Halophilic Archaeon Haloarcula Japonica Research Articles

Characterization of a GlgC homolog from extremely halophilic archaeon Haloarcula japonica.

Glycogen synthesis in bacteria is mainly organized by the products of glgB, glgC, and glgA genes comprising the widely known glg operon. On the genome of extremely halophilic archaeon Haloarcula japonica, there was a gene cluster analogous to the bacterial glg operon. In this study, we focused on a GlgC homolog of Ha. japonica, and its recombinant enzyme was prepared and characterized. The enzyme showed highest activity toward GTP and glucose-1-phosphate as substrates in the presence of 2.6 m KCl and predicted to be work as "GDP-glucose pyrophosphorylase" in Ha. japonica.

Halophilic mechanism of the enzymatic function of a moderately halophilic dihydrofolate reductase from Haloarcula japonica strain TR-1.

Dihydrofolate (DHF) reductase coded by a plasmid of the extremely halophilic archaeon Haloarcula japonica strain TR-1 (HjDHFR P1) shows moderate halophilicity on enzymatic activity at pH 6.0, although there is no significant effect of NaCl on its secondary structure. To elucidate the salt-activation and -inactivation mechanisms of this enzyme, we investigated the effects of pH and salt concentration, deuterium isotope effect, steady-state kinetics, and rapid-phase ligand-binding kinetics. Enzyme activity was increased eightfold by the addition of 500mM NaCl at pH 6.0, fourfold by 250mM at pH 8.0, and became independent of salt concentration at pH 10.0. Full isotope effects observed at pH 10.0 under 0-1000mM NaCl indicated that the rate of hydride transfer, which was the rate-determining step at the basic pH region, was independent of salt concentration. Conversely, rapid-phase ligand-binding experiments showed that the amplitude of the DHF-binding reaction increased and the tetrahydrofolate (THF)-releasing rate decreased with increasing NaCl concentration. These results suggested that the salt-activation mechanism of HjDHFR P1 is via the population change of the anion-unbound and anion-bound conformers, which are binding-incompetent and -competent conformations for DHF, respectively, while that of salt inactivation is via deceleration of the THF-releasing rate, which is the rate-determining step at the neutral pH region.

Pyrophosphate hydrolysis in the extremely halophilic archaeon Haloarcula japonica is catalyzed by a single enzyme with a broad ionic strength range.

The soluble protein fraction of the extremely halophilic archaeon Haloarcula japonica exhibits substantial inorganic pyrophosphate (PPi) hydrolysis activity in the presence of 2-4M NaCl (Wakai et al, J Biol Chem 288:29247-29251, 2013), which provides high ionic strength (2-4). In this study, much higher PPi hydrolysis activity was unexpectedly detected, even with 0M NaCl in the presence of 100-200mM MgSO4, providing a much lower ionic strength of 0.4-0.8, in the same protein fraction. Na+ and Mg2+ ions were required for activity under high and low ionic strength conditions, respectively. A recombinant H. japonica pyrophosphatase (HjPPase) exhibited PPi hydrolysis activity with the same broad ionic strength range, indicating that the activity associated with such a broad ionic strength range could be attributed to a single enzyme. Thus, we concluded that the broad ionic strength range of HjPPase may contribute to adaptation for both Na+ and Mg2+ which are abundant but variable in the unstable living environments of H. japonica.

Identification of carotenoids from the extremely halophilic archaeon Haloarcula japonica

The carotenoids produced by extremely halophilic archaeon Haloarcula japonica were extracted and identified by their chemical, chromatographic, and spectroscopic characteristics (UV-Vis and mass spectrometry). The composition (mol%) was 68.1% bacterioruberin, 22.5% monoanhydrobacterioruberin, 9.3% bisanhydrobacterioruberin, <0.1% isopentenyldehydrorhodopin, and trace amounts of lycopene and phytoene. The in vitro scavenging capacity of a carotenoid, bacterioruberin, extracted from Haloarcula japonica cells against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was evaluated. The antioxidant capacity of bacterioruberin was much higher than that of β -carotene.

