Genetics Techniques for Thermococcus kodakarensis

Thermococcus kodakarensis (T. kodakarensis) has emerged as a premier model system for studies of archaeal biochemistry, genetics, and hyperthermophily. This prominence is derived largely from the natural competence of T. kodakarensis and the comprehensive, rapid, and facile techniques available for manipulation of the T. kodakarensis genome. These genetic capacities are complemented by robust planktonic growth, simple selections, and screens, defined in vitro transcription and translation systems, replicative expression plasmids, in vivo reporter constructs, and an ever-expanding knowledge of the regulatory mechanisms underlying T. kodakarensis metabolism. Here we review the existing techniques for genetic and biochemical manipulation of T. kodakarensis. We also introduce a universal platform to generate the first comprehensive deletion and epitope/affinity tagged archaeal strain libraries

Association of a Multi-Synthetase Complex with Translating Ribosomes in the Archaeon \u3cem\u3eThermococcus kodakarensis\u3c/em\u3e

In archaea and eukaryotes aminoacyl-tRNA synthetases (aaRSs) associate in multi-synthetase complexes (MSCs), however the role of such MSCs in translation is unknown. MSC function was investigated in vivo in the archaeon Thermococcus kodakarensis, wherein six aaRSs were affinity co-purified together with several other factors involved in protein synthesis, suggesting that MSCs may interact directly with translating ribosomes. In support of this hypothesis, the aminoacyltRNA synthetase (aaRS) activities of the MSC were enriched in isolated T. kodakarensis polysome fractions. These data indicate that components of the archaeal protein synthesis machinery associate into macromolecular assemblies in vivo and provide the potential to increase translation efficiency by limiting substrate diffusion away from the ribosome, thus facilitating rapid recycling of tRNAs

Competition between auditory and visual spatial cues during visual task performance

There is debate in the crossmodal cueing literature as to whether capture of visual attention by means of sound is a fully automatic process. Recent studies show that when visual attention is endogenously focused sound still captures attention. The current study investigated whether there is interaction between exogenous auditory and visual capture. Participants preformed an orthogonal cueing task, in which, the visual target was preceded by both a peripheral visual and auditory cue. When both cues were presented at chance level, visual and auditory capture was observed. However, when the validity of the visual cue was increased to 80% only visual capture and no auditory capture was observed. Furthermore, a highly predictive (80% valid) auditory cue was not able to prevent visual capture. These results demonstrate that crossmodal auditory capture does not occur when a competing predictive visual event is presented and is therefore not a fully automatic process

The Nuclear Pore Complex Mediates Binding of the Mig1 Repressor to Target Promoters

All eukaryotic cells alter their transcriptional program in response to the sugar glucose. In Saccharomyces cerevisiae, the best-studied downstream effector of this response is the glucose-regulated repressor Mig1. We show here that nuclear pore complexes also contribute to glucose-regulated gene expression. NPCs participate in glucose-responsive repression by physically interacting with Mig1 and mediating its function independently of nucleocytoplasmic transport. Surprisingly, despite its abundant presence in the nucleus of glucose-grown nup120Δ or nup133Δ cells, Mig1 has lost its ability to interact with target promoters. The glucose repression defect in the absence of these nuclear pore components therefore appears to result from the failure of Mig1 to access its consensus recognition sites in genomic DNA. We propose that the NPC contributes to both repression and activation at the level of transcription

Archaeal Nucleosome Positioning In Vivo and In Vitro is Directed by Primary Sequence Motifs

Background: Histone wrapping of DNA into nucleosomes almost certainly evolved in the Archaea, and predates Eukaryotes. In Eukaryotes, nucleosome positioning plays a central role in regulating gene expression and is directed by primary sequence motifs that together form a nucleosome positioning code. The experiments reported were undertaken to determine if archaeal histone assembly conforms to the nucleosome positioning code. Results: Eukaryotic nucleosome positioning is favored and directed by phased helical repeats of AA/TT/AT/TA and CC/GG/CG/GC dinucleotides, and disfavored by longer AT-rich oligonucleotides. Deep sequencing of genomic DNA protected from micrococcal nuclease digestion by assembly into archaeal nucleosomes has established that archaeal nucleosome assembly is also directed and positioned by these sequence motifs, both in vivo in Methanothermobacter thermautotrophicus and Thermococcus kodakarensis and in vitro in reaction mixtures containing only one purified archaeal histone and genomic DNA. Archaeal nucleosomes assembled at the same locations in vivo and in vitro, with much reduced assembly immediately upstream of open reading frames and throughout the ribosomal rDNA operons. Providing further support for a common positioning code, archaeal histones assembled into nucleosomes on eukaryotic DNA and eukaryotic histones into nucleosomes on archaeal DNA at the same locations. T. kodakarensis has two histones, designated HTkA and HTkB, and strains with either but not both histones deleted grow normally but do exhibit transcriptome differences. Comparisons of the archaeal nucleosome profiles in the intergenic regions immediately upstream of genes that exhibited increased or decreased transcription in the absence of HTkA or HTkB revealed substantial differences but no consistent pattern of changes that would correlate directly with archaeal nucleosome positioning inhibiting or stimulating transcription. Conclusions: The results obtained establish that an archaeal histone and a genome sequence together are sufficient to determine where archaeal nucleosomes preferentially assemble and where they avoid assembly. We confirm that the same nucleosome positioning code operates in Archaea as in Eukaryotes and presumably therefore evolved with the histone-fold mechanism of DNA binding and compaction early in the archaeal lineage, before the divergence of Eukaryotes

