Biochem Biophys Res Commun, 2003 Mar 21, 302(4), 728 - 34 Multiple replication origins of the archaeon Halobacterium species NRC-1; Zhang R et al.; The genomic sequence of the halophilic archaeon Halobacterium NRC-1 has been analyzed by the Z curve method . The Z curve is a three-dimensional curve that uniquely represents a given DNA sequence . Based on the known behaviors of the Z curves for the archaea whose replication origins have been identified, the analysis of the Z curve for the genome of Halobacterium NRC-1 strongly suggests that the large genome has two replication origins, oriC1 (921,863-922,014) and oriC2 (1,806,444-1,807,229), which are located at two sharp peaks of the Z curve . These two regions are next to the cdc6 genes and contain multiple copies of stretches of G and C, i.e., ggggtgggg and ccccacccc, which may also be regarded as direct and inverted repeats . Based on the above analysis, a model of replication of Halobacterium NRC-1 with two replication origins and two termini has been proposed . The experimental confirmation of this model would constitute the first example of multiple replication origins of archaea, which will finally provide much insight into the understanding of replication mechanisms of eukaryotic organisms, including human . In addition, the potential multiple replication origins of the archaeon Sulfolobus solfataricus are suggested by the analysis based on the Z curve method. Biochemistry, 2003 Mar 25, 42(11), 3319 - 25 Site-specific mutations provide new insights into the origin of pH effects and alternative spectral forms in the photoactive yellow protein from Halorhodospira halophila; Meyer TE et al.; Acid/base titrations of wild-type PYP and mutants, either in buffer or in the presence of chaotropes such as thiocyanate, establish the presence of four spectral forms including the following: a neutral form (446-476 nm), an acidic form (350-355 nm), an alkaline form (430-440 nm), and an intermediate wavelength form (355-400 nm) . The acidic species is formed by protonation of the oxyanion of the para-hydroxy-cinnamyl cysteine chromophore as a secondary result of acid denaturation (with pK(a) values of 2.8-5.4) and often results in precipitation of the protein, and in the case of wild-type PYP, eventual hydrolysis of the chromophore thioester bond at pH values below 2 . Thus, the large and complex structural changes associated with the acidic species make it a poor model for the long-lived photocycle intermediate, I(2), which undergoes more moderate structural changes . Mutations at E46, which is hydrogen-bonded to the chromophore, have only two spectral forms accessible to them, the neutral and the acidic forms . Thus, an intact E46 carboxyl group is essential for observation of either intermediate or alkaline wavelength forms . The alkaline form is likely to be due to ionization of E46 in the folded protein . We postulate that the intermediate wavelength form is due to a conformational change that allows solvent access to E46 and formation of a hydrogen-bond from a water molecule to the carboxylic acid group, thus weakening its interaction with the chromophore . Increasing solvent access to the intermediate spectral form with denaturant concentration results in a continuously blue-shifted wavelength maximum. J Mol Biol, 2003 Mar 21, 327(2), 347 - 57 Unique amino acid composition of proteins in halophilic bacteria; Fukuchi S et al.; The amino acid compositions of proteins from halophilic archaea were compared with those from non-halophilic mesophiles and thermophiles, in terms of the protein surface and interior, on a genome-wide scale . As we previously reported for proteins from thermophiles, a biased amino acid composition also exists in halophiles, in which an abundance of acidic residues was found on the protein surface as compared to the interior . This general feature did not seem to depend on the individual protein structures, but was applicable to all proteins encoded within the entire genome . Unique protein surface compositions are common in both halophiles and thermophiles . Statistical tests have shown that significant surface compositional differences exist among halophiles, non-halophiles, and thermophiles, while the interior composition within each of the three types of organisms does not significantly differ . Although thermophilic proteins have an almost equal abundance of both acidic and basic residues, a large excess of acidic residues in halophilic proteins seems to be compensated by fewer basic residues . Aspartic acid, lysine, asparagine, alanine, and threonine significantly contributed to the compositional differences of halophiles from meso- and thermophiles . Among them, however, only aspartic acid deviated largely from the expected amount estimated from the dinucleotide composition of the genomic DNA sequence of the halophile, which has an extremely high G+C content (68%) . Thus, the other residues with large deviations (Lys, Ala, etc.) from their non-halophilic frequencies could have arisen merely as "dragging effects" caused by the compositional shift of the DNA, which would have changed to increase principally the fraction of aspartic acid alone. Indian J Exp Biol, 2002 Feb, 40(2), 220 - 2 Potentiality of a new compound for in vitro differentiation between halophilic and non-halophilic vibrios; Ray R et al.; Sensitivity of 21 halophilic vibrios and 16 clinical isolates of non-halophilic vibrios was determined against a new possible antivibrio agent, a pyrimidine analogue, 4, 6-dimethylpyrimidine -2-thiol (4,6-DMPT) . It appeared to be a vibriocidal agent, having a mean MIC and MBC of 32 microg/ml for halophilic strains and 64 microg/ml for non-halophilic strains and an LD50 of 300 mg/Kg body weight of mice . Thus, 4,6-DMPT may help an in vitro distinction between halophilic and non-halophilic vibrios . Sensitivity of these strains was also studied with respect to pteridine, crystal violet and Tween 80 hydrolysis as further markers distinguishing between these 2 groups which could also be differentiated by their growth on TCBS or/and CLED media. Acta Crystallogr D Biol Crystallogr, 2003 Mar, 59(Pt 3), 566 - 8 Epub 2003 Feb 21. Crystallization and preliminary X-ray crystallographic studies of salt-tolerant glutaminase from Micrococcus luteus K-3; Chantawannakul P et al.; Glutaminase from the marine bacterium Micrococcus luteus K-3 (Micrococcus glutaminase) is a salt-tolerant protein which shows equivalent activities both in the absence and the presence of 3 M sodium chloride and is distinct from halophilic proteins, which are inactivated in the absence of salt . To investigate the mechanisms of the salt-tolerant adaptation of Micrococcus glutaminase, the glutaminase and its major fragment containing about 80% of the protein were crystallized using the hanging-drop vapour-diffusion method . The glutaminase crystals belong to space group P622, with unit-cell parameters a = b = 111.4, c = 210.9 A, alpha = beta = 90, gamma = 120 degrees, and diffract to 2.6 A resolution . The fragment crystals belong to space group F222, with unit-cell parameters a = 115.7, b = 116.4, c = 144.9 A, alpha = beta = gamma = 90 degrees, and diffract to 2.4 A resolution . Data from selenomethionine (SeMet) substituted glutaminase crystals and from SeMet-substituted fragment crystals were collected to 2.6 and 2.4 A resolution, respectively . Structural analyses of the glutaminase and its fragment are currently being attempted using the multiwavelength anomalous diffraction (MAD) phasing method. FEMS Microbiol Lett, 2003 Jan 28, 218(2), 239 - 44 A variant of the hyperthermophile Archaeoglobus fulgidus adapted to grow at high salinity; Goncalves LG et al.; A variant of Archaeoglobus fulgidus VC-16 was isolated from cultures obtained after a stepwise transfer from media containing 1.8-6.3% NaCl by a plating-independent, selected-cell cultivation technique, using a laser microscope . This variant, A . fulgidus VC-16S, had a higher growth rate throughout the salt range of the parental strain, but was also able to grow in media containing NaCl up to 6.3%, whereas the parental strain could not grow above 4.5% NaCl . Diglycerol phosphate (DGP), only encountered in the Archaeoglobales, was the major solute accumulated under supra-optimal salinities, whereas at supra-optimal growth temperatures di-myo-inositol phosphate was the predominant solute . The accumulation of compatible solutes during growth of variant VC-16S was lower than in the parental strain within 1.8-4.5% NaCl, but the levels of compatible solutes, including DGP, increased sharply in the variant at higher salinities (5.5 and 6.0%) . This variant represents, at this time, one of the most halophilic hyperthermophiles known, and its ability to grow at high salinity appears to be due to the massive accumulation of DGP. J Mol Biol, 2003 Feb 21, 326(3), 859 - 73 The Oligomeric states of Haloarcula marismortui malate dehydrogenase are modulated by solvent components as shown by crystallographic and biochemical studies; Irimia A et al.; The three-dimensional crystal structure of the (R207S, R292S) mutant of malate dehydrogenase from Haloarcula marismortui was solved at 1.95A resolution in order to determine the role of salt bridges and solvent ions in halophilic adaptation and quaternary structure stability . The mutations, located at the dimer-dimer interface, disrupt two inter-dimeric salt bridge clusters that are essential for wild-type tetramer stabilisation . Previous experiments in solution, performed on the double mutant, had shown a tetrameric structure in 4M NaCl, which dissociated into active dimers in 2M NaCl . In order to establish if the active dimeric form is a product of the mutation, or if it also exists in the wild-type protein, complementary studies were performed on the wild-type enzyme by analytical centrifugation and small angle neutron scattering experiments . They showed the existence of active dimers in NaF, KF, Na(2)SO(4), even in the absence of NADH, and in the presence of NADH at concentrations of NaCl below 0.3M . The crystal structure shows a tetramer that, in the absence of the salt bridge clusters, appears to be stabilized by a network of ordered water molecules and by Cl(-) binding at the dimer-dimer interface . The double mutant and wild-type dimer folds are essentially identical (the r.m.s . deviation between equivalent C(alpha) positions is 0.39A) . Chloride ions are also observed at the monomer-monomer interfaces of the mutant, contributing to the stability of each dimer against low salt dissociation . Our results support the hypothesis that extensive binding of water and salt is an important feature of adaptation to a halophilic environment. Extremophiles, 2003 Feb, 7(1), 71 - 7 Epub 2002 Oct 03. Isolation of fusion proteins containing SecY and SecE, components of the protein translocation complex from the halophilic archaeon Haloferax volcanii; Irihimovitch V et al.; By exploiting the salt-insensitive interaction of the cellulose-binding domain (CBD) of the Clostridium thermocellum cellulosome with cellulose, purification of CBD-fused versions of SecY and SecE, components of the translocation apparatus of the halophilic archaeon Haloferax volcanii, was undertaken . Following transformation of Haloferax volcanii cells with CBD-SecY- or -SecE-encoding plasmids, cellulose-based purification led to the capture of stably expressed, membrane-bound 68 and 25 kDa proteins, respectively . Both fusion proteins were recognized by antibodies raised against the CBD . Thus, CBD-cellulose interactions can be employed as a salt-insensitive affinity purification system for the capture of complexes containing the Haloferax volcanii translocation apparatus components SecY and SecE. Extremophiles, 2003 Feb, 7(1), 29 - 34 Epub 2002 Sep 28. Salinisphaera shabanensis gen . nov., sp . nov., a novel, moderately halophilic bacterium from the brine-seawater interface of the Shaban Deep, Red Sea; Antunes A et al.; A novel, moderately halophilic bacterium was isolated from the brine-seawater interface of the Shaban Deep, northern Red Sea . A polyphasic approach was used for the taxonomic characterization of this isolate, with the phenotypic and phylogenetic data clearly showing the distinctiveness of this bacterium . Cells of isolate E1L3A were Gram-negative, monotrichous cocci that showed a remarkable physiological flexibility, as could be seen by the quite broad growth ranges for oxygen, temperature, NaCl, and, to a smaller degree, pH . In addition, it was able to grow from atmospheric pressure up to 15 MPa, making it a piezotolerant bacterium . Phylogenetically, strain E1L3A represents a new, deeply branching lineage within the gamma-Proteobacteria, as determined by 16S rRNA gene sequence analysis . No close relatives are known so far, with sequence similarity to other cultivated members of the gamma-Proteobacteria being lower than 88% . The creation of the new genus Salinisphaera and the new species Salinisphaera shabanensis (DSM 14853; JCM 11575) for this new and highly versatile microorganism is therefore proposed. Extremophiles, 2003 Feb, 7(1), 1 - 7 Epub 2002 Sep 03. Differential expression of genes influenced by changing salinity using RNA arbitrarily primed PCR in the archaeal halophile Haloferax volcanii; Bidle KA; Extreme halophiles belonging to the domain Archaea require a minimum of approximately 10% NaCl for growth . Many of these obligate halophiles will continue to grow even as NaCl concentrations approach saturation . The haloarchaeon Haloferax volcanii is a model organism in which to study the effects of changes in medium salinity on gene expression, as this organism grows over a wide range of NaCl concentrations, between 12% and 23%, with little effect on growth rate . An RNA arbitrarily primed PCR (RAP-PCR) approach has been applied to identify those genes that are differentially expressed in response to changing salinity . Differences in gene expression can be detected using this methodology, as each sample generates its own unique