Deletion of alternative pathways for reductant recycling in Thermococcus kodakarensis increases hydrogen production.

Hydrogen (H₂) production by Thermococcus kodakarensis compares very favourably with the levels reported for the most productive algal, fungal and bacterial systems. T. kodakarensis can also consume H₂ and is predicted to use several alternative pathways to recycle reduced cofactors, some of which may compete with H₂ production for reductant disposal.

Affinity purification of an archaeal DNA replication protein network.

Nineteen Thermococcus kodakarensis strains have been constructed, each of which synthesizes a different His(6)-tagged protein known or predicted to be a component of the archaeal DNA replication machinery. Using the His(6)-tagged proteins, stable complexes assembled in vivo have been isolated directly from clarified cell lysates and the T. kodakarensis proteins present have been identified by mass spectrometry.

Archaeal intrinsic transcription termination in vivo.

Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) strains have been constructed with synthetic and natural DNA sequences, predicted to function as archaeal transcription terminators, identically positioned between a constitutive promoter and a beta-glycosidase-encoding reporter gene (TK1761). Expression of the reporter gene was almost fully inhibited by the upstream presence of 5'-TTTTTTTT (T(8)) and was reduced >70% by archaeal intergenic sequences that contained oligo(T) sequences. An archaeal intergenic sequence (t(mcrA)) that conforms to the bacterial intrinsic terminator motif reduced TK1761 expression approximately 90%, but this required only the oligo(T) trail sequence and not the inverted-repeat and loop region.

Shuttle vector expression in Thermococcus kodakaraensis: contributions of cis elements to protein synthesis in a hyperthermophilic archaeon.

Shuttle vectors that replicate stably and express selectable phenotypes in both Thermococcus kodakaraensis and Escherichia coli have been constructed. Plasmid pTN1 from Thermococcus nautilis was ligated to the commercial vector pCR2.1-TOPO, and selectable markers were added so that T.

Polarity in archaeal operon transcription in Thermococcus kodakaraensis.

An in vivo archaeal gene reporter system has been established based on TK1761, a gene that encodes a nonessential beta-glycosidase in Thermococcus kodakaraensis. Following the introduction of nonsense codons into promoter-proximal genes, polarity in operon expression in this archaeon has been established by both microarray hybridization assays and a reporter gene expression system.

Spontaneous trpY mutants and mutational analysis of the TrpY archaeal transcription regulator.

Over 90% of Methanothermobacter thermautotrophicus mutants isolated as spontaneously resistant to 5-methyl tryptophan had mutations in trpY. Most were single-base-pair substitutions that identified separate DNA- and tryptophan-binding regions in TrpY. In vivo and in vitro studies revealed that DNA binding was sufficient for TrpY repression of trpY transcription but that TrpY must bind DNA and tryptophan to assemble a complex that represses trpEGCFBAD.

Isolation and characterization of an amiloride-resistant mutant of Methanothermobacter thermautotrophicus possessing a defective Na+/H+ antiport.

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to the Na+/H+ antiporter inhibitor amiloride was isolated. The Na+/H+ exchanger activity in the mutant cells was remarkably decreased in comparison with wild-type cells. Methanogenesis rates in the mutant strain were higher than wild-type cells and resistant to the inhibitory effect of 2 mM amiloride.

TFB1 or TFB2 is sufficient for Thermococcus kodakaraensis viability and for basal transcription in vitro.

Archaeal RNA polymerases (RNAPs) are most similar to eukaryotic RNAP II (Pol II) but require the support of only two archaeal general transcription factors, TBP (TATA-box binding protein) and TFB (archaeal homologue of the eukaryotic general transcription factor TFIIB) to initiate basal transcription. However, many archaeal genomes encode more than one TFB and/or TBP leading to the hypothesis that different TFB/TBP combinations may be employed to direct initiation from different promoters in Archaea. As a first test of this hypothesis, we have determined the ability of RNAP purified from Thermococcus kodakaraensis (T.

Biochemical analysis of neomycin-resistance in the methanoarchaeon Methanothermobacter thermautotrophicus and some implications for energetic processes in this strain.

Methanogenesis-driven ATP synthesis in a neomycin-resistant mutant of Methanothermobacter thermautotrophicus (formerly Methanobacterium thermoautotrophicum strain DeltaH) was strongly inhibited at both pH 6.8 and pH 8.5 by the uncoupler 3,3',4',5 -tetrachlorosalicylanilide (TCS) in the presence of either 1 or 10 mM NaCl.

Histones in crenarchaea.

Archaeal histone-encoding genes have been identified in marine Crenarchaea. The protein encoded by a representative of these genes, synthesized in vitro and expressed in Escherichia coli, binds DNA and forms complexes with properties typical of an archaeal histone. The discovery of histones in Crenarchaea supports the argument that histones evolved before the divergence of Archaea and Eukarya.

Isolation and characterization of an uncoupler-resistant mutant of Methanothermobacter thermautotrophicus.

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to the protonophorous uncoupler TCS was isolated. The mutant strain exhibited increased CH(4) formation and elevated level of ATPase activity under non-growing conditions. ATP synthesis driven by methanogenic electron transport as well as by potassium diffusion potential in the presence of either H(+) or Na(+) ions was markedly diminished in the mutant strain.