Thermococcus kodakarensis encodes three MCM homologs but only one is essential

The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea and eukaryotes. In eukaryotes, this complex is an assembly of six different but related polypeptides (MCM2-7) but, in most archaea, one MCM protein assembles to form a homohexameric complex. Atypically, the Thermococcus kodakarensis genome encodes three archaeal MCM homologs, here designated MCM1-3, although MCM1 and MCM2 are unusual in having long and unique N-terminal extensions. The results reported establish that MCM2 and MCM3 assemble into homohexamers and exhibit DNA binding, helicase and ATPase activities in vitro typical of archaeal MCMs. In contrast, MCM1 does not form homohexamers and although MCM1 binds DNA and has ATPase activity, it has only minimal helicase activity in vitro. Removal of the N-terminal extension had no detectable effects on MCM1 but increased the helicase activity of MCM2. A T. kodakarensis strain with the genes TK0096 (MCM1) and TK1361 (MCM2) deleted has been constructed that exhibits no detectable defects in growth or viability, but all attempts to delete TK1620 (MCM3) have been unsuccessful arguing that that MCM3 is essential and is likely the replicative helicase in T. kodakarensis. The origins and possible function(s) of the three MCM proteins are discussed

Kinetic Investigation of Escherichia coli RNA Polymerase Mutants That Influence Nucleotide Discrimination and Transcription Fidelity

Recent RNA polymerase (RNAP) structures led to a proposed three-step model of nucleoside triphosphate (NTP) binding, discrimination, and incorporation. NTPs are thought to enter through the secondary channel, bind to an E site, rotate into a pre-insertion (PS) site, and ultimately align in the catalytic (A) site. We characterized the kinetics of correct and incorrect incorporation for several Escherichia coli RNAPs with substitutions in the proposed NTP entry pore (secondary channel). Substitutions of the semi-conserved residue betaAsp(675), which is >10A away from these sites, significantly reduce fidelity; however, substitutions of the totally conserved residues betaArg(678) and betaAsp(814) do not significantly alter the correct or incorrect incorporation kinetics, even though the corresponding residues in RNAPII crystal structures appear to be interacting with the NTP phosphate groups and coordinating the second magnesium ion in the active site, respectively. Structural analysis suggests that the lower fidelity of the betaAsp(675) mutants most likely results from reduction of the negative potential of a small pore between the E and PS sites and elimination of several structural interactions around the pore. We suggest a mechanism of nucleotide discrimination that is governed both by rotation of the NTP through this pore and subsequent rearrangement or closure of RNAP to align the NTP in the A site

A novel DNA nuclease is stimulated by association with the GINS complex

Chromosomal DNA replication requires the spatial and temporal coordination of the activities of several complexes that constitute the replisome. A previously uncharacterized protein, encoded by TK1252 in the archaeon Thermococcus kodakaraensis, was shown to stably interact with the archaeal GINS complex in vivo, a central component of the archaeal replisome. Here, we document that this protein (TK1252p) is a processive, single-strand DNA-specific exonuclease that degrades DNA in the 5′ → 3′ direction. TK1252p binds specifically to the GINS15 subunit of T. kodakaraensis GINS complex and this interaction stimulates the exonuclease activity in vitro. This novel archaeal nuclease, designated GINS-associated nuclease (GAN), also forms a complex in vivo with the euryarchaeal-specific DNA polymerase D. Roles for GAN in replisome assembly and DNA replication are discussed

A global fit to determine the pseudoscalar mixing angle and the gluonium content of the eta' meson

We update the values of the eta-eta' mixing angle and of the eta' gluonium content by fitting our measurement R_phi = BR(phi to eta' gamma)/ BR(phi to eta gamma) together with several vector meson radiative decays to pseudoscalars (V to P gamma), pseudoscalar mesons radiative decays to vectors (P to V gamma) and the eta' to gamma gamma, pi^0 to gamma gamma widths. From the fit we extract a gluonium fraction of Z^2_G = 0.12 +- 0.04, the pseudoscalar mixing angle psi_P = (40.4 +- 0.6) degree and the phi-omega mixing angle psi_V = (3.32 +- 0.09) degree. Z^2_G and psi_P are fairly consistent with those previously published. We also evaluate the impact on the eta' gluonium content determination of future experimental improvements of the eta' branching ratios and decay width.Comment: 13 pages, 7 figures to submit to JHE

Mammalian cell entry genes in Streptomyces may provide clues to the evolution of bacterial virulence

Understanding the evolution of virulence is key to appreciating the role specific loci play in pathogenicity. Streptomyces species are generally non-pathogenic soil saprophytes, yet within their genome we can find homologues of virulence loci. One example of this is the mammalian cell entry (mce) locus, which has been characterised in Mycobacterium tuberculosis. To investigate the role in Streptomyces we deleted the mce locus and studied its impact on cell survival, morphology and interaction with other soil organisms. Disruption of the mce cluster resulted in virulence towards amoebae (Acanthamoeba polyphaga) and reduced colonization of plant (Arabidopsis) models, indicating these genes may play an important role in Streptomyces survival in the environment. Our data suggest that loss of mce in Streptomyces spp. may have profound effects on survival in a competitive soil environment, and provides insight in to the evolution and selection of these genes as virulence factors in related pathogenic organisms