Rie Matsumi

Formate-driven growth coupled with H2 production

Although a common reaction in anaerobic environments, the conversion of formate and water to bicarbonate and H2 (with a change in Gibbs free energy of ΔG° = +1.3 kJ mol−1) has not been considered energetic enough to support growth of microorganisms. Recently, experimental evidence for growth on formate was reported for syntrophic communities of Moorella sp. strain AMP and a hydrogen-consuming Methanothermobacter species and of Desulfovibrio sp. strain G11 and Methanobrevibacter arboriphilus strain AZ. The basis of the sustainable growth of the formate-users is explained by H2 consumption by the methanogens, which lowers the H2 partial pressure, thus making the pathway exergonic. However, it has not been shown that a single strain can grow on formate by catalysing its conversion to bicarbonate and H2. Here we report that several hyperthermophilic archaea belonging to the Thermococcus genus are capable of formate-oxidizing, H2-producing growth. The actual ΔG values for the formate metabolism are calculated to range between −8 and −20 kJ mol−1 under the physiological conditions where Thermococcus onnurineus strain NA1 are grown. Furthermore, we detected ATP synthesis in the presence of formate as a sole energy source. Gene expression profiling and disruption identified the gene cluster encoding formate hydrogen lyase, cation/proton antiporter and formate transporter, which were responsible for the growth of T. onnurineus NA1 on formate. This work shows formate-driven growth by a single microorganism with protons as the electron acceptor, and reports the biochemical basis of this ability.

Reverse Gyrase Is Not a Prerequisite for Hyperthermophilic Life

We disrupted the reverse gyrase gene from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. An apparent positive supercoiling activity that was observed in the host strain was not found in the disruptant strain. We found that a lack of reverse gyrase led to a retardation in growth that was more striking at higher temperatures. However, the disruption of the reverse gyrase gene did not lead to a lethal phenotype at 90 degrees C. This study provides experimental evidence that reverse gyrase is not a prerequisite for hyperthermophilic life.

Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance

We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg(Tk)) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu(Tk)) or a gene cluster which includes apu(Tk) and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 microM simvastatin were isolated. The transformants exhibited growth in the presence of 20 microM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg(Tk) locus when the endogenous hmg(Tk) gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg(Pf)) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The Deltaapu(Tk) strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu(Tk) is a major polysaccharide-degrading enzyme in T. kodakaraensis.

Isoprenoid biosynthesis in Archaea - Biochemical and evolutionary implications

Isoprenoids are indispensable for all types of cellular life in the Archaea, Bacteria, and Eucarya. These membrane-associated molecules are involved in a wide variety of vital biological functions, ranging from compartmentalization and stability, to protection and energy-transduction. In Archaea, isoprenoid compounds constitute the hydrophobic moiety of the typical ether-linked membrane lipids. With respect to stereochemistry and composition, these archaeal lipids are very different from the ester-linked, fatty acid-based phospholipids in bacterial and eukaryotic membranes. This review provides an update on isoprenoid biosynthesis pathways, with a focus on the archaeal enzymes. The black-and-white distribution of fundamentally distinct membrane lipids in Archaea on the one hand, and Bacteria and Eucarya on the other, has previously been used as a basis for hypothetical evolutionary scenarios, a selection of which will be discussed here.

Crystal structure of HypA, a nickel-binding metallochaperone for [NiFe] hydrogenase maturation.

HypA is one of the auxiliary proteins involved in the maturation of [NiFe] hydrogenases. By an unknown mechanism, HypA functions as a metallochaperone in the insertion of the Ni atom into hydrogenases. We have determined the crystal structures of HypA from Thermococcus kodakaraensis KOD1 in both monomeric and dimeric states. The structure of the HypA monomer consists of Ni- and Zn-binding domains. The relative arrangement of the two metal-binding domains has been shown to be associated with local conformations of the conserved Ni-binding motif, suggesting a communication between the Ni- and Zn-binding sites. The HypA dimer has been shown to be stabilized by unexpected domain swapping through archaea-specific linker helices. In addition, the hexameric structure of HypA is formed in the crystal packing. Several hydrogen bonds and hydrophobic interactions stabilize the hexamer interface. These findings suggest the functional diversity of HypA proteins.

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.

Isolation and characterization of a novel poly(vinyl alcohol)-degrading bacterium, Sphingopyxis sp. PVA3

We have isolated a poly(vinyl alcohol) (PVA)-degrading bacterium from an activated sludge sample obtained from the drainage of a dyeing factory. Enrichment cultures were performed in media containing PVA as the sole or major carbon source. After several rounds of cultivation on liquid and solid media, we were able to isolate a single colony with PVA-degrading ability (strain PVA3). The bacterium could degrade PVA in the absence of symbionts or cofactors such as pyrroloquinoline quinone (PQQ). Over 90% of PVA, at an initial concentration of 0.1%, was degraded within a 6-day cultivation. Degradation was confirmed by both iodometric methods and gel permeation chromatography. Examination of the PVA attached to the cells revealed a large increase in carbonyl groups, suggesting the oxidation of hydroxyl groups of the polymer on the surfaces of cells. Addition of PQQ to the culture medium did not enhance the growth and the PVA-degrading rates of strain PVA3. Furthermore, we found that cells grown on PVA generated hydrogen peroxide upon the addition of PVA. The results strongly suggest that the initial oxidation of PVA is mediated via a PVA oxidase, and not a PQQ-dependent dehydrogenase. A biochemical and phylogenetic characterization of the bacterium was performed. The sequence of the 16S ribosomal RNA gene of the bacterium indicated a phylogenetic position of the strain within the genus Sphingopyxis, and the strain was therefore designated Sphingopyxis sp. PVA3.

