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Nucleoside Diphosphate Kinase of Halobacteria - Amino Acid Sequence and Salt-Response Pattern

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Nucleoside Diphosphate Kinase of Halobacteria - Amino Acid Sequence and Salt-Response Pattern գ؈೰ি൝ԙӔࠠ Vol.3, 2004ۙئ Journal of Japanese Society for Extremophiles (2004), Vol. 3, 18-27 ଙཉ൫ۍ Mizuki T1, Kamekura M2, Ishibashi M3, Usami R1, Yoshida Y1, Tokunaga M3, Horikoshi K1 Nucleoside diphosphate kinase of halobacteria - Amino acid sequence and salt-response pattern - 1 Department of Applied Chemistry, Faculty of Engineering, Toyo University, Kawagoe, Saitama 350-8585, Japan 2 Noda Institute for Scientific Research, Noda, Chiba 278-0037, Japan 3 Department of Agricultural Chemistry, Kagoshima University, Kagoshima 890-0065, Japan Corresponding author: Kamekura M, [email protected] Phone: +81-4-7123-5573. Fax: +81-4-7123-5953. Received: Oct. 15, 2003 / Accepted: Nov. 27, 2003 Abstract Nucleotide diphosphate kinase (NDK) was purified exchange g-phosphates between nucleoside triphosphates and from 12 strains of halobacteria using ATP-agarose diphosphates, thus playing a key role in maintaining cellular chromatography and their N-terminal amino acid sequences pools of all nucleoside triphosphates 1). NDK of halobacteria were determined. The electrophoretic mobilities of these was first studied as a major cytosolic protein of Natrialba enzymes differed significantly on the native-PAGE or the SDS- magadii (formerly Natronobacterium magadii) that bound to PAGE when the samples were not heat treated. Comparison of ATP-agarose and eluted with 5 mM ATP (adenosine seven complete amino acid sequences from seven species of triphosphate) in the presence of 3.5 M NaCl 16). This enzyme Haloarcula and those from Har. californiae and Har. japonica, was active over a wide range of NaCl concentrations; from whose N-terminal 3 amino acids have not been determined yet, 0.09 M to 3.5 M. Independently, Ishibashi et al. 5) purified revealed that they are very similar and differed at only 1 to 4 NDK from a strain of Halobacterium salinarum as a protein residues in 153 residues. NDKs from Haloarcula quadrata and that bound to ATP-agarose in the absence of salt and eluted Har. sinaiiensis differed only at the 30th amino acid (arginine with ATP. Further detailed experiments have demonstrated that vs. cysteine), yet they showed a remarkable difference in their NDK is one of the few halobacterial proteins that do not salt-response patterns, suggesting that a single amino acid require high salt concentrations to maintain their stability 6, 17). substitution can cause a one molar shift in the optimal NaCl An alignment of more than 100 published NDK amino acid concentration. sequences has shown clear conservation of primary structure throughout Eucarya, Bacteria, and Archaea (Kamekura, Key words extremely halophilic, Archaea, haloarchaea unpublished data). Although the NDK from the strain of Halobacterium salinarum is the only halobacterial NDK Introduction whose complete amino acid sequence has been published 5), Halobacteria are extremely halophilic Archaea that require three more sequences have been deduced from the genome high salt concentrations for growth. They are currently sequence of Halobacterium sp. NRC-1 14) and from two contig classified in 18 genera belonging to the family sequences publicly available, contig 131 of Haloarcula Halobacteriaceae 3, 10). The majority of strains grow best at marismortui and contig 3063 of Haloferax volcanii (consult NaCl concentrations of 3.5-4.5 M. To compensate for the high http://zdna2.umbi.umd.edu/~haloweb/). The sequence of the NaCl concentration in the environment, halobacteria NDK gene from Halobacterium sp. NRC-1 is identical to that accumulate intracellular KCl as high as 5 M. Most of the Hbt. salinarum strain 5). The three NDKs consist of 160 halobacterial proteins are adapted to function in the presence of amino acids (Hbt. salinarum) or 153 amino acids (Har. high salt concentrations and lose their activities at low salt marismortui and Hfx. volcanii) (see Results), and can be concentrations 12). aligned without gaps or insertions. The Hbt. salinarum NDK Nucleoside diphosphate kinase (NDK, EC: 2.7.4.6) has possesses one additional residue at N-terminus and 6 additional been shown to be almost ubiquitous in living organisms, with residues at C-terminus. The pair-wise similarities between the just a few exceptions 15). The main function of NDK is to three NDKs in the overlapping 153 residues are high, ranging 18 գ؈೰ি൝ԙӔࠠ Vol.3, 2004ۙئ from 78 to 88 %. Given the close similarity of these NDKs, a debris, the supernatant was applied to a small column (0.7 ml comparison of their salt response patterns and stabilities may bed volume) of ATP-agarose (Sigma, 4% cross-linked, C-8) provide insights into the way these enzymes have adapted to equilibrated with SB. After washing four times with 1.5 ml function at different salt concentrations. aliquots