The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis.

Many aspects of and factors required for DNA replication are conserved across all three domains of life, but there are some significant differences surrounding lagging-strand synthesis. In Archaea, a 5'-to-3' exonuclease, related to both bacterial RecJ and eukaryotic Cdc45, that associates with the replisome specifically through interactions with GINS was identified and designated GAN (for GINS-associated nuclease). Despite the presence of a well-characterized flap endonuclease (Fen1), it was hypothesized that GAN might participate in primer removal during Okazaki fragment maturation, and as a Cdc45 homologue, GAN might also be a structural component of an archaeal CMG (Cdc45, MCM, and GINS) replication complex. We demonstrate here that, individually, either Fen1 or GAN can be deleted, with no discernible effects on viability and growth. However, deletion of both Fen1 and GAN was not possible, consistent with both enzymes catalyzing the same step in primer removal from Okazaki fragments in vivo RNase HII has also been proposed to participate in primer processing during Okazaki fragment maturation. Strains with both Fen1 and RNase HII deleted grew well. GAN activity is therefore sufficient for viability in the absence of both RNase HII and Fen1, but it was not possible to construct a strain with both RNase HII and GAN deleted. Fen1 alone is therefore insufficient for viability in the absence of both RNase HII and GAN. The ability to delete GAN demonstrates that GAN is not required for the activation or stability of the archaeal MCM replicative helicase.IMPORTANCE The mechanisms used to remove primer sequences from Okazaki fragments during lagging-strand DNA replication differ in the biological domains. Bacteria use the exonuclease activity of DNA polymerase I, whereas eukaryotes and archaea encode a flap endonuclease (Fen1) that cleaves displaced primer sequences. RNase HII and the GINS-associated exonuclease GAN have also been hypothesized to assist in primer removal in Archaea Here we demonstrate that in Thermococcus kodakarensis, either Fen1 or GAN activity is sufficient for viability. Furthermore, GAN can support growth in the absence of both Fen1 and RNase HII, but Fen1 and RNase HII are required for viability in the absence of GAN.

Archaeal DNA polymerase D but not DNA polymerase B is required for genome replication in Thermococcus kodakarensis.

Three evolutionarily distinct families of replicative DNA polymerases, designated polymerase B (Pol B), Pol C, and Pol D, have been identified. Members of the Pol B family are present in all three domains of life, whereas Pol C exists only in Bacteria and Pol D exists only in Archaea. Pol B enzymes replicate eukaryotic chromosomal DNA, and as members of the Pol B family are present in all Archaea, it has been assumed that Pol B enzymes also replicate archaeal genomes. Here we report the construction of Thermococcus kodakarensis strains with mutations that delete or inactivate key functions of Pol B. T. kodakarensis strains lacking Pol B had no detectable loss in viability and no growth defects or changes in spontaneous mutation frequency but had increased sensitivity to UV irradiation. In contrast, we were unable to introduce mutations that inactivated either of the genes encoding the two subunits of Pol D. The results reported establish that Pol D is sufficient for viability and genome replication in T. kodakarensis and argue that Pol D rather than Pol B is likely the replicative DNA polymerase in this archaeon. The majority of Archaea contain Pol D, and, as discussed, if Pol D is the predominant replicative polymerase in Archaea, this profoundly impacts hypotheses for the origin(s), evolution, and distribution of the different DNA replication enzymes and systems now employed in the three domains of life.

Thermococcus kodakarensis has two functional PCNA homologs but only one is required for viability.

Proliferating cell nuclear antigen (PCNA) monomers assemble to form a ring-shaped clamp complex that encircles duplex DNA. PCNA binding to other proteins tethers them to the DNA providing contacts and interactions for many other enzymes essential for DNA metabolic processes. Most eukarya and euryarchaea have only one PCNA homolog but Thermococcus kodakarensis uniquely has two, designated PCNA1 and PCNA2, encoded by TK0535 and TK0582, respectively. Here, we establish that both PCNA1 and PCNA2 form homotrimers that stimulate DNA synthesis by archaeal DNA polymerases B and D and ATP hydrolysis by the replication factor C complex. In exponentially growing cells, PCNA1 is abundant and present at an ~100-fold higher concentration than PCNA2 monomers. Deletion of TK0582 (PCNA2) had no detectable effects on viability or growth whereas repeated attempts to construct a T. kodakarensis strain with TK0535 (PCNA1) deleted were unsuccessful. The implications of these observations for PCNA1 function and the origin of the two PCNA-encoding genes in T. kodakarensis are discussed.