RNA fingerprint for each growth condition examined . RNA was prepared from H . volcanii cultures grown with two different NaCl concentrations in the medium, RAP-PCR was performed, and seven differentially expressed transcripts were identified . These fragments were cloned, sequenced, and subjected to transcript analysis to confirm their regulation . One of the sequences identified in this study displays homology to the eukaryotic Ser/Thr protein kinase Ire1p, a sensor of protein unfolding in yeast and mammalian cells . Evidence for serine phosphorylation in H . volcanii is also presented. Int J Toxicol, 2003 Jan-Feb, 22(1), 9 - 23 Short-term repeated-dose toxicity profile of archaeosomes administered to mice via intravenous and oral routes; Omri A et al.; Archaeosomes, liposomes made from polar ether lipids of archaea, show promise for vaccine and drug delivery applications . The potential toxicity of intravenously (14, 70, or 140 mg/kg/day for 5 consecutive days) and orally (gavaged at 55, 275, or 550 mg/kg/day for 10 consecutive days) administered unilamellar archaeosomes, prepared from the total polar lipids (TPLs) extracted from several species of archaea, was assessed in female BALB/c mice . Liposomes prepared from an ester phospholipid composition were included for comparative purposes . Control groups of mice were administered 0.1 ml phosphate-buffered saline (PBS) by either route . Animals were monitored at least once daily for temperature, body weight, and clinical signs of adverse reactions . One day after the last dose, the mice were sacrificed . Blood was collected for selected biochemical/enzyme analyses, and the major organs (heart, lungs, liver, spleen, kidneys) were weighed and examined macroscopically . In addition, the spleens were examined histologically . At the two lower dosages of intravenously administered vesicles, there were no significant indications of toxicity, as compared with the PBS-administered control group . At the highest intravenous dose of 140 mg/kg/day, archaeosomes prepared from the TPL of the extreme halophiles, Halobacterium salinarum and Natronobacterium magadii, indicated potential toxicity, as evidenced by clinical signs (hyperactivity and/or piloerection), drop in body temperature, and loss in body weight . Spleens from mice administered some archaeosomes types, primarily at the highest intravenous dose tested, were enlarged, had increased organ weight, and microscopic examination revealed mild to moderate expansion of the red pulp with increased numbers of hematopoietic cells, but no changes in the white pulp . There were similar clinical signs at one or more of the higher oral doses of the ester liposomes and some of the archaeosome types; however, no other apparent toxicity was observed . Based on this limited mouse study, archaeosomes were generally well tolerated after intravenous or oral delivery at the dosages so indicated in this study. J Biol Chem, 2003 Apr 11, 278(15), 12881 - 7 Epub 2003 Feb 03. Post-translational secretion of fusion proteins in the halophilic archaea Haloferax volcanii; Irihimovitch V et al.; Although protein secretion occurs post-translationally in bacteria and is mainly a cotranslational event in Eukarya, the relationship between the translation and translocation of secreted proteins in Archaea is not known . To address this question, the signal peptide-encoding region of the surface layer glycoprotein gene from the Haloarchaea Haloferax volcanii was fused either to the cellulose-binding domain of the Clostridium thermocellum cellulosome or to the cytoplasmic enzyme dihydrofolate reductase from H . volcanii . Signal peptide-cleaved mature versions of both the cellulose-binding domain and dihydrofolate reductase could be detected in the growth medium of transformed H . volcanii cells . Immunoblot analysis revealed, however, the presence of full-length signal peptide-bearing forms of both proteins inside the cytoplasm of the transformed cells . Proteinase accessibility assays confirmed that the presence of cell-associated signal peptide-bearing proteins was not due to medium contamination . Moreover, the pulse-radiolabeled signal peptide cellulose-binding domain chimera could be chased from the cytoplasm into the growth medium even following treatment with anisomycin, an antibiotic inhibitor of haloarchaeal protein translation . Thus, these results provide evidence that, in Archaea, at least some secreted proteins are first synthesized inside the cell and only then translocated across the plasma membrane into the medium. Proc Natl Acad Sci U S A, 2003 Feb 18, 100(4), 1649 - 54 Epub 2003 Jan 31. Crystal structure of a photoactive yellow protein from a sensor histidine kinase: conformational variability and signal transduction; Rajagopal S et al.; Photoactive yellow protein (E-PYP) is a blue light photoreceptor, implicated in a negative phototactic response in Ectothiorhodospira halophila, that also serves as a model for the Per-Arnt-Sim superfamily of signaling molecules . Because no biological signaling partner for E-PYP has been identified, it has not been possible to correlate any of its photocycle intermediates with a relevant signaling state . However, the PYP domain (Ppr-PYP) from the sensor histidine kinase Ppr in Rhodospirillum centenum, which regulates the catalytic activity of Ppr by blue light absorption, may allow such issues to be addressed . Here we report the crystal structure of Ppr-PYP at 2 A resolution . This domain has the same absorption spectrum and similar photocycle kinetics as full length Ppr, but a blue-shifted absorbance and considerably slower photocycle than E-PYP . Although the overall fold of Ppr-PYP resembles that of E-PYP, a novel conformation of the beta 4-beta 5 loop results in inaccessibility of Met-100, thought to catalyze chromophore reisomerization, to the chromophore . This conformation also exposes a highly conserved molecular surface that could interact with downstream signaling partners . Other structural differences in the alpha 3-alpha 4 and beta 4-beta 5 loops are consistent with these regions playing significant roles in the control of photocycle dynamics and, by comparison to other sensory Per-Arnt-Sim domains, in signal transduction . Because of its direct linkage to a measurable biological output, Ppr-PYP serves as an excellent system for understanding how changes in photocycle dynamics affect signaling by PYPs. Wei Sheng Wu Xue Bao, 2002 Aug, 42(4), 406 - 10 {Molecular cloning and amino acid composition analysis of a halophilic thiolase gene}; Liu T et al.; 5' and 3' end sequence of acaBl gene as primers, the gene of halophilic thiolase from haloarchae, Halobacterium sp . ZP-6 was cloned and its amino acid composition was calculated . Compared with non-halophilic thiolase, the halophilic thiolase contains more negative charge amino acid, less positive amino acid and less strong hydrophobic amino acid, and use preferably small side-chain amino acid . Those suggest that electrostatic screen, hydrophobic effect and surface tension all contribute to halophilic properties of thiolase. Wei Sheng Wu Xue Bao, 2002 Apr, 42(2), 133 - 7 {Analysis of 16S rDNA sequences and DNA-DNA hybridization of moderately halophilic bacteria from Xinjiang region}; Zeng J et al.; Based on the previous studies on numerical taxonomy and 16S rDNA PCR-RFLP analysis, the moderately halophilic bacteria isolated from Xinjiang Region constituted a new cluster, and the phylogenetic tree was constructed by comparing with the 16S rDNA sequences of the other moderately halophilic bacteria species . In the phylogenetic tree, most of the reference strains were clustered in a group, and the similarity values of 16S rDNA sequence were above 96% . However, AI-3, Alcanivorax borkumensis and Halobacillus litoralis were clustered in another group, and the similarity value of 16S rDNA sequences between AI-3 and Alcanivorax borkumensis was 96%, and that of 16S rDNA sequences between AI-3 and Halobacillus litoralis was 99% . The results indicated that AI-3 was different from the reference strains in phylogeny . The values of DNA homology in the new cluster were more than 70%, but the value between AI-3 and Halomonas elongata was less than 50% . Thus, the strain AI-3 possibly represent a new moderately halophilic bacteria species. Wei Sheng Wu Xue Bao, 1999 Jun, 39(3), 220 - 5 {Different protein forms of purple membrane produced by Halobacterium halobium under various nutritional condition}; Xin H et al.; The extremely halophilic bacterium, purple membrane producing Halobacterium halobium strain R1 were cultured in four liquid media which consist of different constituents . After the purple membrane is harvested by ultracentrification and purified by sucrose density gradient ultracentrification, and run on SDS-PAGE, the result of Coomassie blue staining showed different bR protein forms: three bands with molecule weight ranging from 26.0 to 27.5 kD in peptone medium, while only one band with MW 26.0 kD in other three media-complex, synthetic and artificial seawater media, which corresponded to the mature form in peptone medium . The result of Western blotting not only confirmed the different protein forms of Coomassie blue staining in the four media, but also gave additional bands that Coomassie blue can not detect due to its lower sensitivity: 1) in the complex, synthetic and artificial seawater media, a faint 28.0 kD band existed, while in the peptone medium it did not exist; and 2) in the four media, it appeared that a faint 23.0-24.0 kD band exited . The different bR protein forms of purple membrane which result from the difference of nutritional constituents in these four media might be caused by different precursor processing enzymes or different activities of precursor processing enzyme(s) under these different nutritional conditions. J Biol Chem, 2003 Apr 4, 278(14), 12000 - 5 Epub 2003 Jan 27. Characterization of an archaeal multidrug transporter with a unique amino acid composition; Ninio S et al.; The Smr family of multidrug transporters consists of small membrane proteins that extrude various drugs in exchange with protons rendering cells resistant to these drugs . Smr proteins identified to date have been found only in Eubacteria . In this work we present the cloning and characterization of an Smr protein from the archaeon Halobacterium salinarum, the first Smr in the archaeal kingdom . The protein, named Hsmr, was identified through sequence similarity to the Smr family, and the DNA sequence was cloned into an Escherichia coli expression system . Hsmr is heterologously expressed in a functional form despite the difference in lipid composition of the membrane and the lower salt in the cell and its environment . Cells harboring the Hsmr plasmid transport ethidium bromide in an uncoupler-sensitive process and gain resistance to ethidium bromide and acriflavine . Hsmr binds tetraphenylphosphonium (TPP(+)) with a relatively low affinity (K(D) approximately 200 nm) at low salt concentration that increases (K(D) approximately 40 nm) upon the addition of 2 m of either NaCl or KCl . The Hsmr protein contains many of the signature sequence elements of the Smr family and also a high content of negative residues in the loops, characteristic of extreme halophiles . Strikingly, Hsmr is composed of over 40% valine and alanine residues . These residues are clustered at certain regions of the protein in domains that are not important for activity, as judged from lack of conservation and from previous studies with other Smr proteins . We suggest that this high content of alanine and valine residues is a reflection of a "natural" alanine and valine scanning necessitated by the high GC content of the gene . This phenomenon reveals significant sequence elements in small multidrug transporters. Biochemistry, 2003 Feb 4, 42(4), 965 - 70 Heterologous production of Halorhodospira halophila holo-photoactive yellow protein through tandem expression of the postulated biosynthetic genes; Kyndt JA et al.; The photoactive yellow protein (PYP) is a bacterial photoreceptor which is the structural prototype for the PAS domain superfamily of regulators and receptors . PYP is known to have a unique p-hydroxycinnamic acid chromophore, covalently attached to a cysteine . To date, it has not been shown how holo-PYP is formed in vivo . Two genes, nearby pyp, were postulated to encode the biosynthetic enzymes, but only one was previously isolated and shown to have the requisite activity . By using a dual plasmid system, one expressing the PYP from Halorhodospira halophila and the other expressing a two-gene operon, consisting of tyrosine ammonia lyase and p-hydroxycinnamic acid ligase, we are able to present evidence that a functionally active holo-PYP can be synthesized in Escherichia coli . Plasmids containing only one of the two enzymes failed to produce holoprotein . Thus, the two genes have been shown to be both necessary and sufficient for production of holoprotein, although the activating group remains unknown . This expression system not only holds great potential for mutagenesis studies but also opens new possibilities in the search for (a) signaling partner(s) of the PYP. Biophys J, 2003 Feb, 84(2 Pt 1), 1180 - 91 Deuterium isotope effects in the photocycle transitions of the photoactive yellow protein; Hendriks J et al.; The Photoactive Yellow Protein (PYP) from Halorhodospira halophila (formerly Ectothiorhodospira halophila) is increasingly used as a model system . As such, a thorough understanding of the photocycle of PYP is essential . In this study we have combined information from pOH- (or pH-) dependence and (kinetic) deuterium isotope effects to elaborate on existing photocycle models . For several characteristics of PYP we were able to make a distinction between pH- and pOH-dependence, a nontrivial distinction when comparing data from samples dissolved in H(2)O and D(2)O . It turns out that most characteristics of PYP are pOH-dependent . We confirmed the existence of a pB' intermediate