Constant Enthalpy Change Value during Pyrophosphate Hydrolysis within the Physiological Limits of NaCl

A decrease in water activity was thought to result in smaller enthalpy change values during PPi hydrolysis, indicating the importance of solvation for the reaction. However, the physiological significance of this phenomenon is unknown. Here, we combined biochemistry and calorimetry to solve this problem using NaCl, a physiologically occurring water activity-reducing reagent. The pyrophosphatase activities of extremely halophilic Haloarcula japonica, which can grow at ∼4 m NaCl, and non-halophilic Escherichia coli and Saccharomyces cerevisiae were maximal at 2.0 and 0.1 m NaCl, respectively. Thus, halophilic and non-halophilic pyrophosphatases exhibit distinct maximal activities at different NaCl concentration ranges. Upon calorimetry, the same exothermic enthalpy change of −35 kJ/mol was obtained for the halophile and non-halophiles at 1.5–4.0 and 0.1–2.0 m NaCl, respectively. These results show that solvation changes caused by up to 4.0 m NaCl (water activity of ∼0.84) do not affect the enthalpy change in PPi hydrolysis. It has been postulated that PPi is an ATP analog, having a so-called high energy phosphate bond, and that the hydrolysis of both compounds is enthalpically driven. Therefore, our results indicate that the hydrolysis of high energy phosphate compounds, which are responsible for biological energy conversion, is enthalpically driven within the physiological limits of NaCl.This work was supported by Grant-in-aid for Scientific Research on Innovative Areas 20118005 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan

Amino acid sequence diversities in TBP, TATA binding protein, of extremely halophilic archaeon Haloarcula japonica strain TR-1

The TATA-box binding protein (TBP) is a basal transcription factor involved in transcription initiation in Eukarya and Archaea. Through exhaustive analyses of the whole geneme of extremely halophilic archaeon, Haloarcula japonica strain TR-1, six TBP genes were found and structurally analyzed. These TBPs were designated as TBP1, TBP2, TBP3, TBP4, TBP5 and TBP6, respectively and these TBPs were diverged from other archaeal TBPs that have been known. TBP1 gene was found to encode a polypeptide consisting of 182 amino acid residues, showing 35.0％ identity to that of H. marismortui. TBP2 gene was found to encode a polypeptide consisting of 182 amino acid residues, showing 36.5％ identity to that of H. marismortui. TBP3 gene was found to encode a polypeptide consisting of 186 amino acid residues, showing 100％ identity to that of H. marismortui. TBP4 gene was found to encode a polypeptide consisting of 185 amino acid residues, showing 42.9％ identity to that of H. marismortui. TBP5 gene was found to encode a polypeptide consisting of 182 amino acid residues, showing 35.4％ identity to that of H. marismortui. TBP6 gene was found to encode a polypeptide consisting of 182 amino acid residues, showing 46.1％ identity to that of H. marismortui. By phylogenetic analyses of these six proteins, TBP3 is conserved in amino acid sequence with other archaeal strains including methanogens and thermophiles, It may suggest that the TBP3 is core TBP function in transcriptional initiation such as housekeeping genes. Six histidine-tagged version of the H. japonica TBPs were produced in Escherichia coli in a denature conditions after construction of overexpression plasmids and purified by means of Ni-chelating chromatography

<Original Papers>Structure analysis of BasS and BasR in rice seedling blight pathogen Burkholderia plantarii

Burkholderia plantani is a plant pathogen causing rice seedling blight. It produces tropolone, the disease causing substance. To elucidate and understand molecular mechanisms of the pathogenesis, we analyzed the genome of B. plantarii. In this study, we focused on two component system, in prokaryotic signal transduction. This system is basically composed of a histidine kinase (HK, sensor) residing in the inner membrane and a cognate response regulator (RR) in the cytoplasm. Here, BasS, a histidine kinase (HK, sensor) and BasR, a response regulator (RR) in this strain, were structurally analysed, showing these are tandemely ordered. Phylogenetic analysis of these prodcuts were also analysed. Construction of plasmids for gene disruption of these genes, basS and basR was also carried out, in terms of future study approach in reverse genetics. Obtaining mutants by transformation will be useful for understanding the relationships between pathogenesis and function of these genes

Molecular cloning and structural analyses of the pyrF gene for orotate monophosphate (OMP)decarboxylase from extremely halophilic archaeon Haloarcula japonica strain TR-1

The orotate monophosphate decarboxylase gene (pyrF) in the pyrimidine biosynthetic pathway have unique characteristics. In this study, the pyrF gene was found to encode a polypetide consisting of 277 amino acid residues, showing 96,0 ％ identity to that of H.marismortui. Southern hybridization of the pyfF gene region with partial sequence probe revealed that single copy number of the gene is distributed on the chromosome of H. japonica genome. By phylogenetic analyses of the H. japonica PyrF, the amino acid sequences of the enzyme is relatively conserved in other archaeal strains including methanogens and thermophiles. Alignments of several sequences containing pyrF gene showed that the genetic organization of pyrF gene was not conserved in several haloarchaeal strains expect for H.marismortui