Mannosylglycerate and Di-myo-Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress

Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities. Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for P. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.     

Vanadate-Resistant Mutants of Neurospora crassa Are Deficient in a High-Affinity Phosphate Transport System

Mutant strains of Neurospora crassa have been selected which grow on media containing vanadate, an inhibitor of the plasma membrane ATPase. The mutations all map to a single region (designated van) on the left arm of linkage group VII. The van mutants are unable to take up vanadate from the medium and are also deficient in the uptake of phosphate via a derepressible, high-affinity phosphate transport system. In the van mutants, the K(m) for phosphate transport is elevated as much as 35-fold, indicating that the van locus may code for a structural component of the high-affinity phosphate transport system.     

Association of a multi-synthetase complex with translating ribosomes in the archaeon Thermococcus kodakarensis

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 aminoacyl-tRNA 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.     

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.     

Regulation of Phosphate Metabolism in NEUROSPORA CRASSA: Isolation of Mutants Deficient in the Repressible Alkaline Phosphatase

Mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase, but, unlike nuc-1 and nuc-2 mutants, are able to make the repressible acid phosphatase and the repressible phosphate permease under conditions of derepression (phosphate deprivation). The new mutants, called pho-2, map in Linkage Group V, and are unlinked to the putative control mutants, nuc-1, nuc-2-pcon(c), and preg(c). Three of the pho-2 mutants do not make detectable amounts of repressible alkaline phosphatase, but the fourth makes about 1% of the level found in wild type. The small amount of alkaline phosphatase made by this strain appears to be qualitatively similar or identical to the wild-type enzyme, as judged by electrophoretic mobility, heat stability, and titration with specific antibody to the wild-type enzyme. Several revertants of this strain have been examined in the same way, and the alkaline phosphatase of these strains also appears to be qualitatively normal. Reversion events can occur at, or near, the pho-2 locus, but also occur in at least two unlinked sites (suppressor mutations). One suppressor maps very close to nuc-1.     

An Uncharacterized Member of the Ribokinase Family in Thermococcus kodakarensis Exhibits myo-Inositol Kinase Activity

Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high K_m values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater k_cat and much lower K_m value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in k_cat/K_m values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate.     

Replication protein A complex in Thermococcus kodakarensis interacts with DNA polymerases and helps their effective strand synthesis

Replication protein A (RPA) is an essential component of DNA metabolic processes. RPA binds to single-stranded DNA (ssDNA) and interacts with multiple DNA-binding proteins. In this study, we showed that two DNA polymerases, PolB and PolD, from the hyperthermophilic archaeon Thermococcus kodakarensis interact directly with RPA in vitro. RPA was expected to play a role in resolving the secondary structure, which may stop the DNA synthesis reaction, in the template ssDNA. Our in vitro DNA synthesis assay showed that the pausing was resolved by RPA for both PolB and PolD. These results supported the fact that RPA interacts with DNA polymerases as a member of the replisome and is involved in the normal progression of DNA replication forks.     

An alternative beads‐on‐a‐string chromatin architecture in Thermococcus kodakarensis

We have applied chromatin sequencing technology to the euryarchaeon Thermococcus kodakarensis, which is known to possess histone‐like proteins. We detect positioned chromatin particles of variable sizes associated with lengths of DNA differing as multiples of 30 bp (ranging from 30 bp to >450 bp) consistent with formation from dynamic polymers of the archaeal histone dimer. T. kodakarensis chromatin particles have distinctive underlying DNA sequence suggesting a genomic particle‐positioning code and are excluded from gene‐regulatory DNA suggesting a functional organization. Beads‐on‐a‐string chromatin is therefore conserved between eukaryotes and archaea but can derive from deployment of histone‐fold proteins in a variety of multimeric forms.     