in the pR to pB transition of the photocycle . In addition, we were able to show that the pR to pB' transition is reversible, which explains the previously observed biexponential character of the pR-to-pB photocycle step . Also, the absorption spectrum of pB' is slightly red-shifted with respect to pB . The recovery of the pG state is accompanied by an inverse kinetic deuterium isotope effect . Our interpretation of this is that before the chromophore can be isomerized, it is deprotonated by a hydroxide ion from solution . From this we propose a new photocycle intermediate, pB(deprot), from which pG is recovered and which is in equilibrium with pB . This is supported in our data through the combination of the observed pOH and pH dependence, together with the kinetic deuterium isotope effect. Microb Ecol, 2003 Feb, 45(2), 183 - 90 Epub 2003 Jan 28. Survival of filamentous fungi in hypersaline Dead Sea water; Kis-Papo T et al.; A variety of filamentous fungi have recently been isolated from the Dead Sea (340 g/L total dissolved salts) . To assess the extent to which such fungi can survive for prolonged periods in Dead Sea water, we examined the survival of both spores and mycelia in undiluted Dead Sea water and in Dead Sea water diluted to different degrees with distilled water . Mycelia of Aspergillus versicolor and Chaetomium globosum strains isolated from the Dead Sea remained viable for up to 8 weeks in undiluted Dead Sea water . Four Dead Sea isolates (A . versicolor, Eurotium herbariorum, Gymnascella marismortui, and C . globosum) retained their viability in Dead Sea water diluted to 80% during the 12 weeks of the experiment . Mycelia of all species survived for the full term of the experiment in Dead Sea water diluted to 50% and 10% of its original salinity . Comparison of the survival of Dead Sea species and closely related isolates obtained from other locations showed prolonged viability of the strains obtained from the Dead Sea . Spores of isolates obtained from the terrestrial shore of the Dead Sea generally proved less tolerant to suspension in undiluted Dead Sea water than spores of species isolated from the water column . Spores of the species isolated from the control sites had lost their viability in undiluted Dead Sea water within 12 weeks . However, with the exception of Emericella spores, which showed poor survival, a substantial fraction of the spores of Dead Sea fungal isolates remained viable for that period . The difference in survival rate between spores and mycelia of isolates of the same species points to the existence of adapted halotolerant and/or halophilic fungi in the Dead Sea. J Appl Microbiol, 2003, 94(2), 295 - 300 Diversity of moderately halophilic bacteria producing extracellular hydrolytic enzymes; Sanchez-Porro C et al.; AIMS: The aim of this study was to determine the diversity of moderately halophilic bacteria with hydrolase activities . METHODS AND RESULTS: Screening bacteria from different hypersaline environments in South Spain led to the isolation of a total of 122 moderately halophilic bacteria able to produce different hydrolases (amylases, DNases, lipases, proteases and pullulanases) . These bacteria are able to grow optimally in media with 5-15% salts and in most cases up to 20-25% salts . In contrast to strains belonging to previously described species, that showed very little hydrolase activities, environmental isolates produced a great variety of hydrolases . These strains were identified as members of the genera: Salinivibrio (55 strains), Halomonas (25 strains), Chromohalobacter (two strains), Bacillus-Salibacillus (29 strains), Salinicoccus (two strains) and Marinococcus (one strain), as well as eight non-identified isolates . CONCLUSIONS: Moderately halophilic bacteria are a source of hydrolytic enzymes such as amylases, DNases, lipases, proteases and pullulanases . SIGNIFICANCE AND IMPACT OF THE STUDY: Although most culture collection strains are not able to produce hydrolases, it has been shown that environmental isolates can produce these potentially biotechnological important enzymes. J Bacteriol, 2003 Feb, 185(3), 772 - 8 Development of a gene knockout system for the halophilic archaeon Haloferax volcanii by use of the pyrE gene; Bitan-Banin G et al.; So far, the extremely halophilic archaeon Haloferax volcanii has the best genetic tools among the archaea . However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies . In this paper we describe the development of pyrE-based positive selection and counterselection systems to generate an efficient gene knockout system . The H . volacanii pyrE1 and pyrE2 genes were isolated, and the pyrE2 gene was shown to code for the physiological enzyme orotate phosphoribosyl transferase . A DeltapyrE2 strain was constructed and used to isolate deletion mutants by the following two steps: (i) integration of a nonreplicative plasmid carrying both the pyrE2 wild-type gene, as a selectable marker, and a cloned chromosomal DNA fragment containing a deletion in the desired gene; and (ii) excision of the integrated plasmid after selection with 5-fluoroorotic acid . Application of this gene knockout system is described. J Microbiol Methods, 2003 Mar, 52(3), 353 - 9 Production of amylase by newly isolated moderate halophile, Halobacillus sp . strain MA-2; Amoozegar MA et al.; Production of extracellular amylase was demonstrated under stress conditions of high temperature and high salinity in aerobically cultivated culture of a newly isolated moderately halophilic bacterium of spore-forming Halobacillus sp . strain MA-2 in medium containing starch, peptone, beef extract, and NaCl . The maximum amylase production was secreted in the presence of 15% (w/v) Na(2)SO(4) (3.2 U ml(-1)) . The isolate was capable of producing amylase in the presence of NaCl, NaCH(3)COOH, or KCl, with the results NaCl>NaCH(3)COOH>KCl . Maximum amylase activity was exhibited in the medium containing 5% (w/v) NaCl (2.4 U ml(-1)) . Various carbon sources induced enzyme production . The potential of different carbohydrates in the amylase production was in the order: dextrin>starch>maltose>lactose>glucose>sucrose . In the presence of sodium arsenate (100 mM), maximum production of the enzyme was observed at 3.0 U ml(-1) . Copper sulfate (0.1 mM) decreased the amylase production considerately, while lead nitrate had no significant enhancement on amylase production (p<0.05) . The pH, temperature, and aeration optima for enzyme production were 7.8, 30 degrees C, and 200 rpm, respectively, while the optimum pH and temperature for enzyme activity was 7.5-8.5 and 50 degrees C, respectively. Mikrobiologiia, 2002 Nov-Dec, 71(6), 786 - 93 {Magnesium orthophosphate, a new form of reserve phosphates in the halophilic archaeon Halobacterium salinarium}; Smirnov AV et al.; The accumulation and utilization of reserve phosphates in the extremely halophilic archaeon Halobacterium salinarium were studied . The growth of H . salinarium was found to depend on the initial concentration of inorganic phosphate (Pi) in the culture medium and its content in the inoculum . Growing cells consumed 85-95% of Pi from the medium . Unlike the reserve phosphates of many other microorganisms, which are mainly polyphosphates, the reserve phosphates of H . salinarium cells contain no more than 15% polyphosphates, the rest being magnesium orthophosphate . The excessive consumption of Pi from the medium changed cell morphology and caused the death of part of the cell population . The cells that remained viable could grow in a Pi-deficient medium, utilizing about 70% of reserve magnesium phosphate as the phosphorus source. Life Sci Space Res, 1973, 11, 63 - 6 On the multiplication of xerophilic micro-organisms under simulated Martian conditions; Imshenetsky AA et al.; The environmental conditions prevailing on Mars would supposedly favour the existence there of micro-organisms belonging to xerophiles, anaerobes, or micro-aerophiles, oligonitrophiles, which are able to grow in wide temperature intervals . From soil samples taken in deserts and tundra, antarctic halophilic bacteria, able to grow in liquid media containing 20-25% of sodium chloride, were isolated . Some of these cultures appeared to be also osmophilic (growth on media with 50% glucose); they grew at temperatures from 5 degrees C to 50 degrees C, and developed on media without a nitrogen source (oligonitrophiles) . Of special interest was the halophilic and osmophilic form of Bacillus megaterium isolated from the Nubian desert . In experiments with this bacterium the following technique was used . A thin film of potato extract agar was prepared on a glass slide and dried over a saturated K2SO4 solution in a closed container up to the level of maximal hygroscopic moisture . The cell suspension was then sprayed on to the agar surface, the film dried again at 45 degrees C, and the glass slide put in the test tube over the saturated solution of K2SO4 . The test tube was evacuated, flushed three times with a gas mixture containing 80% CO2 plus 20% Ar, and sealed . Under these conditions the water content of the agar film was equal to the maximal hygroscopic moisture; only the xerophilic form of bacteria are able to develop at this moisture level . This halophilic strain of Bac . megaterium grew satisfactorily under these conditions, as did a halophilic and osmophilic strain of Mycococcus ruber isolated in Antarctica . Both the halophilic strain of Bac . megaterium and that of M . ruber were able to grow under simulated Martian conditions . Xerophily and halophily may be linked . This assumption was supported by relatively high incidence of xerophilic forms among halophilic bacteria isolated from different soils of both high and low salt content as well as from salty muds. Protein Expr Purif, 2003 Jan, 27(1), 128 - 33 Cloning, expression, and efficient purification in Escherichia coli of a halophilic nucleoside diphosphate kinase from the moderate halophile Halomonas sp . #593; Yonezawa Y et al.; Most typical halophilic enzymes from extremely halophilic archaea require high concentrations of salt for their activity and stability . These enzymes are inactive in Escherichia coli unless refolded in the presence of salts in vitro . In this report, we describe cloning of the ndk gene of nucleoside diphosphate kinase from a moderately halophilic eubacterium and overexpression of the protein in E . coli as an N-terminal hexa-His fusion to facilitate its purification on Ni-NTA affinity resin . We demonstrate evidence that the protein is properly folded and exhibits the same specific activity and stability as the native protein from Halomonas cells. Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2271 - 80 Characterization of a novel halophilic archaeon, Halobiforma haloterrestris gen . nov., sp . nov., and transfer of Natronobacterium nitratireducens to Halobiforma nitratireducens comb . nov; Hezayen FF et al.; Strain 135(T), a novel red-pigmented, aerobic, extremely halophilic member of the Archaea showing rod, coccus and slightly pleomorphic morphology, was isolated from hypersaline soil close to Aswan (Egypt) . This organism is neutrophilic, motile and requires at least 2.2 M NaCl, but no MgCl2, for growth and exhibits optimal growth at 42 degrees C . Polar lipid analysis revealed the presence of sulfated triglycosyl diether and triglycosyl diether as the sole glycolipids as well as the absence of the glycerol diether analogue of phosphatidyl glycerosulfate . C20:C20 and C20:C25 core lipids are present in almost equal proportions . The G+C content of the DNA is 66.9 mol% . 16S rDNA analysis revealed that strain 135(T) was a member of the phyletic group defined by the family Halobacteriaceae, but there was a low degree of similarity to other members of this family . Highest similarity values of 96.4 and 93.8-94.3% were obtained to the 16S rDNA of Natronobacterium nitratireducens and Natronobacterium gregoryi, Natronococcus occultus and Natronococcus amylolyticus . Strain 135(T) is able to accumulate polyhydroxybutyrate as intracellular reserve material . On the basis of the data presented, strain 135(T) should be placed in a new genus, Halobiforma gen . nov . as Halobiforma haloterrestris sp . nov . The type strain is strain 135(T) (= DSM 13078(T) = JCM 11627(T)) . Moreover, the transfer of Natronobacterium nitratireducens to Halobiforma nitratireducens comb . nov . is proposed. Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2175 - 82 Thioalkalispira microaerophila gen . nov., sp . nov., a novel lithoautotrophic, sulfur-oxidizing bacterium from a soda lake; Sorokin DY et al.; An anaerobic enrichment medium (pH 10) with thiosulfate as electron donor and nitrate as electron acceptor was inoculated with sediment from soda lake Fazda (Wadi Natrun, Egypt); a novel strain, ALEN 1(T), was isolated from the subsequent enrichment culture . Cells of strain ALEN 1(T) had a spiral morphology (0.3-0.45 x 1-4 microm), were motile and had a single polar flagellum . Sphaeroplasts were formed by the cells and were rapidly lysed during prolonged aerobic incubation of cultures . Cells of strain ALEN 1(T) contained a membrane-associated yellow pigment . The metabolism of this novel organism was obligately chemolithoautotrophic, and thiosulfate or sulfide were utilized as electron donors . Washed cells of strain ALEN 1(T) oxidized thiosulfate, sulfide, polysulfide and elemental sulfur to sulfate . Best growth was observed when the strain was grown under micro-oxic conditions (1-2% O2 in gas phase), whereas growth was inhibited under fully oxic conditions . Nitrate was reduced to nitrite without growth of the novel organism, but other nitrogen oxides were not utilized as electron acceptors . Strain ALEN 1(T) was alkaliphilic and moderately halophilic . It grew between pH 8 and 10.4 (optimum around pH 10) with a salt concentration of between 0.3 and 1.5 M Na+ (optimum 0-5 M) . The maximum growth rate (0.08 h(-1)) of the organism was achieved in a thiosulfate-limited micro-oxic continuous culture (pH 10) . Phylogenetic analyses of the 16S rDNA sequences of strain ALEN 1(T) and its closest relatives demonstrated that this strain formed a deep branch within the gamma-Proteobacteria, with no obvious association to any described cluster of species/genera . On the basis of its unique physiological properties and distinct phylogenetic position, it is proposed that strain ALEN 1(T) (= DSM 14786(T) = UNICEM 212(T)) represents a novel genus within the gamma-Proteobacteria, for which the name Thioalkalispira is proposed . It is also proposed that the type species of this novel genus be named Thioalkalispira microaerophila. Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2049 - 54 Halomonas muralis sp . nov., isolated from microbial biofilms colonizing the walls and murals of the Saint-Catherine chapel (Castle Herberstein, Austria); Heyrman J et al.; A group of seven halophilic strains (optimal growth at 2.5-10.0% NaCl) was isolated from samples of a wall and a mural painting, both heavily contaminated by microbial growth, inside the Saint-Catherine chapel of Castle Herberstein (Austria) . The strains were subjected to a polyphasic taxonomic study that included DNA-DNA relatedness studies, DNA base-ratio determinations, 16S rDNA sequence analysis, rep-PCR genomic fingerprinting, fatty acid analysis and phenotypic and biochemical characterization . The data obtained indicate that the strains belong to the genus Halomonas and represent a novel species, for which the name Halomonas muralis sp . nov . is proposed . The type strain is strain LMG 20969(T) ( = DSM 14789(T)). Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2043 - 8 Lentibacillus salicampi gen . nov., sp . nov., a moderately halophilic bacterium isolated from a salt field in Korea; Yoon JH et al.; A Gram-variable, aerobic, endospore-forming, rod-shaped bacterial strain, SF-20(T), which was isolated from a salt field in Korea, was subjected to a polyphasic taxonomic study . Cells of this organism were motile by means of single flagella . Strain SF-20(T) grew optimally in the presence of 4-8% NaCl . The cell wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid . The predominant menaquinone is MK-7 . Strain SF-20(T) has a cellular fatty acid profile containing major amounts of branched fatty acids . The major fatty acids are anteiso-C15:0 and iso-C16:0 . The cellular phospholipids are phosphatidylglycerol and diphosphatidylglycerol . The G+C content of the DNA is 44 mol% . Strain SF-20(T) is phylogenetically closely related to the genus Bacillus and some related genera and, particularly, formed a coherent cluster with the genera Salibacillus and Virgibacillus . The clustering fidelity between strain SF-20(T) and the cluster comprising these two genera was supported by bootstrap analysis at a confidence level of 67.2% . Strain SF-20(T) exhibited levels of 16S rDNA similarity of 93.0-94.7% to the genus Salibacillus and 94.0-94.1% to the genus Virgibacillus . On the basis of phenotypic and phylogenetic data, strain SF-20(T) should be classified in a novel genus and species, for which the name Lentibacillus salicampi gen . nov., sp . nov . is proposed . The type strain is strain SF-20(T) (= KCCM 41560(T) = JCM 11462(T)). J Gen Appl Microbiol, 2002 Oct, 48(5), 269 - 79 Paraliobacillus ryukyuensis gen . nov., sp . nov., a new Gram-positive, slightly halophilic, extremely halotolerant, facultative anaerobe isolated from a decomposing marine alga; Ishikawa M et al.; A slightly halophilic, extremely halotolerant, alkaliphilic, and facultatively anaerobic rod bacterium was isolated from a decomposing marine alga collected in Okinawa, Japan . The isolate, designated O15-7(T), was Gram-positive, endospore-forming, catalase-positive, menaquinone-7-possessing bacterium that is motile by peritrichous flagella . The isolate was an inhabitant of marine environments; the optimum NaCl concentration for growth was 0.75-3.0% (w/v) with a range of 0-22.0%, and the optimum pH was 7.0-8.5 with a range of 5.5-9.5 . Catalase was produced in aerobic cultivation but not in anaerobic cultivation . Carbohydrate, sugar alcohol or a related carbon compound was required for growth . In aerobic cultivation, the isolate produced pyruvate, acetate and CO(2) from glucose, and in anaerobic cultivation, it produced lactate, formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1 for the last three products . No gas was produced anaerobically . Lactate yield per consumed glucose was markedly affected by the pH of the fermentation medium: 51% at pH 6.5 and 8% at pH 9.0 . The cell-wall peptidoglycan contained meso-diaminopimelic acid . Phylogenetically, the isolate occupied an independent lineage within the group composed of the halophilic/halotolerant/alkaliphilic and/or alkalitolerant species in Bacillus rRNA group 1 with the highest 16S rRNA gene sequence similarity of 95.2% to the genus Gracilibacillus . For this isolate, Paraliobacillus ryukyuensis gen . nov., sp . nov . was proposed . The type strain, O15-7(T) (G+C535.6 mol%), has been deposited in the DSMZ, IAM, NBRC, and NRIC (DSM 15140(T)=IAM 15001(T)=NBRC 10001(T)=NRIC 0520(T)). J Gen Appl Microbiol, 1998 Aug, 44(4), 235 - 241 A Class II fructose-1,6-bisphosphate aldolase from a halophilic archaebacterium Haloferax mediterranei; D'Souza SE et al.; Fructose-1,6-bisphosphate (FBP) aldolase (EC 4.1.2.13) was purified 97-fold from a halophilic archaebacterium Haloferax mediterranei, with a specific activity of 2.8 . The enzyme was characterized as a Class II aldolase on the basis of its inhibition by EDTA and other metal chelators . The enzyme had a specific requirement for divalent metal Fe(2+) for activity . Sulfhydryl compounds enhanced aldolase activity. J Gen Appl Microbiol, 1997 Jun, 43(3), 163 - 167 Measurement of in situ halophilic glyceraldehyde-3-phosphate dehydrogenase activity from the permeabilized cells of archaebacterium Haloarcula vallismortis; Patil AR et al.; The cells of Haloarcula vallismortis, an extreme halophilic archaebacterium, were permeabilized by various chemical, physical, and biological treatments . Biological permeabilization by lysozyme and papain showed effective results as observed by studying the in situ activity of halophilic glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) as the model enzyme . Detergents N-cetyl-N, N, N-trimethyl ammonium bromide (CTAB) and digitonin also showed significant results . Other strains of halobacteria could also be permeabilized by lysozyme . The cell morphology did not show any significant change after permeabilization as observed by phase contrast microscopy . The enzyme characteristics of hGAPDH were studied in situ using permeabilized H . vallismortis cells . The properties, like optimum pH, Km for GAP and NAD(+), inhibition by heavy metals, sulphydryl reagents, and other compounds, showed remarkable similarity with those studied in vitro. Extremophiles, 2002 Dec, 6(6), 491 - 8 Epub 2002 Aug 24. Intracellular ion and organic solute concentrations of the extremely halophilic bacterium Salinibacter ruber; Oren A et al.; Salinibacter ruber is a red obligatory aerobic chemoorganotrophic extremely halophilic Bacterium, related to the order Cytophagales . It was isolated from saltern crystallizer ponds, and requires at least 150 g l(-1) salt for growth . The cells have an extremely high potassium content, the ratio K(+)/protein being in the same range as in halophilic Archaea of the order Halobacteriales . X-ray microanalysis in the electron microscope of cells grown in medium of 250 g l(-1) salt confirmed the high intracellular K(+)concentrations, and showed intracellular chloride to be about as high as the cation concentrations within the cells . A search for intracellular organic osmotic solutes, using (13)C-NMR and HPLC techniques, showed glutamate, glycine betaine, and N-alpha-acetyllysine to be present in low concentrations only, contributing very little to the overall osmotic balance . The results presented suggest that the extremely halophilic Bacterium Salinibacteruses a similar mode of haloadaptation to that of the Archaea of the order Halobacteriales, and does not accumulate organic osmotic solutes such as are used by all other known halophilic and halotolerant aerobic Bacteria. Extremophiles, 2002 Dec, 6(6), 479 - 83 Epub 2002 Jul 30. Taxonomic characterization of Haloferax sp . (" H . alicantei") strain Aa 2.2: description of Haloferax lucentensis sp . nov; Gutierrez MC et al.; An extremely halophilic archaeon, previously named as Haloferax sp . strain Aa 2.2 or "Haloferax alicantei" that has been extensively used for genetic studies with halobacteria, was taxonomically characterized by using phenotypic tests (including morphological, physiological, biochemical and nutritional features), DNA-DNA hybridization and 16S rRNA sequence phylogenetic analysis . This organism was isolated in 1986 by Torreblanca et al . from a pond of a Spanish saltern located in Alicante . The cells were pleomorphic, Gram negative and grew optimally at 25% NaCl . The polar lipid composition was similar to that of species of the genus Haloferax . The DNA G+C content of this strain was 64.5 mol% . Phylogenetic analysis based on 16S rRNA sequence comparison confirmed that this archaeon is a member of the genus Haloferax and was most closely related to Haloferax volcanii . DNA-DNA hybridization between strain Aa 2.2 and the type strain of all named species of the genus Haloferax revealed low levels of relatedness (25-2%), supporting the placement of this organism in a new species . On the basis of the phenotypic characteristics, molecular data and phylogenetic analysis we propose to name strain Aa 2.2 as a new species, Haloferax lucentensis sp . nov . The type strain is Aa 2.2 (=JCM 9276=NCIMB 13854=CIP 107410=DSM 14919=CECT 5871=CCM 7023). Extremophiles, 2002 Dec, 6(6), 453 - 62 Epub 2002 Jul 13. Novel insights into the role of potassium for osmoregulation in Halomonas elongata; Kraegeloh A et al.; The role of K(+) in osmoregulation of the halophilic bacterium Halomonas elongata was investigated . At lower salinities (0.51 M NaCl), K(+) was the predominant cytoplasmic solute (1.25 micro mol mg protein(-1)) . At higher salinities (1.03 M NaCl) ectoine became the main cytoplasmic solute (1.57 micro mol mg protein(-1)), while the K(+) content remained unchanged . In response to osmotic upshock, cells of H . elongata simultaneously accumulated ectoine and K(+) glutamate . The ectoine and K(+) glutamate levels in osmotically stressed cells exceeded the level of cells adapted to high salinities . The increase in K(+) glutamate was long lasting (>120 min) and not transient, as described for non-halophiles . Regulation of the synthesis of ectoine and glutamate was proven to occur mainly at the level of enzyme activity . Limitation of K(+) inhibited the growth of salt-adapted H . elongata cells, especially at high salinities, and caused a decrease of the intracellular organic solute content, inhibition of respiration, and an abolition of the cell's ability to respond to osmotic stress . The saturation constant K(S) for K(+) was estimated to be 105 micro M at a salinity of 0.51 M NaCl, indicating that an uptake system of medium affinity is responsible for K(+) accumulation in H . elongata. Extremophiles, 2002 Dec, 6(6), 445 - 52 Epub 2002 Aug 13. Halosimplex carlsbadense gen . nov., sp . nov., a unique halophilic archaeon, with three 16S rRNA genes, that grows only in defined medium with glycerol and acetate or pyruvate; Vreeland RH et al.; A halophilic archaeon has been isolated from unsterilized salt crystals taken from the 250-million-year-old Salado formation in southeastern New Mexico . This microorganism grows only on defined media supplemented with either a combination of acetate and glycerol, glycerol and pyruvate, or pyruvate alone . The archaeon is unable to grow on complex media or to use carbohydrates, amino acids, fats, proteins, or nucleic acids for growth . Unlike other halophilic microbes, this organism possesses four glycolipids, two of which may be novel . The microbe is unique in that it has three dissimilar 16S rRNA genes . Two of the three genes show only 97% similarity to one another, while the third gene possesses only 92%-93% similarity to the other two . Inferred phylogenies indicate that the organism belongs to a deep branch in the line of Haloarcula and Halorhabdus . All three lines of taxonomic evidence: phenotype, lipid patterns, and phylogeny, support creation of a new genus and species within the halophilic Archaea . The name suggested for this new genus and species is Halosimplex carlsbadense . The type strain is 2-9-1(T) (= ATCC BAA-75 and JCM 11222) as written in the formal description. Extremophiles, 2002 Dec, 6(6), 437 - 44 Epub 2002 Jul 30. Presence of two novel cardiolipins in the halophilic archaeal community in the crystallizer brines from the salterns of Margherita di Savoia (Italy) and Eilat (Israel); Lattanzio VM et al.; Two novel cardiolipin derivatives were recently detected in Halobacterium salinarum, namely an archaeal analog of bisphosphatidylglycerol (BPG) and a glycocardiolipin (GlyC) . GlyC was found to be tightly bound to bacteriorhodopsin . To obtain information on the presence and distribution of these archaeal cardiolipins, we have analyzed the lipids extracted from the crystallizer ponds of the salterns of Margherita di Savoia (Italy) and Eilat (Israel) and from cultures of representative species of the Halobacteriaceae by electrospray ionization mass spectrometry . BPG was present as a minor lipid component in the lipids extracted from the biomass of the Margherita di Savoia and the Eilat salterns, while GlyC was detected only in the extract of the biomass of Margherita di Savoia . Both compounds were enriched in the membrane fraction obtained by dialysis of the cells against distilled water . We detected BPG in all members of the Halobacteriaceae tested, but GlyC has so far been found only in the genera Halobacterium and Haloarcula . A sulfated diglycosyl diether