Gene expression and characterization of a novel GH family 18 chitinase from extremely halophilic archaeon Halobacterium salinarum NRC-1

An open reading frame encoding a chitinase homolog (ChiN1) was found in the genome of extremely halophilic archaeon Halobacterium salinarum NRC-1. ChiN1 is a multidomain enzyme consisting of a chitin-binding domain, a polycystic kidney disease domain and a catalytic domain belonging to glycoside hydrolase family 18. chiN1 gene was successfully expressed in extremely halophilic archaeon Haloarcula japonica TR-1 by employing the promoter sequence of its cell surface glycoprotein gene. A large amount of recombinant ChiN1 was secreted into the culture supernatant. The Ha. japonica-produced ChiN1 was purified and characterized. The optimal pH and temperature of ChiN1 are pH 4.5 and 55°C, respectively. ChiN1 was most active at 1.0 M NaCl and stable over a wide range of NaCl concentration from 1.0 to 4.5 M. This is the first report on a chitinase from extremely halophilic archaeon.

Molecular cloning of transducer gene hjtB from extremely halophilic archaeon Haloarcula japonica

A transducer gene, hjtB, was cloned from genomic DNA of extremely halophilic archaeon Haloarcula japonica. The structural gene consisted of an open reading frame of 984 nucleotides encoding 328 amino acids. RT-PCR analysis revealed that this gene was transcribed in Ha. japonica.

Gene cloning, expression and partial characterization of cell division protein FtsZ1 from extremely halophilic archaeon Haloarcula japonica strain TR-1

The gene encoding a cell division protein FtsZ1 was cloned from an extremely halophilic archaeon, Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the ftsZ1 gene revealed that the structural gene consisted of an open reading frame of 1,158 nucleotides encoding 386 amino acids. Transcription of the ftsZ1 gene in Ha. japonica was confirmed by RT-PCR. A modified ftsZ1 gene was inserted into the shuttle vector pWL102 and used to transform Ha. japonica. The recombinant FtsZ1 was produced as a fusion with hexahistidine-tag in Ha. japonica host cells and purified. Purified recombinant FtsZ1 exhibited GTP-dependent polymerization activity and GTP-hydrolyzing activity in the presence of high concentrations of KCl.

Role of an N-terminal domain found in the ferredoxin from extremely halophilic archaeon Haloarcula japonica

The ferredoxin (Fd) from Haloarcula japonica possesses a plant-type [2Fe-2S] cluster and is stable at high salt concentrations. Ha. japonica Fd (HjFd) includes an N-terminal additional domain rich in acidic amino acids, as well as a common core domain that contains the Fe-S cluster. The N-terminally HAT-tagged intact HjFd (HAT/HjFd) and spinach/Ha. japonica chimeric Fd (HAT/Sp/HjFd) were prepared and characterized. Escherichia coli-produced HAT/Sp/HjFd and Ha. japonica-produced HAT/HjFd were produced as holoproteins. On the other hand, E. coli-produced HAT/HjFd did not incorporate the Fe-S cluster. These results suggested that the N-terminal domain of HjFd contributed to the polypeptide folding and successive Fe-S cluster incorporation under high salt conditions. Both Ha. japonica-produced HAT/HjFd and E. coli-produced HAT/Sp/HjFd were stable at high salt concentrations (≥1.5 M NaCl), although a reduction in stability was observed at lower concentrations. Lack of the N-terminal domain did not affect the stability of HjFd, indicating that the core domain mainly contributed to the stability of HjFd at high salt concentrations. Solubility of E. coli-produced HAT/Sp/HjFd under high salt conditions was significantly lower than that of Ha. japonica-produced HAT/HjFd. It was revealed that substitution of the N-terminal domain of HjFd to that of spinach Fd injured the solubility of HjFd. Thus, it was concluded that the N-terminal domain of HjFd should perform the essential functions for halophilic adaptation from the folding process through the folded state.

Molecular cloning of transducer genes hjtA and hjtC from extremely halophilic archaeon Haloarcula japonica

The genes encoding putative taxis transducers hjtA and hjtC were cloned from triangular disc-shaped extremely halophilic archaeon Haloarcula japonica strain TR-1. Nucleotide sequencing analysis revealed that HjtA and HjtC consisted of 438 and 630 amino acids, respectively. HjtA and HjtC have the highly conserved sequence which is commonly found in signaling domains of bacterial and archaeal transducers.