Distinct physiological roles of the three [NiFe]-hydrogenase orthologs in the hyperthermophilic archaeon Thermococcus kodakarensis

Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H₂) and play a key role in the energy metabolism of microorganisms in anaerobic environments. The hyperthermophilic archaeon Thermococcus kodakarensis KOD1, which assimilates organic carbon coupled with the reduction of elemental sulfur (S⁰) or H₂ generation, harbors three gene operons encoding [NiFe]-hydrogenase orthologs, namely, Hyh, Mbh, and Mbx. In order to elucidate their functions in vivo, a gene disruption mutant for each [NiFe]-hydrogenase ortholog was constructed. The Hyh-deficient mutant (PHY1) grew well under both H₂S- and H₂-evolving conditions. H₂S generation in PHY1 was equivalent to that of the host strain, and H₂ generation was higher in PHY1, suggesting that Hyh functions in the direction of H₂ uptake in T. kodakarensis under these conditions. Analyses of culture metabolites suggested that significant amounts of NADPH produced by Hyh are used for alanine production through glutamate dehydrogenase and alanine aminotransferase. On the other hand, the Mbh-deficient mutant (MHD1) showed no growth under H₂-evolving conditions. This fact, as well as the impaired H₂ generation activity in MHD1, indicated that Mbh is mainly responsible for H₂ evolution. The copresence of Hyh and Mbh raised the possibility of intraspecies H₂ transfer (i.e., H₂ evolved by Mbh is reoxidized by Hyh) in this archaeon. In contrast, the Mbx-deficient mutant (MXD1) showed a decreased growth rate only under H₂S-evolving conditions and exhibited a lower H₂S generation activity, indicating the involvement of Mbx in the S⁰ reduction process. This study provides important genetic evidence for understanding the physiological roles of hydrogenase orthologs in the Thermococcales.     

Nalidixic Acid-Resistant Mutants of Escherichia coli Deficient in Isocitrate Dehydrogenase

icd Mutants of Escherichia coli K-12, selected for their resistance to nalidixic acid, are deficient in isocitrate dehydrogenase.     

New lv Mutants of Pea Are Deficient in Phytochrome B

The lv-1 mutant of pea (Pisum sativum L.) is deficient in responses regulated by phytochrome B (phyB) in other species but has normal levels of spectrally active phyB. We have characterized three further lv mutants (lv-2, lv-3, and lv-4), which are all elongated under red (R) and white light but are indistinguishable from wild type under far-red light. The phyB apoprotein present in the lv-1 mutant was undetectable in all three new lv mutants. The identification of allelic mutants with and without phyB apoprotein suggests that Lv may be a structural gene for a B-type phytochrome. Furthermore, it indicates that the lv-1 mutation results specifically in the loss of normal biological activity of this phytochrome. Red-light-pulse and fluence-rate-response experiments suggest that lv plants are deficient in the low-fluence response (LFR) but retain a normal very-low-fluence-rate-dependent response for leaflet expansion and inhibition of stem elongation. Comparison of lv alleles of differing severity indicates that the LFR for stem elongation can be mediated by a lower level of phyB than the LFR for leaflet expansion. The retention of a strong response to continuous low-fluence-rate R in all four lv mutants suggests that there may be an additional phytochrome controlling responses to R in pea. The kinetics of phytochrome destruction and reaccumulation in the lv mutant indicate that phyB may be involved in the light regulation of phyA levels.     

Genetic Examination and Mass Balance Analysis of Pyruvate/Amino Acid Oxidation Pathways in the Hyperthermophilic Archaeon Thermococcus kodakarensis