was the major glycolipid detected in the biomass of both salterns. J Bacteriol, 2003 Jan, 185(1), 311 - 6 The cobY gene of the archaeon Halobacterium sp . strain NRC-1 is required for de novo cobamide synthesis; Woodson JD et al.; Genetic and nutritional analyses of mutants of the extremely halophilic archaeon Halobacterium sp . strain NRC-1 showed that open reading frame (ORF) Vng1581C encodes a protein with nucleoside triphosphate:adenosylcobinamide-phosphate nucleotidyltransferase enzyme activity . This activity was previously associated with the cobY gene of the methanogenic archaeon Methanobacterium thermoautotrophicum strain DeltaH, but no evidence was obtained to demonstrate the direct involvement of this protein in cobamide biosynthesis in archaea . Computer analysis of the Halobacterium sp . strain NRC-1 ORF Vng1581C gene and the cobY gene of M . thermoautotrophicum strain DeltaH showed the primary amino acid sequence of the proteins encoded by these two genes to be 35% identical and 48% similar . A strain of Halobacterium sp . strain NRC-1 carrying a null allele of the cobY gene was auxotrophic for cobinamide-GDP, a known intermediate of the late steps of cobamide biosynthesis . The auxotrophic requirement for cobinamide-GDP was corrected when a wild-type allele of cobY was introduced into the mutant strain, demonstrating that the lack of cobY function was solely responsible for the observed block in cobamide biosynthesis in this archaeon . The data also show that Halobacterium sp . strain NRC-1 possesses a high-affinity transport system for corrinoids and that this archaeon can synthesize cobamides de novo under aerobic growth conditions . To the best of our knowledge this is the first genetic and nutritional analysis of cobalamin biosynthetic mutants in archaea. J Bacteriol, 2003 Jan, 185(1), 165 - 74 Subunit topology of two 20S proteasomes from Haloferax volcanii; Kaczowka SJ et al.; Haloferax volcanii, a halophilic archaeon, synthesizes three different proteins (alpha1, alpha2, and beta) which are classified in the 20S proteasome superfamily . The alpha1 and beta proteins alone form active 20S proteasomes; the role of alpha2, however, is not clear . To address this, alpha2 was synthesized with an epitope tag and purified by affinity chromatography from recombinant H . volcanii . The alpha2 protein copurified with alpha1 and beta in a complex with an overall structure and peptide-hydrolyzing activity comparable to those of the previously described alpha1-beta proteasome . Supplementing buffers with 10 mM CaCl(2) stabilized the halophilic proteasomes in the absence of salt and enabled them to be separated by native gel electrophoresis . This facilitated the discovery that wild-type H . volcanii synthesizes more than one type of 20S proteasome . Two 20S proteasomes, the alpha1-beta and alpha1-alpha2-beta proteasomes, were identified during stationary phase . Cross-linking of these enzymes, coupled with available structural information, suggested that the alpha1-beta proteasome was a symmetrical cylinder with alpha1 rings on each end . In contrast, the alpha1-alpha2-beta proteasome appeared to be asymmetrical with homo-oligomeric alpha1 and alpha2 rings positioned on separate ends . Inter-alpha-subunit contacts were only detected when the ratio of alpha1 to alpha2 was perturbed in the cell using recombinant technology . These results support a model that the ratio of alpha proteins may modulate the composition and subunit topology of 20S proteasomes in the cell. FEBS Lett, 2002 Dec 18, 532(3), 309 - 12 Properties of the chaperonin complex from the halophilic archaeon Haloferax volcanii; Large AT et al.; The halophilic archaeon Haloferax volcanii has three genes encoding type II chaperonins, named cct1, cct2 and cct3 . We show here that the three CCT proteins are all expressed but not to the same level . All three proteins are further induced on heat shock . The CCT proteins were purified by ammonium sulphate precipitation, sucrose gradient centrifugation and hydrophobic interaction chromatography . This procedure yields a high molecular mass complex (or complexes) . The complex has ATPase activity, which is magnesium dependent, low salt-sensitive and stable to at least 75 degrees C . Activity requires high levels of potassium ions and was reduced in the presence of an increasing concentration of sodium ions. J Gen Appl Microbiol, 2002 Aug, 48(4), 201 - 9 Lactic acid bacteria isolated from soy sauce mash in Thailand; Tanasupawat S et al.; Fourteen sphere-shaped and 30 rod-shaped lactic acid bacteria were isolated from soy sauce mash of two factories in Thailand . These strains were separated into two groups, Group A and Group B, by cell shape and DNA-DNA similarity . Group A contained 14 tetrad-forming strains, and these strains were identified as Tetragenococcus halophilus by DNA similarity . Group B contained 30 rod-shaped bacteria, and they were further divided into four Subgroups, B1, B2, B3, and B4, and three ungrouped strains by phenotypic characteristics and DNA similarity . Subgroup B1 contained 16 strains, and these strains were identified as Lactobacillus acidipiscis by DNA similarity . Subgroup B2 included two strains, and the strains were identified as Lactobacillus farciminis by DNA similarity . Subgroup B3 contained five strains . The strains had meso-diaminopimelic acid in the cell wall, and were identified as Lactobacillus pentosus by DNA similarity . The strains tested produced DL-lactic acid from D-glucose . Subgroup B4 contained four strains . The strains had meso-diaminopimelic acid in the cell wall, and they were identified as Lactobacillus plantarum by DNA similarity . Two ungrouped strains were homofermentative, and one was heterofermentative . They showed a low degree of DNA similarity with the type strains tested, and were left unnamed . The distribution of lactic acid bacteria in soy sauce mash in Thailand is discussed. J Gen Appl Microbiol, 2002 Apr, 48(2), 109 - 16 Haliangium ochraceum gen . nov., sp . nov . and Haliangium tepidum sp . nov.: novel moderately halophilic myxobacteria isolated from coastal saline environments; Fudou R et al.; Phenotypic and phylogenetic studies were performed on two myxobacterial strains, SMP-2 and SMP-10, isolated from coastal regions . The two strains are morphologically similar, in that both produce yellow fruiting bodies, comprising several sessile sporangioles in dense packs . They are differentiated from known terrestrial myxobacteria on the basis of salt requirements (2-3% NaCl) and the presence of anteiso-branched fatty acids . Comparative 16S rRNA gene sequencing studies revealed that SMP-2 and SMP-10 are genetically related, and constitute a new cluster within the myxobacteria group, together with the Polyangium vitellinum Pl vt1 strain as the closest neighbor . The sequence similarity between the two strains is 95.6% . Based on phenotypic and phylogenetic evidence, it is proposed that these two strains be assigned to a new genus, Haliangium gen . nov., with SMP-2 designated as Haliangium ochraceum sp . nov . (= JCM 11303(T) = DSM 14365(T)), and SMP-10 as Haliangium tepidum sp . nov . (= JCM 11304(T)= DSM 14436(T)). Astrobiology, 2001 Summer, 1(2), 161 - 4 Martian water: are there extant halobacteria on Mars? Landis GA. On Earth, life exists in all niches where water exists in liquid form for at least a portion of the year . On Mars, any liquid water would have to be a highly concentrated brine solution . It is likely, therefore, that any present-day Martian microorganisms would be similar to terrestrial halophiles . Even if present-day life does not exist on Mars, it is an interesting speculation that ancient bacteria preserved in salt deposits could be retrieved from an era when the climate of Mars was more conducive to life. Phytother Res, 2002 Dec, 16(8), 751 - 3 Screening of some indigenous Qatari medicinal plants for antimicrobial activity; Mahasneh AM; Aqueous, ethanol and butanol crude extracts of the aerial parts of ten plants exhibited variable degrees of antimicrobial activity against four bacterial and two fungal species . Aqueous extracts had low antimicrobial activity against E.coli, P.aeruginosa, B . cerreus, S.aureus, C.albicans and A.flavus . Avicennia marina (AM) aqueous extract exhibited a moderate antifungal activity . Ethanol and butanol crude extracts exhibited an improved antimicrobial activity . However, butanol exhibited a superior antimicrobial activity compared with aqueous and ethanol crudes . Compared with the standard antibiotics tested the butanol extract had the highest activity . Butanol extracts at 2000 microg/disc of AM, Lotus halophilus (LA), Pulicaria gnaphaloides (PG) and Capparis spinosa (CS) had a very good antibacterial activity against both gram-positive and -negative bacteria as well as moderate to good antifungal activity against C . albicans and A . flavus . Medicago laciniata (ML), Limonium axillare (LA) and (PG) butanol crude extract compared with standard chloramphenicol, tetracycline and nalidixic acid exhibited a superior antifungal activity . Acta Crystallogr D Biol Crystallogr, 2002 Dec, 58(Pt 12), 2125 - 6 Epub 2002 Nov 23. Crystallization of an alpha-amylase, AmyA, from the thermophilic halophile Halothermothrix orenii; Li N et al.; This report is the first crystallographic study of an amylase from an organism that is both thermophilic and halophilic . alpha-Amylase from the thermophilic halophile Halothermothrix orenii (AmyA) is a 515-residue protein . It is stable and significantly active at 338 K in starch solution containing NaCl {up to 25%(w/v)} . Purified recombinant AmyA protein crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 55.126, b = 61.658, c = 147.625 A, using the hanging-drop vapour-diffusion method . The crystal diffracts X-rays to a resolution limit of 1.89 A. Prikl Biokhim Mikrobiol, 2002 Nov-Dec, 38(6), 616 - 24 {Characteristics of cyclodextrin production using cyclodextrin glucanotransferases of various groups of microorganisms}; Abelian VA et al.; Cyclodextrin glucanotransferases (CGTases; EC 2.4.1.19) from newly isolated mesophilic, thermophilic, alkalophilic, and halophilic bacilli, as well as from thermoactinomycetes, have been purified to homogeneity, and some of their physicochemical and biochemical characteristics (cyclizing, disproportionating, and hydrolytic activities) have been studied . Cyclodextrin (CD) production in the presence and absence of compounds favoring formation of complexes had certain specific features . We were able to demonstrate that CG-Tases of mesophilic and thermophilic strains form mixtures of alpha-, beta-, and gamma-CDs, whereas the enzymes from halophilic and alkalophilic microorganisms preferentially catalyze the formation of beta-CDs. Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 61 - 72 The cell membrane plays a crucial role in survival of bacteria and archaea in extreme environments; Konings WN et al.; The cytoplasmic membrane of bacteria and archaea determine to a large extent the composition of the cytoplasm . Since the ion and in particular the proton and/or the sodium ion electrochemical gradients across the membranes are crucial for the bioenergetic conditions of these microorganisms, strategies are needed to restrict the permeation of these ions across their cytoplasmic membrane . The proton and sodium permeabilities of all biological membranes increase with the temperature . Psychrophilic and mesophilic bacteria, and mesophilic, (hyper)thermophilic and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains low and constant (homeo-proton permeability) . Thermophilic bacteria, however, have more difficulties to restrict the proton permeation across their membrane at high temperatures and these organisms have to rely on the less permeable sodium ions for maintaining a high sodium-motive force for driving their energy requiring membrane-bound processes . Transport of solutes across the bacterial and archaeal membrane is mainly catalyzed by primary ATP driven transport systems or by proton or sodium motive force driven secondary transport systems . Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary ATP-driven uptake systems for their carbon and energy sources . Several high-affinity ABC transporters for sugars from hyperthermophiles have been identified and characterized . The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments. Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 51 - 9 The molecular basis of sensing and responding to light in microorganisms; Hellingwerf KJ; Photon absorption in biological signal transfer is mediated by a limited number of photoreceptor families, each characterised by binding of chromophore from a particular class of chemical compounds . Most photosensors become activated upon light-induced E/Z (i.e., trans/cis) isomerization of a double bond in their chromophore . This change in configuration of the chromophore subsequently must be translated into a change in the conformation of the photosensor protein, and transmitted to the downstream partner in the signal transduction chain . Particularly in archaea the molecular mechanism of signal transfer from the photosensor all the way to its target, i.e., the motility machinery, specific promoters and/or specific enzymes, is well understood for selected examples . In most of these, this flow of information makes use of a mechanism that is based on the so-called 'two-component paradigm' . Best know among these are the light-induced behavioural responses in Halobacterium salinarum, i.e., attraction by green- and repulsion by blue light . Regarding eukaryotic microorganisms our understanding of light-induced signal transfer, beyond the photoreceptor proteins, is restricted . This is due to their much more complex motility apparatus, the involvement of various secondary messengers and their compartmentalisation . The latter may require translocation of transcriptional activators to the nucleus and may form the basis of the sensing of the direction of the light . For a limited number of photoreceptor proteins we begin to understand the intra-molecular transition required to bring about the change in conformation of the protein that initiates signal transfer, i.e., the structure of the so-called 'signalling state' . This insight is most advanced in the photoactive yellow protein from Ectothiorhodospira halophila, a photoreceptor initiating a repellent response upon blue light excitation . In some well-established examples of sensor proteins involved in the transmission of chemical signals, formation of the signalling state appears to be just a shift in the equilibrium between two states that both are already present in the absence of signals . In photoreceptor proteins, however, this situation appears to be much more complex. Water Res, 2002 Nov, 36(19), 4811 - 20 Kinetics of phenol biodegradation in high salt solutions; Peyton BM et al.; Biological treatment of high-salinity industrial wastewaters using halophilic bacteria can be used to remove organic compounds without first decreasing the salt concentration . While halophilic degradation of phenol and other organics has been investigated, there exists a general absence of kinetic data in current literature to allow evaluation of this treatment alternative . Liquid, soil and sediment samples were collected from three distinct saline environments in the western United States . Samples were enriched in media containing 10% (w/v) NaCl at pH 7.0, with phenol as a substrate . Mixed culture batch experiments were conducted at 30 degrees C with initial phenol concentrations of 50 mg/L . Evaluation of phenol degradation and corresponding cell growth data with Monod and Andrews models indicated that the kinetics were zero-order with respect to phenol . Zero-order specific growth rates ranged from 0.22 to 0.32 h(-1), while observed cell yields were 0.18-0.28 mg cell protein/mg phenol for the five cultures . For one of the cultures, phenol degradation rates were also quantified at concentrations of up to 320 mg/L . Under these conditions, specific growth rates ranged from 0.09 to 0.22 h(-1), decreasing with increasing initial phenol concentrations . Cell yields at these higher initial phenol concentrations ranged from 0.20 to 0.29 mg cell protein/mg phenol. FEMS Microbiol Lett, 2002 Nov 5, 216(2), 235 - 41 Secondary and quaternary structural transition of the halophilic archaeon nucleoside diphosphate kinase under high- and low-salt conditions; Ishibashi M et al.; Most halophilic enzymes from extremely halophilic archaea are denatured immediately after transfer from high-salt to low-salt medium . However, nucleoside diphosphate kinase (HsNDK) from the extremely halophilic archaeon Halobacterium salinarum seems to be exceptional, since the enzyme exhibited catalytic activity even under the low-salt condition . Here we show the mechanism how HsNDK is active under both high- and low-salt conditions that the HsNDK hexamer in high-salt medium dissociates into a dimer in the low-salt medium without denaturation . The observed change of the subunit structure was accompanied by a large decrease of alpha-helical content and lowered thermal sensitivity, yet keeping the conformations . This novel hexamer to dimer conversion under high- and low-salt conditions, respectively, seems to be the mechanism by which HsNDK is avoided from the irreversible denaturation. FEMS Microbiol Lett, 2002 Nov 5, 216(2), 193 - 9 The HOG pathway in the halophilic black yeast Hortaea werneckii: isolation of the HOG1 homolog gene and activation of HwHog1p; Turk M et al.; The mitogen-activated protein kinase (MAPK) Hog1p plays an essential role in the yeast hyperosmotic response . A homolog of the HOG1 gene was isolated from the halophilic black yeast Hortaea werneckii encoding a putative 359 amino acid protein, HwHog1p, with high homology to Saccharomyces cerevisiae Hog1p and to other eukaryotic Hog1p homologs . HwHog1p contains a TGY motif within a protein kinase catalytic domain and a C-terminal common docking (CD) motif . Its activation by increased salinity is regulated at the posttranscriptional level . HwHog1p is located on the plasma membrane under nonstress conditions . Upon increased external salinity it is translocated from the membrane, presumably to the nucleus. FEMS Microbiol Lett, 2002 Oct 29, 216(1), 105 - 9 Genetic evidence for a novel thymidylate synthase in the halophilic archaeon Halobacterium salinarum and in Campylobacter jejuni; Giladi M et al.; A search of the complete genome sequence of the halophilic archaeon Halobacterium salinarum failed to identify a gene homologous to the thymidylate synthase (thyA) gene present in the closely related Haloferax volcanii . To understand the source of thymidine synthesis in Hbt . salinarum, a genomic library of Hbt . salinarum was constructed and used to complement a Hfx . volcanii thyA deletion mutation . The Hbt . salinarum ORF that complemented the thyA mutation shares sequence homology with ORFs found in numerous microorganisms that lack a thyA gene, including the recently discovered thyX of Helicobacter pylori . We also show that a homolog of the Hbt . salinarum ORF is present in Campylobacter jejuni and is able to complement an Escherichia coli thyA mutant under oxygen-limiting conditions. Syst Appl Microbiol, 2002 Oct, 25(3), 360 - 75 A detailed phenotypic characterisation of the type strains of Halomonas species; Mata JA et al.; We have made a detailed phenotypic characterisation of the type strains of 21 species within the genus Halomonas and have also studied any possible intraspecific variation of strains within H . eurihalina, H . halophila, H . maura and H . salina . We used 234 morphological, physiological, biochemical, nutritional and antimicrobial susceptibility tests . Nutritional assays were carried out using both classical and miniaturized (BIOLOG system) identification methods . Two different numerical analyses were made using the TAXAN program; the first included the differential data from all the tests carried out whilst the second used only the 57 tests with the highest diagnostic scores (> or = 0.5) . The results of both analyses were quite similar and demonstrated the phenotypic heterogeneity of the Halomonas species in question . At a 62% similarity level the type species were grouped into three phena, the main difference between them being the capacity of those included within phenon A (H . aquamarina, H . meridiana, H . cupida, H . pantelleriensis and H . halmophila) to produce acids from sugars . The species grouped in phenon C (H . campisalis, H . desiderata and H . subglasciescola) used fewer organic substrates than the others . The remaining strains were included in phenon B . H . marisflavi was clearly distinct and thus was not included in any of the three phena . High phenotypic similarity (more than 88%) was found between Halomonas campisalis and Halomonas desiderata . The results of our work should allow researchers to minimise the tests required to arrive at a reliable phenotypic characterisation of Halomonas isolates and to select those of most use to differentiate Halomonas species from each other. Proc Natl Acad Sci U S A, 2002 Nov 12, 99(23), 14913 - 8 Epub 2002 Oct 28. Coordinate regulation of energy transduction modules in Halobacterium sp . analyzed by a global systems approach; Baliga NS et al.; The extremely halophilic archaeon Halobacterium NRC-1 can switch from aerobic energy production (energy from organic compounds) to anaerobic phototrophy (energy from light) by induction of purple membrane biogenesis . The purple membrane is made up of multiple copies of a 1:1 complex of bacterioopsin (Bop) and retinal called bacteriorhodopsin that functions as a light-driven proton pump . A light- and redox-sensing transcription regulator, Bat, regulates critical genes encoding the biogenesis of the purple membrane . To better understand the regulatory network underlying this physiological state, we report a systems approach using global mRNA and protein analyses of four strains of Halobacterium sp.: the wild-type, NRC-1; and three genetically perturbed strains: S9 (bat+), a purple membrane overproducer, and two purple membrane deficient strains, SD23 (a bop knockout) and SD20 (a bat knockout) . The integrated DNA microarray and proteomic data reveal the coordinated coregulation of several interconnected biochemical pathways for phototrophy: isoprenoid synthesis, carotenoid synthesis, and bacteriorhodopsin assembly . In phototrophy, the second major biomodule for ATP production, arginine fermentation, is repressed . The primary systems level insight provided by this study is that two major energy production pathways in Halobacterium sp., phototrophy and arginine fermentation, are inversely regulated, presumably to achieve a balance in ATP production under anaerobic conditions. Biochemistry, 2002 Nov 5, 41(44), 13245 - 52 Link between protein-solvent and weak protein-protein interactions gives insight into halophilic adaptation; Costenaro L et al.; Malate dehydrogenase (Hm MalDH) from the extreme halophile Haloarcula marismortui is a very acidic protein with extensive ion binding properties . It is a good model for the study of solvation-solubility relationships . We measured the small-angle neutron or X-ray scattering profiles of folded and stable Hm MalDH at various protein concentrations and derived the second virial coefficients A(2) . In NaCl, CsCl, KF, KCl, and NaCH(3)CO(2), A(2) values are positive, indicating globally repulsive protein-protein interactions . Below 1 M MgCl(2) and MgSO(4) or above 2 M (NH(4))(2)SO(4), A(2) rapidly decreases . From structure factor modeling with DLVO (Derjaguin, Landau, Verwey, and Overbeek)-like potentials, an effective diameter of 80-82 A is found for the protein particle in solution, compatible with its structural dimensions; the effective charge of the particle is undefined because of the high salt concentration . The strong variations of the protein-protein interaction are correlated to an attractive potential whose depth evolves with the salinity but in an opposite way in Mg salts and (NH(4))(2)SO(4) . A repulsive Donnan term, corresponding to counterion dissociation, and an attractive term related to previously measured preferential salt binding parameters are discussed from well-established thermodynamics considerations and qualitatively account for the behavior of the protein-protein interactions in the various solutions . Because a solvation shell with a composition different from bulk induces protein-protein attraction, molecular adaptation to high salt would be directed to allow protein-salt interactions in order to avoid water or salt enrichment at the surface of the protein and thus preserve its solubility. Biochemistry, 2002 Nov 5, 41(44), 13234 - 44 Solvent interactions of halophilic malate dehydrogenase; Ebel C et al.; Malate dehydrogenase from the extreme halophilic Haloarcula marismortui (Hm MalDH) is an acidic protein that is unstable below molar salt concentrations . The solvated folded protein was studied by small-angle neutron scattering in solvents containing salt: NaCl, NaCH(3)CO(2), KF, NH(4)Cl, NH(4)CH(3)CO(2), (NH(4))(2)SO(4), MgCl(2), and MgSO(4) . It was found that the global solvent interactions depend mainly on the nature of the cation . Complementary mass density measurements in MgCl(2), NaCl, NaCH(3)CO(2), and (NH(4))(2)SO(4) allowed determining the partial molal volumes of the protein, which were found to increase slightly with the salt, and the preferential salt binding parameters for each solvent condition . These are strongly dependent on the cation type and salt concentration . Hm MalDH can be modeled as an invariant particle binding 4100 water molecules in MgCl(2) and 2000 +/- 200 in NaCl, NaCH(3)CO(2), or (NH(4))(2)SO(4) . The number of salt molecules associated to the particle decreases from about 85 to 0 in the order MgCl(2) > NaCl = NaCH(3)CO(2) > (NH(4))(2)SO(4) . Alternatively, we considered exchangeable sites for water and salt with the effects of solvent nonideality . It does not change the description of the solvent interactions . Solvent anions act on Hm MalDH stability through a limited number of strong binding sites, as those seen at the interfaces of Hm MalDH by crystallography . Cations would act through some strong and numerous weak binding sites defined on the folded protein, in possible addition to nonspecific hydration effects. J Bacteriol, 2002 Nov, 184(22), 6207 - 15 Chloride, a new environmental signal molecule involved in gene regulation in a moderately halophilic bacterium, Halobacillus halophilus; Roessler M et al.; The gram-positive, aerobic, moderately halophilic bacterium Halobacillus halophilus is challenged in its environment by frequently changing salt (NaCl) concentrations . Recently, H . halophilus was shown to be the first prokaryote that is dependent on Cl(-) for growth . In a search for the biological function of Cl(-) in this prokaryote, we identified different Cl(-)-dependent processes, which suggests a more general role for Cl(-) in the metabolism of H . halophilus . To analyze the effect of Cl(-) in more detail, we concentrated on one model system, the Cl(-)-dependent production of flagella, and aimed to identify the molecular basis for the Cl(-) dependence of flagellum production . Here, we report that synthesis of the major subunit of the flagellum, FliC, is dependent on the Cl(-) concentration of the medium, as determined by Western blot analyses . The gene encoding FliC was cloned and sequenced, and Northern blot as well as reverse transcriptase PCR analyses revealed that expression of fliC is Cl(-) dependent . FliC is the first protein of known function demonstrated to be synthesized in a Cl(-)-dependent manner in a prokaryote . Two-dimensional gel electrophoresis of cells grown under different conditions revealed five more Cl(-)-induced proteins; these were identified by N-terminal sequencing and database searches to be orthologs of proteins involved in stress response in Bacillus