of SB, NDK was eluted with 1.5 ml of the SB In this study, we a) purified NDKs from strains of containing 3 mM ATP, yielding homogeneous preparation (see halobacteria and investigated their electrophoretic behavior on Results), which was stored at - 30ò. polyacrylamide gels, b) determined N-terminal amino acid sequences and PCR-amplified gene sequences, allowing a Amino acid sequence determination detailed comparison of their protein sequences, c) studied the N-terminal amino acid sequences of the purified NDK salt response patterns of NDKs from some halobacteria. preparations were determined with an Applied Biosystems 492 Protein Sequencer. Forty five µl of the preparation was loaded Materials and methods on to a ProSorb membrane and washed with 500 µl of 0.1% trifluoroacetic acid to remove the Tris in the elution buffer. Cultivation of strains and purification of NDKs The following halobacterial strains were used in this study: Polyacrylamide gel electrophoreses Halobacterium sp. NRC-1 JCM 11081, Haloarcula Native and sodium dodecyl sulfate (SDS)-PAGE were vallismortisT ATCC 29715T, Har. aidinensis JCM 10024T, Har. performed using precast 15 -25 % polyacrylamide gels argentinensis JCM 9737T, “Har. californiae” ATCC 33799, (Daiichi Pure Chemicals, Tokyo) according to the methods Har. japonica JCM 7785T, Har. hispanica ATCC 33960T, Har. recommended by the manufacturer. For SDS-PAGE, 8 µl of marismortui JCM 8966T, Har. quadrata JCM 11048T, “Har. NDK preparation was mixed with 8 µl of a sample buffer sinaiiensis” ATCC 33800, Haloferax volcaniiT WFD11 (from containing 2% SDS, 30% glycerol, 0.125 M Tris-HCl, pH 6.8, Dr. WF Doolittle, Dalhousie Univ., Canada), 10 % 2-mercaptoethanol, and 0.01 % BPB. The mixture was Halogeometricum borinquenseT JCM 10706T, Halomicrobium either boiled for 3 min or not heat-treated before loading to the mukohataeiT JCM 9738T, Halorubrum saccharovorumT JCM gel. In some experiments the mixtures were heated at various 8865T, Haloterrigena sp. GSL-11 (from Dr. FJ Post, Utah State temperatures and for various periods. After electrophoresis Univ., USA), Natrialba asiaticaT JCM 9576T, Nab. magadii with a constant current of 40 mA at 5ò, the gels were stained NCIMB 2190T, Natrinema pellirubrumT JCM 10476T, and with Coomassie Brilliant Blue. Natronomonas pharaonisT JCM 8858T. Most of the neutrophilic strains were cultivated in 20 ml Extraction of genomic DNA, PCR amplification and (for the extraction of genomic DNA) or 200 ml (for the sequencing of NDK genes preparation of NDK) of a medium containing 1% casamino Halobacterial cells grown up in 20 ml medium were harvested, acids, 1% yeast extract, 0.1 % sodium glutamate_H2O, 0.3 % washed, and suspended in 25% NaCl, 10mM Tris-HCl, pH 7.6. trisodium citrate_2H2O, 0.2% KCl, 2% MgSO4_7H2O, and Cell lysis was accomplished by adding N-lauroyl sarcosine at a 20% NaCl, pH 6.5. Some strains required a medium with pH final concentration of 1 % (w/v), and DNA was purified as adjusted to 7.1. The alkaliphilic strains, Nab. magadii and Nmn. described previously 8). pharaonis were cultivated in a medium consisting of 1% NDK genes were amplified by PCR using Platinum Taq Bacteriological Peptone (Oxoid), 0.3% trisodium citrate_2H2O, DNA Polymerase High Fidelity (Invitrogen). Amplification of 0.1% MgSO4_7H2O, 0.2% KCl, 22.5% NaCl, and 1.6 % the genes from Haloarcula strains was done with a primer set sodium carbonate. Incubation was at 37ºC in 150 ml or 500ml of a sense primer, 5’-GAG CGC ACG TTC GTG ATG GTC Erlenmeyer flask with orbital shaking at 150 strokes per AAI CCI GAC G (see Results) and an antisense primer-1, 5’- minute. GAG TTC GTC CAG TCG GTC GGC AAC CTT CTC A or The cells in a 200 ml culture were harvested at late an antisense primer-2, 5’-AGC CGT CGA GCT GGT AGA exponential phase, suspended in 10 to 15 ml, depending on the GCG TCA GGC TCA. The antisense primer-1 and 2 were cell density, of 3 M NaCl sonication buffer (SB) containing 2 synthesized from the sequence of nucleotide 138-108 and mM MgCl2 and 50mM Tris-HCl, pH 8.0 and disrupted by nucleotide 97-68 downstream from the stop codon of the ndk sonication with a Branson sonifier for 10 to 15 min on ice. gene of Har. marismortui delineated in the contig 131. The After spinning at 10,000 rpm for 20 min to get rid of cell expected lengths of amplified products were 591bp and 550bp, 19 գ؈೰ি൝ԙӔࠠ Vol.3, 2004ۙئ respectively. The NDK reaction was started by adding 3 µl of the For the gene amplification, though partial, of strains of enzyme preparation, which had been diluted and kept on ice, other genera, an antisense primer-3, 5’-GTC CCA GTC CAC to the 97 µl of the NDK-RM and incubating at 37ò in a water GAG CTC IIC GTC GTC GAA GAA, was designed from the bath. After 1 min incubation, 1 µl aliquot was taken and two sequences (AB036344 and contig 131) encoding a injected into the 616 µl LL-RM to measure a relative relatively conserved amino acid sequence, FFDD(or E)D(or luminescence (RLU).
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