The present study investigated the simultaneous oxidation of pyruvate and amino acids during H₂-evolving growth of the hyperthermophilic archaeon Thermococcus kodakarensis. The comparison of mass balance between a cytosolic hydrogenase (HYH)-deficient strain (the ΔhyhBGSL strain) and the parent strain indicated that NADPH generated via H₂ uptake by HYH was consumed by reductive amination of 2-oxoglutarate catalyzed by glutamate dehydrogenase. Further examinations were done to elucidate functions of three enzymes potentially involved in pyruvate oxidation: pyruvate formate-lyase (PFL), pyruvate:ferredoxin oxidoreductase (POR), and 2-oxoisovalerate:ferredoxin oxidoreductase (VOR) under the HYH-deficient background in T. kodakarensis. No significant change was observed by deletion of pflDA, suggesting that PFL had no critical role in pyruvate oxidation. The growth properties and mass balances of ΔporDAB and ΔvorDAB strains indicated that POR and VOR specifically functioned in oxidation of pyruvate and branched-chain amino acids, respectively, and the lack of POR or VOR was compensated for by promoting the oxidation of another substrate driven by the remaining oxidoreductase. The H₂ yields from the consumed pyruvate and amino acids were increased from 31% by the parent strain to 67% and 82% by the deletion of hyhBGSL and double deletion of hyhBGSL and vorDAB, respectively. Significant discrepancies in the mass balances were observed in excess formation of acetate and NH₃, suggesting the presence of unknown metabolisms in T. kodakarensis grown in the rich medium containing pyruvate.     

磷胁迫对烤烟高亲和磷转运蛋白基因表达及磷素吸收利用的影响

为探明缺磷胁迫对烤烟磷吸收的影响机理,以烤烟品种‘豫烟10号’为材料,采用盆栽试验设置不施磷(-P)和正常供磷(+P)2个处理,分析了烟草生育后期根系和叶片中高亲和磷转运蛋白基因NtPht1;1、NtPht1;2(简称PT1、PT2)的表达差异性及其表达量与烟草磷含量、磷积累量的关系。结果表明:(1)随生育期的推进,烤烟根系和叶片的PT1基因相对表达量、PT2基因相对表达量、干物质重和磷素积累量逐渐增加,烟叶的磷含量逐渐降低,根系磷含量无明显的变化规律。(2)磷胁迫促使PT1、PT2基因的表达量上调,其中叶片PT1基因的相对表达量高于根系,叶片PT2基因的相对表达量显著低于根系。(3)磷胁迫限制了烤烟对干物质和磷素的积累,在移栽后90d,-P处理烤烟根系和叶片的干物质重、磷素积累量不到+P处理的一半,处理间差异极显著。研究认为,在缺磷环境下,烟草高亲和磷转运蛋白基因PT1和PT2表达量的增加,是由磷胁迫下烟草体内磷积累量降低所引起的系统性调控。     

Biochemical and genetic characterization of the three metabolic routes in Thermococcus kodakarensis linking glyceraldehyde 3-phosphate and 3-phosphoglycerate

In the classical Embden-Meyerhof (EM) pathway for glycolysis, the conversion between glyceraldehyde 3-phosphate (GAP) and 3-phosphoglycerate (3-PGA) is reversibly catalysed by phosphorylating GAP dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK). In the Euryarchaeota Thermococcus kodakarensis and Pyrococcus furiosus, an additional gene encoding GAP:ferredoxin oxidoreductase (GAPOR) and a gene similar to non-phosphorylating GAP dehydrogenase (GAPN) are present. In order to determine the physiological roles of the three routes that link GAP and 3-PGA, we individually disrupted the GAPOR, GAPN, GAPDH and PGK genes (gor, gapN, gapDH and pgk respectively) of T. kodakarensis. The Δgor strain displayed no growth under glycolytic conditions, confirming its proposed function to generate reduced ferredoxin for energy generation in glycolysis. Surprisingly, ΔgapN cells also did not grow under glycolytic conditions, suggesting that GAPN plays a key role in providing NADPH under these conditions. Disruption of gor and gapN had no effect on gluconeogenic growth. Growth experiments with the ΔgapDH and Δpgk strains indicated that, unlike their counterparts in the classical EM pathway, GAPDH/PGK play a major role only in gluconeogenesis. Biochemical analyses indicated that T. kodakarensis GAPN did not recognize aldehyde substrates other than d-GAP, preferred NADP(+) as cofactor and was dramatically activated with glucose 1-phosphate.