complex controls dosage compensation and inactivation of the X chromosome genes.     

Hydrogenase-independent uptake and metabolism of electrons by the archaeon Methanococcus maripaludis

Direct, shuttle-free uptake of extracellular, cathode-derived electrons has been postulated as a novel mechanism of electron metabolism in some prokaryotes that may also be involved in syntrophic electron transport between two microorganisms. Experimental proof for direct uptake of cathodic electrons has been mostly indirect and has been based on the absence of detectable concentrations of molecular hydrogen. However, hydrogen can be formed as a transient intermediate abiotically at low cathodic potentials (<−414 mV) under conditions of electromethanogenesis. Here we provide genetic evidence for hydrogen-independent uptake of extracellular electrons. Methane formation from cathodic electrons was observed in a wild-type strain of the methanogenic archaeon Methanococcus maripaludis as well as in a hydrogenase-deletion mutant lacking all catabolic hydrogenases, indicating the presence of a hydrogenase-independent mechanism of electron catabolism. In addition, we discovered a new route for hydrogen or formate production from cathodic electrons: Upon chemical inhibition of methanogenesis with 2-bromo-ethane sulfonate, hydrogen or formate accumulated in the bioelectrochemical cells instead of methane. These results have implications for our understanding on the diversity of microbial electron uptake and metabolism.     

Isolation of a coenzyme M-auxotrophic mutant and transformation by electroporation in Methanococcus voltae.

An auxotrophic mutant of Methanococcus voltae was isolated that required coenzyme M (CoM) for growth. With the mutant as a recipient, conditions were developed that allowed the introduction of wild-type CoM+ DNA into the mutant methanogen via electroporation. This method also allowed the rescue of both a histidine and purine auxotroph as well as the introduction of DNA determining resistance to the CoM analog 2-bromoethanesulfonic acid. Electroporation of the CoM(+)-determining DNA was 50- to 80-fold more efficient than natural transformation.     

Localization of the F420-reducing hydrogenase in Methanococcus voltae cells by immuno-gold technique

The F₄₂₀-reducing hydrogenase of Methanococcus voltae, which takes part in the terminal reduction step of methanogenesis, was localized in situ in ultrathin sections. This result was obtained by the immuno-gold technique using a high titer antiserum raised against the purified enzyme. Its specifity for the hydrogenase was shown by Western blot analysis. The hydrogenase of M. voltae was found to be membrane-associated.Abbreviations ELISA Enzyme linked immuno sorbent assay - F₄₂₀ 8-hydroxy-5-deazaflavin     

Subcellular localization of the Bacillus subtilis structural maintenance of chromosomes (SMC) protein

The Bacillus subtilis structural maintenance of chromosomes (SMC) protein is a member of a large family of proteins involved in chromosome organization. We found that SMC is a moderately abundant protein ( approximately 1000 dimers per cell). In vivo cross-linking and immunoprecipitation assays revealed that SMC binds to many regions on the chromosome. Visualization of SMC in live cells using a fusion to the green fluorescent protein (GFP) and in fixed cells using immunofluorescence microscopy indicated that a portion of SMC localizes as discrete foci in positions similar to that of the DNA replication machinery (replisome). When visualized simultaneously, SMC and the replisome were often in similar regions of the cell but did not always co-localize. Persistence of SMC foci did not depend on ongoing replication, but did depend on ScpA and ScpB, two proteins thought to interact with SMC. Our results indicate that SMC is bound to many sites on the chromosome and a concentration of SMC is localized near replication forks, perhaps there to bind and organize newly replicated DNA.