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. To explore the reductant flux and possible competition for H2 production in vivo, T. kodakarensis TS517 was mutated to precisely delete each of the alternative pathways of reductant disposal, H2 production and consumption. The results obtained establish that H2 is generated predominantly by the membrane-bound hydrogenase complex (Mbh), confirm the essential role of the SurR (TK1086p) regulator in vivo, delineate the roles of sulfur (S°) regulon proteins and demonstrate that preventing H2 consumption results in a substantial net increase in H2 production. Constitutive expression of TK1086 (surR) from a replicative plasmid restored the ability of T. kodakarensis TS1101 (ΔTK1086) to grow in the absence of S° and stimulated H2 production, revealing a second mechanism to increase H2 production. Transformation of T. kodakarensis TS1101 with plasmids that express SurR variants constructed to direct the constitutive synthesis of the Mbh complex and prevent expression of the S° regulon was only possible in the absence of S° and, under these conditions, the transformants exhibited wild-type growth and H2 production. With S° present, they grew slower but synthesized more H2 per unit biomass than T. kodakarensis TS517.

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. Based on the results obtained, a network of interactions among the archaeal replication proteins has been established that confirms previously documented and predicted interactions, provides experimental evidence for previously unrecognized interactions between proteins with known functions and with unknown functions, and establishes a firm experimental foundation for archaeal replication research. The proteins identified and their participation in archaeal DNA replication are discussed and related to their bacterial and eukaryotic counterparts.

Thermococcus kodakarensis genetics: TK1827-encoded beta-glycosidase, new positive-selection protocol, and targeted and repetitive deletion technology.

Inactivation of TK1761, the reporter gene established for Thermococcus kodakarensis, revealed the presence of a second beta-glycosidase that we have identified as the product of TK1827. This enzyme (pTK1827) has been purified and shown to hydrolyze glucopyranoside but not mannopyranoside, have optimal activity at 95 degrees C and from pH 8 to 9.5, and have a functional half-life of approximately 7 min at 100 degrees C. To generate a strain with both TK1761 and TK1827 deleted, a new selection/counterselection protocol has been developed, and the levels of beta-glycosidase activity in T. kodakarensis strains with TK1761 and/or TK1827 deleted and with these genes expressed from heterologous promoters are described. Genetic tools and strains have been developed that extend the use of this selection/counterselection procedure to delete any nonessential gene from the T. kodakarensis chromosome. Using this technology, TK0149 was deleted to obtain an agmatine auxotroph that grows on nutrient-rich medium only when agmatine is added. Transformants can therefore be selected rapidly, and replicating plasmids can be maintained in this strain growing in rich medium by complementation of the TK0149 deletion.

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. Template DNAs were amplified from each T. kodakarensis strain, and transcription in vitro by T. kodakarensis RNA polymerase was terminated by sequences that reduced TK1761 expression in vivo. Termination occurred at additional sites on these linear templates, including at a 5'-AAAAAAAA (A(8)) sequence that did not reduce TK1761 expression in vivo. When these sequences were transcribed on supercoiled plasmid templates, termination occurred almost exclusively at oligo(T) sequences. The results provide the first in vivo experimental evidence for intrinsic termination of archaeal transcription and confirm that archaeal transcription termination is stimulated by oligo(T) sequences and is different from the RNA hairpin-dependent mechanism established for intrinsic bacterial termination.