Isolation of crtI homolog from extremely halophilic archaeon Haloarcula japonica strain TR-1

Halobacterium sp. strain NRC-1 possesses phytoene dehydrogenase genes (crtIs). A crtI2 homolog was cloned from Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the possible crtI2 revealed that the structural gene consisted of an open reading frame of 1,521 nucleotides encoding 507 amino acids. Transcription of crtI2 in Ha. japonica was confirmed by RT-PCR.

Gene cloning of ftsZ1 homolog from extremely halophilic archaeon Haloarcula japonica.

The gene encoding FtsZ1 was cloned from triangular disc-shaped extremely halophilic archaeon Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the possible ftsZ1 gene revealed that the structural gene consisted of an open reading frame of 1,038 nucleotides encoding 346 amino acids. Transcription of the ftsZ1 gene in Ha. japonica was confirmed by RT-PCR.

Cloning and expression of the ferredoxin gene from extremely halophilic archaeon Haloarculajaponica strain TR-1.

The gene encoding a ferredoxin (Fd) from Haloarculajaponica strain TR-1 was cloned and sequenced. Sequence analysis of the cloned Ha. japonica Fd gene revealed that the structural gene consisted of an open reading frame of 387 nucleotides encoding 129 amino acids. The deduced amino acid sequence of Ha. japonica Fd showed 84 to 98% identity with corresponding sequences in other extremely halophilic archaea. The Ha. japonica Fd gene was inserted into the shuttle vector pWL102 and used to transform Ha. japonica. Ha. japonica Fd could then be produced as a fusion with His-Tag (6xHis) in Ha. japonica host cells. The absorption and ESR spectra of the Fd/His Tag fusion protein revealed the presence of a [2Fe-2S] cluster which is characteristic of native Ha. japonica Fd.

The gene encoding a novel halorhodopsin-like protein of extremely halophilic archaeon Haloarcula japonica strain TR-1.

The gene encoding the halorhodopsin (hR)-like protein was cloned and sequenced from Haloarcula japonica strain TR-1. The structural gene consisted of an open reading frame of 828 nucleotides encoding 276 amino acids. The deduced amino acid sequence of the Ha. japonica hR-like protein showed the highest homology to those of cruxhalorhodopsins. Furthermore, we have demonstrated that the Ha. japonica chR gene is transcriptionally induced by light.

Transcriptional regulation of cruxrhodopsin gene from extremely halophilic archaeon Haloarcula japonica strain TR-1.

Transcription of the cruxrhodopsin (cR) gene in extremely halophilic archaeon Haloarcula japonica strain TR-1 was investigated using Northern analysis to quantify message level. In the cell cultures growing in the dark, cR transcript level remained very low. In contrast, exposure of the cell cultures to light stimulated transcription of the cR gene. In addition, cR gene transcription was also induced when Ha. japonica was grown under high oxygen tension and then shifted to low oxygen tension in the dark. These results suggested that transcription of the cR gene is regulated by high light intensity and low oxygen tension.

Cloning and sequencing of the gene encoding the cell surface glycoprotein of Haloarcula japonica strain TR-1.

The triangular disk-shaped halophilic archaeon Haloarcula japonica strain TR-1 has a glycoprotein on its cell surface. The complete gene encoding the cell surface glycoprotein (CSG) was cloned and sequenced. The gene has an open reading frame of 2586 bp, and a potential archaeal promoter sequence approximately 150 bp upstream of the ATG initiation codon. The mature CSG is composed of 828 amino acids and is preceded by a signal sequence of 34 amino acid residues. A hydropathy analysis showed a hydrophobic stretch at the C-terminus, that probably serves as a transmembrane domain. The amino acid sequence of the Ha. japonica CSG showed 52.1% and 43.2% identities to those from the Halobacterium halobium and Haloferax volcanii CSGs, respectively. Five potential N-glycosylation sites were found in the mature Ha. japonica CSG, sites that were distinctly different from those in Hb. halobium and Hf. volcanii. The Ha. japonica CSG gene was expressed in Escherichia coli.

Purification and partial characterization of cell surface glycoprotein from extremely halophilic archaeon Haloarcula japonica strain TR-1

Haloarculajaponica has a glycoprotein on its cell surface. The cell surface glycoprotein of H. japonica was purified and characterized. It had an apparent molecular mass of 180 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carbohydrate content was about 12% (wt/wt). The polypeptide portion contained a large proportion of acidic amino acids, and the sequence of 18 N-terminal amino acids was determined.