Thermococcus kodakarensis mutants deficient in di-myo-inositol phosphate use aspartate to cope with heat stress

Many of the marine microorganisms which are adapted to grow at temperatures above 80 degrees C accumulate di-myo-inositol phosphate (DIP) in response to heat stress. This led to the hypothesis that the solute plays a role in thermoprotection, but there is a lack of definitive experimental evidence. Mutant strains of Thermococcus kodakarensis (formerly Thermococcus kodakaraensis), manipulated in their ability to synthesize DIP, were constructed and used to investigate the involvement of DIP in thermoadaptation of this archaeon. The solute pool of the parental strain comprised DIP, aspartate, and alpha-glutamate. Under heat stress the level of DIP increased 20-fold compared to optimal conditions, whereas the pool of aspartate increased 4.3-fold in response to osmotic stress. Deleting the gene encoding the key enzyme in DIP synthesis, CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, abolished DIP synthesis. Conversely, overexpression of the same gene resulted in a mutant with restored ability to synthesize DIP. Despite the absence of DIP in the deletion mutant, this strain exhibited growth parameters similar to those of the parental strain, both at optimal (85 degrees C) and supraoptimal (93.7 degrees C) temperatures for growth. Analysis of the respective solute pools showed that DIP was replaced by aspartate. We conclude that DIP is part of the strategy used by T. kodakarensis to cope with heat stress, and aspartate can be used as an alternative solute of similar efficacy. This is the first study using mutants to demonstrate the involvement of compatible solutes in the thermoadaptation of (hyper)thermophilic organisms.

Biochemical Properties of a Putative Signal Peptide Peptidase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

We have performed the first biochemical characterization of a putative archaeal signal peptide peptidase (SppA(Tk)) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. SppA(Tk), comprised of 334 residues, was much smaller than its counterpart from Escherichia coli (618 residues) and harbored a single predicted transmembrane domain near its N terminus. A truncated mutant protein without the N-terminal 54 amino acid residues (deltaN54SppA(Tk)) was found to be stable against autoproteolysis and was examined further. DeltaN54SppA(Tk) exhibited peptidase activity towards fluorogenic peptide substrates and was found to be highly thermostable. Moreover, the enzyme displayed a remarkable stability and preference for alkaline pH, with optimal activity detected at pH 10. DeltaN54SppA(Tk) displayed a K(m) of 240 +/- 18 microM and a V(max) of 27.8 +/- 0.7 micromol min(-1) mg(-1) towards Ala-Ala-Phe-4-methyl-coumaryl-7-amide at 80 degrees C and pH 10. The substrate specificity of the enzyme was examined in detail with a FRETS peptide library. By analyzing the cleavage products with liquid chromatography-mass spectrometry, deltaN54SppA(Tk) was found to efficiently cleave peptides with a relatively small side chain at the P-1 position and a hydrophobic or aromatic residue at the P-3 position. The positively charged Arg residue was preferred at the P-4 position, while substrates with negatively charged residues at the P-2, P-3, or P-4 position were not cleaved. When predicted signal sequences from the T. kodakaraensis genome sequence were examined, we found that the substrate specificity of deltaN54SppA(Tk) was in good agreement with its presumed role as a signal peptide peptidase in this archaeon.

Identification of the amino acid residues essential for proteolytic activity in an archaeal signal peptide peptidase.

Signal peptide peptidases (SPPs) are enzymes involved in the initial degradation of signal peptides after they are released from the precursor proteins by signal peptidases. In contrast to the eukaryotic enzymes that are aspartate peptidases, the catalytic mechanisms of prokaryotic SPPs had not been known. In this study on the SPP from the hyperthermophilic archaeon Thermococcus kodakaraensis (SppATk), we have identified amino acid residues that are essential for the peptidase activity of the enzyme. ΔN54SppATk, a truncated protein without the N-terminal 54 residues and putative transmembrane domain, exhibits high peptidase activity, and was used as the wild-type protein. Sixteen residues, highly conserved among archaeal SPP homologue sequences, were selected and replaced by alanine residues. The mutations S162A and K214A were found to abolish peptidase activity of the protein, whereas all other mutant proteins displayed activity to various extents. The results indicated the function of Ser162 as the nucleophilic serine and that of Lys214 as the general base, comprising a Ser/Lys catalytic dyad in SppATk. Kinetic analyses indicated that Ser184, His191 Lys209, Asp215, and Arg221 supported peptidase activity. Intriguingly, a large number of mutations led to an increase in activity levels of the enzyme. In particular, mutations in Ser128 and Tyr165 not only increased activity levels but also broadened the substrate specificity of SppATk, suggesting that these residues may be present to prevent the enzyme from cleaving unintended peptide/protein substrates in the cell. A detailed alignment of prokaryotic SPP sequences strongly suggested that the majority of archaeal enzymes, along with the bacterial enzyme from Bacillus subtilis, adopt the same catalytic mechanism for peptide hydrolysis.