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. kodakaraensis transformants could be selected by DeltatrpE complementation and/or mevinolin resistance. Based on Western blot measurements, shuttle vector expression of RpoL-HA, a hemagglutinin (HA) epitope-tagged subunit of T. kodakaraensis RNA polymerase (RNAP), was approximately 8-fold higher than chromosome expression. An idealized ribosome binding sequence (5'-AGGTGG) was incorporated for RpoL-HA expression, and changes to this sequence reduced expression. Changing the translation initiation codon from AUG to GUG did not reduce RpoL-HA expression, but replacing AUG with UUG dramatically reduced RpoL-HA synthesis. When functioning as translation initiation codons, AUG, GUG, and UUG all directed the incorporation of methionine as the N-terminal residue of RpoL-HA synthesized in T. kodakaraensis. Affinity purification confirmed that an HA- plus six-histidine-tagged RpoL subunit (RpoL-HA-his(6)) synthesized ectopically from a shuttle vector was assembled in vivo into RNAP holoenzymes that were active and could be purified directly from T. kodakaraensis cell lysates by Ni(2+) binding and imidazole elution.

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. In contrast, methanogenesis in wild-type cells was completely inhibited by the same amiloride concentration. ATP synthesis driven by methanogenic electron transport or by an electrogenic potassium efflux in the presence of sodium ions was significantly enhanced in the mutant cells. ATP synthesis driven by potassium diffusion potential was profoundly inhibited in wild-type cells by the presence of uncoupler 3,3',4',5- tetrachlorosalicylanilide and sodium ions, whereas c. 50% inhibition was observed in the mutant cells under the same conditions.

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.k.) to initiate transcription from a variety of T.k. promoters in vitro when provided with T.k. TBP and either TFB1 or TFB2, the two TFBs encoded in the T.k. genome. With every promoter active in vitro, transcription initiation occurred with either TFB1 or TFB2 although the optimum salt concentration for initiation was generally higher for TFB2 (approximately 250 mM K(+)) than for TFB1 (approximately 200 mM K(+)). Consistent with this functional redundancy in vitro, T.k. strains have been constructed with the TFB1- (tfb1; TK1280) or TFB2- (tfb2; TK2287) encoding gene deleted. These mutants exhibit no detectable growth defects under laboratory conditions. Domain swapping between TFB1 and TFB2 has identified a central region that contributes to the salt sensitivity of TFB activity, and deleting residues predicted to form the tip of the B-finger region of TFB2 had no detectable effects on promoter recognition or transcription initiation but did eliminate the production of very short (< or =5 nt) abortive transcripts.

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. An abundant membrane-associated protein complex with molecular mass approximately 670 kDa was detected in the mutant strain after native PAGE. The results indicate that TCS resistance in this mutant has arisen as a consequence of mutation(s) that affects a specific locus coding for an uncoupler binding protein(s) and/or modulate the activity of unidentified ATPase.

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. The generation of a membrane potential in the mutant cells at pH 6.8 was also strongly inhibited by TCS in the presence of 1 or 10 mM NaCl. On the other hand, at pH 8.5 in the presence of 10mM NaCl, a protonophore-resistant membrane potential of approximately 150 mV was found. These results indicate that in the mutant cells the process of energy transduction between methanogenesis and membrane potential generation is not impaired. In contrast to the wild-type strain, ATP synthesis in the mutant cells was driven by an electrochemical gradient of H(+) under alkaline conditions. Unlike wild-type cells, the mutant lacks the capacity to transduce an uncoupler-resistant membrane potential energy at pH 8.5 into ATP synthesis. Na(+)/H(+) exchange was comparable in the wild type and the mutant cells. Western blots of sub-cellular fractions with polyclonal antiserum reactive to the B-subunit of the halobacterial A-type H(+)-translocating ATPase confirmed the presence of A-type ATP synthase in the mutant cells. Furthermore, in the mutant cells a protein band of molecular mass about 45 kDa is absent but there was an abundant protein band at about 67 kDa. Based on the observed bioenergetic features of the mutant cells, neither the A(1)A(o) ATP synthase alone nor together with the Na(+)/H(+) antiporter seems to be responsible for ATP synthesis driven by sodium motive force. Rather, some other links between neomycin-resistance and failure of sodium motive force-dependent ATP synthesis in the neomycin resistant mutant are discussed.