subtilis . The data indicate that Cl(-) is an important environmental signal in this moderate halophile and regulates protein synthesis and gene expression . Furthermore, the data may suggest that Cl(-) plays a role in the signal transduction involved in salt perception by this bacterium. Extremophiles, 2002 Oct, 6(5), 427 - 30 Epub 2002 May 28. The search for traces of life: the protective effect of salt on biological macromolecules; Tehei M et al.; Trapping malate dehydrogenase from the extremely halophilic archaeon Haloarcula marismortui in "dry" salt crystals protects the enzyme against thermal denaturation . Similar protection was not observed for the homologous mesophilic enzyme . In the case of transfer RNA molecules, high salt concentration plays a protective role against thermal degradation allowing activity to be recovered . The results are discussed in the context of exploring the fate of cell-free biological macromolecules in the environment and that of orienting the search for traces of life in planetary exploration. J Mol Biol, 2002 Oct 18, 323(2), 327 - 44 The effect of salts on the activity and stability of Escherichia coli and Haloferax volcanii dihydrofolate reductases; Wright DB et al.; The extremely halophilic Archae require near-saturating concentrations of salt in the external environment and in their cytoplasm, potassium being the predominant intracellular cation . The proteins of these organisms have evolved to function in concentrations of salt that inactivate or precipitate homologous proteins from non-halophilic species . It has been proposed that haloadaptation is primarily due to clustering of acidic residues on the surface of the protein, and that these clusters bind networks of hydrated ions . The dihydrofolate reductases from Escherichia coli (ecDHFR) and two DHFR isozymes from Haloferax volcanii (hvDHFR1 and hvDHFR2) have been used as a model system to compare the effect of salts on a mesophilic and halophilic enzyme . The KCl-dependence of the activity and substrate affinity was investigated . ecDHFR is largely inactivated above 1M KCl, with no major effect on substrate affinity . hvDHFR1 and hvDHFR2 unfold at KCl concentrations below approximately 0.5M . Above approximately 1M, the KCl dependence of the hvDHFR activities can be attributed to the effect of salt on substrate affinity . The abilities of NaCl, KCl, and CsCl to enhance the stability to urea denaturation were determined, and similar efficacies of stabilization were observed for all three DHFR variants . The DeltaG degrees (H(2)O) values increased linearly with increasing KCl and CsCl concentrations . The increase of DeltaG degrees (H(2)O) as a function of the smallest cation, NaCl, is slightly curved, suggesting a minor stabilization from cation binding or screening of electrostatic repulsion . At their respective physiological ionic strengths, the DHFR variants exhibit similar stabilities . Salts stabilize ecDHFR and the hvDHFRs by a common mechanism, not a halophile-specific mechanism, such as the binding of hydrated salt networks . The primary mode of salt stabilization of the mesophilic and halophilic DHFRs appears to be through preferential hydration and the Hofmeister effect of salt on the activity and entropy of the aqueous solvent . In support of this conclusion, all three DHFRs are similarly stabilized by the non-ionic cosolute, sucrose. Arch Microbiol, 2002 Nov, 178(5), 315 - 24 Epub 2002 Jul 26. Characterization of three spiral-shaped purple nonsulfur bacteria isolated from coastal lagoon sediments, saline sulfur springs, and microbial mats: emended description of the genus Roseospira and description of Roseospira marina sp . nov., Roseospira navarrensis sp . nov., and Roseospira thiosulfatophila sp . nov; Guyoneaud R et al.; Three new spirilloid phototrophic purple nonsulfur bacteria were isolated in pure culture from three different environments: strain CE2105 from a brackish lagoon in the Arcachon Bay (Atlantic coast, France), strain SE3104 from a saline sulfur spring in the Pyrenees (Navarra, Spain), and strain AT2115 a microbial mat (Tetiaroa Atoll, Society Islands) . Single cells of the three strains were spiral-shaped and highly motile . Their intracellular photosynthetic membranes were of the vesicular type . Bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series were present as photosynthetic pigments . Optimal growth occurred under photoheterotrophic conditions and in the presence of 0.5-4% w/v NaCl . These features are similar to those described for Roseospira mediosalina . Comparative sequence analysis of their 16S rRNA genes placed these strains within the alpha-subclass of Proteobacteria, in a cluster together with Roseospira mediosalina and Rhodospira trueperi . They form a closely related group of slightly to moderately halophilic spiral-shaped purple nonsulfur bacteria.However, the three new isolates exhibited some differences in their physiology and genetic characteristics . Consequently, we propose that they are members of three new species within the genus Roseospira, Roseospira marina sp . nov., Roseospira navarrensis sp . nov., and Roseospira thiosulfatophila sp . nov., with strains CE2105, SE3104, and AT2115 as the type strains, respectively . As a consequence, an emended description of the genus Roseospira is also given. Curr Protein Pept Sci, 2002 Feb, 3(1), 67 - 78 High-resolution structures of large ribosomal subunits from mesophilic eubacteria and halophilic archaea at various functional States; Yonath A; Structural analysis of the recently determined high resolution structures of the small and the large ribosomal subunits from three bacterial sources, assisted by the medium resolution structure of a complex of the entire ribosome with three tRNAs, led to a quantum jump in our understanding of the process of the translation of the genetic code into proteins . Results of these studies highlighted dynamic aspects of protein biosynthesis; illuminated the modes of action of several antibiotics; indicated strategies adopted by ribosomes for maximizing their functional activity and revealed a wealth of architectural elements, including long tails of proteins penetrating the particle s cores and stabilizing the intricate folds of the RNA chains . Binding of substrate analogues showed that the decoding and the peptide-bond formation are accomplished mainly by RNA . However, several proteins may be functionally relevant in directing the mRNA and in mediating the proper orientation of the tRNA molecules within the ribosomal rRNA frame . Elements involved in intersubunit contacts or in substrate binding are inherently flexible, but maintain well-ordered characteristic conformations in unbound particles . The ribosomes utilize this conformational variability for optimizing their efficiency and minimizing non-productive interactions, hence disorder of functionally relevant features may be linked to less active conformations or to far from physiological conditions . Clinically relevant antibiotics bind almost exclusively to rRNA . In the small subunit they affect the decoding accuracy or limit conformational mobility and in the large subunit they either interfere with substrate binding, by interacting with components of the peptidyl transferase cavity, or hinder the progression of the growing peptide chain. Nucleic Acids Res, 2002 Oct 1, 30(19), 4166 - 75 Reconstitution of the signal recognition particle of the halophilic archaeon Haloferax volcanii; Tozik I et al.; The signal recognition particle (SRP) is a ribonucleoprotein complex involved in the recognition and targeting of nascent extracytoplasmic proteins in all three domains of life . In Archaea, SRP contains 7S RNA like its eukaryal counterpart, yet only includes two of the six protein subunits found in the eukaryal complex . To further our understanding of the archaeal SRP, 7S RNA, SRP19 and SRP54 of the halophilic archaeon Haloferax volcanii have been expressed and purified, and used to reconstitute the ternary SRP complex . The availability of SRP components from a haloarchaeon offers insight into the structure, assembly and function of this ribonucleoprotein complex at saturating salt conditions . While the amino acid sequences of H.volcanii SRP19 and SRP54 are modified presumably as an adaptation to their saline surroundings, the interactions between these halophilic SRP components and SRP RNA appear conserved, with the possibility of a few exceptions . Indeed, the H.volcanii SRP can assemble in the absence of high salt . As reported with other archaeal SRPs, the limited binding of H.volcanii SRP54 to SRP RNA is enhanced in the presence of SRP19 . Finally, immunolocalization reveals that H.volcanii SRP54 is found in the cytosolic fraction, where it is associated with the ribosomal fraction of the cell. Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1807 - 14 Halococcus dombrowskii sp . nov., an archaeal isolate from a Permian alpine salt deposit; Stan-Lotter H et al.; Several extremely halophilic coccoid archaeal strains were isolated from pieces of dry rock salt that were obtained three days after blasting operations in an Austrian salt mine . The deposition of the salt is thought to have occurred during the Permian period (225-280 million years ago) . On the basis of their polar-lipid composition, 16S rRNA gene sequences, cell shape and growth characteristics, the isolates were assigned to the genus Halococcus . The DNA-DNA reassociation values of one isolate, strain H4T, were 35 and 38% with Halococcus salifodinae and Halococcus saccharolyticus, respectively, and 65.8-67.8% with Halococcus morrhuae . The polar lipids of strain H4T were C20-C25 derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate . Whole-cell protein patterns, menaquinone content, enzyme composition, arrangements of cells, usage of carbon and energy sources, and antibiotic susceptibility were sufficiently different between strain H4T and H . morrhuae to warrant designation of strain H4T as a new species within the genus Halococcus . It is proposed that the isolate be named Halococcus dombrowskii, and the type strain is H4T (= DSM 14522T = NCIMB 13803T = ATCC BAA-364T). Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1767 - 72 Halomonas halocynthiae sp . nov., isolated from the marine ascidian Halocynthia aurantium; Romanenko LA et al.; The marine bacterium strain KMM 1376T was isolated from gill tissue of the ascidian Halocynthia aurantium, an inhabitant of the coastal waters of the Sea of Japan . Strain KMM 1376T is an aerobic, gram-negative, non-motile, encapsulated, non-pigmented, slightly halophilic bacterium that is characterized by fimbria-like structures, growth in 0.5-15% NaCl at 7-35 degrees C and absence of acid production from many carbohydrates . The DNA G+C content is 54 mol% . The main fatty acids are C16:0, C16.1omega7c and C18:1omega9c . Comparative 16S rDNA sequence analysis of strain KMM 1376T revealed 92.7-95.3% sequence similarity to members of Halomonas . Based on phenotypic and phylogenetic characteristics, it is proposed that the novel isolate be classified as Halomonas halocynthiae sp . nov., with the type strain KMM 1376T (= DSM 14573T). Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1455 - 9 Psychromonas marina sp . nov., a novel halophilic, facultatively psychrophilic bacterium isolated from the coast of the Okhotsk Sea; Kawasaki K et al.; A facultatively psychrophilic bacterium, strain 4-22T, was isolated from a cold current off the Monbetsu coast of the Okhotsk Sea in Hokkaido, Japan . The isolate was a rod-shaped facultative anaerobe that reduced nitrate to nitrite and hydrolysed starch, DNA and alginic acid, but not chitin or gelatin . The isolate grew at 0 degrees C, but not at 26 degrees C; the optimum growth temperature was 14-16 degrees C . NaCl was required for growth . The DNA G+C content was 43.5 mol% . The whole-cell fatty acids consisted of significant amounts of an unsaturated fatty acid, C16:1, and a saturated fatty acid, C16:0 . A polyunsaturated fatty acid, docosahexaenoic acid (C22:6), was also detected (1.6%) . Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 4-22T was closely related to Psychromonas antarctica (95.7% similarity) . DNA-DNA hybridization revealed a relatedness of 31% between strain 4-22T and P . antarctica . Based on physiological and biochemical characteristics and the phylogenetic position as determined by 16S rRNA gene analysis and DNA-DNA relatedness, it is concluded that the isolate represents a novel species, for which the name Psychromonas marina sp . nov . is proposed . The type strain is 4-22T (= JCM 10501T = IAM 14899T = NCIMB 13792T). J Nat Prod, 2002 Sep, 65(9), 1340 - 3 New C(40)-carotenoid acyl glycoside as principal carotenoid in Salinibacter ruber, an extremely halophilic eubacterium; Lutnaes BF et al.; The principal (>96% of total) carotenoid in the novel, extremely halophilic eubacterium Salinibacter ruber, here called salinixanthin (1), has been assigned the structure (all-E,2'S)-2'-hydroxy-1'-{6-O-(13-methyltetradecanoyl)-beta-D-glycopyranosyloxy}-3',4'-didehydro-1',2'-dihydro-beta,psi-caroten-4-one by spectrometric (vis, EIMS, (1)HNMR, CD, GCMS) and chemical methods. Curr Opin Biotechnol, 2002 Aug, 13(4), 385 - 9 Extreme halophilic enzymes in organic solvents; Marhuenda-Egea FC et al.; The use of halophilic extremozymes in organic media has been limited by the lack of enzymological studies in these media . To explore the behaviour of these extremozymes in organic media, different approaches have been adopted, including the dispersal of the lyophilised enzyme or the use of reverse micelles . The use of reverse micelles in maintaining high activities of halophilic extremozymes under unfavourable conditions could open new fields of application such as the use of these enzymes as biocatalysts in organic media. Plant Physiol, 1995 Oct, 109(2), 549 - 556 Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L; Barkla BJ et al.; Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species . In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport . Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient . Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls . Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 {mu}M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride . The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M . crystallinum is energized by the H+ electrochemical gradient across the tonoplast. Extremophiles, 2002 Aug, 6(4), 267 - 74 Epub 2002 Mar 09. Diversity of Archaea in hypersaline environments characterized by molecular-phylogenetic and cultivation studies; Ochsenreiter T et al.; The diversity of Archaea from three different hypersaline environments was analyzed and compared by polymerase chain reaction (PCR)-based molecular phylogenetic techniques and cultivation approaches . The samples originated from a crystallization pond of a solar saltern in Spain (FC); an alkaline lake in Nevada, USA, (EMF); and a small pond from a slag heap of a potassium mine in Germany (DIE) . Except for two 16S rDNA sequences that were related to crenarchaeota from soil and did not apparently belong to the indigenous halophilic community, all sequences recovered from environmental DNA or cultivated strains grouped within the Halobacteriaceae . Mostly 16S rDNA sequences related to the genera Halorubrum and Haloarcula were detected in sample FC, and organisms belonging to these genera were also recovered by cultivation . In contrast, sequences related to five different groups of halophilic archaea were amplified from sample DIE (including novel lineages with only uncultivated phylotypes), but the organisms that were cultivated from this sample fell into different groups (i.e., Natronococcus, Halorubrum, or unaffiliated) and did not overlap with those predicted using the culture-independent approach . With respect to the highly alkaline sample, EMF, four groups were predicted from the environmental 16S rDNA sequences, two of which ( Natronomonas and Haloarcula) were also recovered through cultivation together with Natronococcus isolates . In summary, we found that halophilic archaea dominate the archaeal populations in these three hypersaline environments and show that culturability of the organisms predicted by molecular surveys might strongly depend on the habitat chosen . While a number of novel halophilic archaea have been isolated, we have not been able to cultivate representatives of the new lineages that were detected in this and several other environmental studies. J Mass Spectrom, 2002 Aug, 37(8), 858 - 66 Mass spectrometric identification of in vivo carbamylation of the amino terminus of Ectothiorhodospira mobilis high-potential iron-sulfur protein, isozyme 1; Van Driessche G et al.; The complete amino acid sequence of a novel high-potential iron-sulfur protein (HiPIP) isozyme 1 from the moderately halophilic phototrophic bacterium Ectothiorhodospira mobilis was determined by a combined approach of chemical and mass spectrometric sequencing techniques . By mass analysis of the apo- and holo-protein in the positive electrospray ionization mode using different electrospray solvents, the protein was found to be post-translationally modified by a moiety of 43 Da . Further analysis showed the nature and location of this modification to be a carbamyl group at the N-terminus of the HiPIP . This rare type of modification has previously been reported to occur in the water-soluble human lens alphaB-crystallin, class D beta-lactamases and some prokaryotic ureases, albeit at an internal lysine residue . In this paper, we discuss the mass spectrometric features of a carbamylated residue at the N-terminus of a peptide or a lysine side-chain during sequence analysis by collision-induced dissociation tandem mass spectrometry . Our data provide evidence for the first case of a prokaryotic carbamylated electron transport protein occurring in vivo . J Biol Inorg Chem, 2002 Sep, 7(7-8), 691 - 703 Epub 2002 Mar 13. Dynamics of wild-type HiPIPs: a Cys77Ser mutant and a partially unfolded HiPIP; Dilg AW et al.; The temperature dependence of the mean square displacement of the (57)Fe nuclei due to motion faster than 100 ns are measured by temperature-dependent Mossbauer spectroscopy for oxidized and reduced HiPIPs from Ectothiorhodospira halophila, Chromatium vinosum WT and a Cys77Ser mutant . The behaviour is interpretable in the frame of the general model of protein dynamics distinguishing two temperature intervals . The character of harmonic and quasi-diffusional modes in HiPIPs is discussed . Dynamic information obtained from Mossbauer spectroscopy and Fe K-edge EXAFS are compared . Structure dynamics of the iron-sulfur cluster in the partially unfolded reduced HiPIP from C . vinosum was investigated by Mossbauer spectroscopy and EXAFS, indicating an intact metal centre and a protein backbone with a largely collapsed secondary structure . The role of the cofactor during protein folding is discussed . Differences in the dynamics between the native protein and the molten globule are found at physiological temperatures only . The structure and dynamic behaviour of the {Fe(4)S(4)}Cys(3)Ser cluster in the Cys77Ser mutant of the HiPIP from C . vinosum are analysed . The temperature dependence of electron relaxation in oxidized HiPIPs is investigated by Mossbauer spectroscopy and analysed theoretically, considering spin-spin and spin-lattice relaxation . The latter consists of contributions from direct phonon bottleneck and Orbach mechanisms . The data agree with former pulsed EPR results . Orbach relaxation is interpreted as due to transitions between electronic isomers of oxidized HiPIPs . With this interpretation, the energetic difference between both isomers equals the energy gap estimated from the temperature dependence of the Orbach relaxation. Biochem Soc Trans, 2002 Aug, 30(4), 710 - 2 Investigations of iron uptake in Halobacterium salinarum; Hubmacher D et al.; The iron transport in the extremely halophilic Euryarchaeon Halobacterium salinarum JW5 was investigated . Experiments to detect endogenous siderophores from H . salinarum failed, but it was able to utilize exogenous siderophores . Measurement of the uptake of (55)Fe and {(14)C}citrate gave evidence only for the accumulation of iron . Two additional membrane proteins could be detected in iron-starved cells, one in iron-repleted membranes and one that is up-regulated there . Respiratory rates of iron-starved membranes after the addition of succinate and NADH differed considerably from iron-repleted ones . Furthermore, both types of membrane exhibited different degrees of inhibition by cyanide. Sheng Wu Gong Cheng Xue Bao, 2002 May, 18(3), 267 - 71 {Development of the genetic transformation system in extremely halophilic archaea}; Zhou MX et al.; The development of the genetic transformation systems in extremely halophilic Archaea was reviewed in this paper . Included are the screening of selectable markers for resistance to antibiotics, the development of gene cloning and expression vectors, and the modifications of the host organisms. Curr Microbiol, 2002 Oct, 45(4), 233 - 9 Analysis of the genome of the moderate halophile Halomonas eurihalina; Llamas I et al.; We have studied the genomic organization of Halomonas eurihalina, a moderately halophilic bacterium that produces an anionic exopolysaccharide with a potentially wide range of applications in industry . To estimate the genome size of strain F2-7, large restriction fragments of genomic DNA were separated by pulsed-field gel electrophoresis . According to the average size of the resolved restriction fragments, the genome size of H . eurihalina strain F2-7 was estimated to be around 2500 kb . The physical map of the chromosome for the endonuclease SwaI has been constructed . The F2-7 strain has two plasmids, pVE1 and pVE2, and in this study we have isolated three new plasmids, pVE3, pVE4, and pVE5, of 5.3, 16, and 6.5 kb, respectively, from strains H-1, H-217, and H-236 of H . eurihalina . We have characterized these plasmids and constructed their physical maps . Curing experiments produced no evidence for the involvement of these plasmids in the expression of the mucous phenotype. Mol Microbiol, 2002 Aug, 45(4), 943 - 50 Adaptation of protein secretion to extremely high-salt conditions by extensive use of the twin-arginine translocation pathway; Rose RW et al.; Halophilic archaea thrive in environments with salt concentrations approaching saturation . However, little is known about the way in which these organisms stabilize their secreted proteins in such 'hostile' conditions . Here, we present data suggesting that the utilization of protein translocation pathways for protein secretion by the Halobacteriaceae differs significantly from that of non-haloarchaea, and most probably represents an adaptation to the high-salt environment . Although most proteins are secreted via the general secretion (Sec) machinery, the twin-arginine translocation (Tat) pathway is mainly used for the secretion of redox proteins and is distinct from the Sec pathway, in that it allows cytoplasmic folding of secreted proteins . tatfind (developed in this study) was used for systematic whole-genome analysis of Halobacterium sp . NRC-1 and several other prokaryotes to identify putative Tat substrates . Our analyses revealed that the vast majority of haloarchaeal secreted proteins were predicted substrates of the Tat pathway . Strikingly, most of these putative Tat substrates were non-redox proteins, the homologues of which in non-haloarchaea were identified as putative Sec substrates . We confirmed experimentally that the secretion of one such putative Tat substrate depended on the twin-arginine motif in its signal sequence . This extensive utilization of the Tat pathway in haloarchaea suggests an evolutionary adaptation to high-salt conditions by allowing cytoplasmic folding of secreted proteins before their secretion. Microbiology, 2002 Aug, 148(Pt 8), 2343 - 9 Cloning, sequencing and expression of an alpha-amylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzyme; Mijts BN et al.; A recombinant clone expressing an amylase was identified from an Escherichia coli generated genomic library of the thermophilic, moderately halophilic, anaerobic bacterium Halothermothrix orenii by activity screening, and the gene encoding the enzyme was designated AmyA . The amyA gene was 1545 bp long, and encoded a 515 residue protein composed of a 25 amino acid putative signal peptide and a 490 amino acid mature protein . It possessed the five consensus regions characteristic of the alpha-amylase family and showed the greatest homology to the Bacillus megaterium group of alpha-amylases . The amyA gene was expressed in E . coli as a hexahistidine-tagged enzyme and purified . The purified recombinant enzyme was optimally active at 65 degrees C in 5% (w/v) NaCl at pH 7.5, with significant activity retained in the presence of up to 25% (w/v) NaCl . It had a specific activity of 22.32 U mg(-1) and required NaCl and CaCl(2) for optimum activity and thermostability . The relatively high proportion of acidic amino acids typically observed for many enzymes from halophiles was absent in H . orenii AmyA. J Appl Microbiol, 2002, 93(3), 438 - 47 Vibrios isolated from the cultured manila clam (Ruditapes philippinarum): numerical taxonomy and antibacterial activities; Castro D et al.; AIMS: A numerical taxonomic study of halophilic Vibrio isolated from healthy and brown ring disease (BRD) affected manila clams (Ruditapes philippinarum), harvested from the Atlantic coast of south-western Spain, was performed . METHODS AND RESULTS: Characterization of 123 presumptive Vibrio spp . was carried out using 94 phenotypic tests . Simple matching and Jaccard similarity coefficients were used for numerical analysis . Cluster analysis by the unweighted pair group method with arithmetic averages yielded 15 phena defined at 0.81 similarity . Large phena corresponded to Vibrio tubiashii, V . splendidus biotype I and V . harveyi (phena 1, 5 and 9, respectively) . The species V.splendidus biotype II, V . natriegens, V . mediterranei and V . alginolyticus were also represented . The inhibitory effect of diffusible extracellular products of the isolates against 27 strains of V.tapetis, the aetiological agent of BRD, was also investigated . Only five V . tubiashii isolates inhibited the growth of V . tapetis strains . The antimicrobial effect was inhibited by heating and depended on the culture medium . CONCLUSIONS: The main Vibrio species associated with manila clams were V . tubiashii, V.spendidus and V . harveyi . The antagonistic relationship established between V . tapetis and the Vibrio spp . clam microbiota may explain the failure of isolation in plating medium of V.tapetis from BRD-affected clams on the south Atlantic coast of Spain . SIGNIFICANCE AND IMPACT OF THE STUDY: Some of the strains isolated from manila clams correspond to agarolytic strains that constitute phenon 7 and they do not fit into any of the currently described Vibrio species. J Biol Chem, 2002 Oct 18, 277(42), 39304 - 11 Epub 2002 Aug 07. Spatial organization of bacteriorhodopsin in model membranes . Light-induced mobility changes; Kahya N et al.; Bacteriorhodopsin is a proton-transporting membrane protein in Halophilic archaea, and it is considered a prototype of membrane transporters and a model for G-protein-coupled receptors . Oligomerization of the protein has been reported, but it is unknown whether this feature is correlated with, for instance, light activation . Here, we have addressed this issue by reconstituting bacteriorhodopsin into giant unilamellar vesicles . The dynamics of the fully active protein was investigated using fluorescence correlation spectroscopy and freeze fracture electron microscopy . At low protein-to-lipid ratios (<1:10 w/w), a decrease in mobility was observed upon protein photoactivation . This process occurred on a second time scale and was fully reversible, i.e . when the dark-adapted state was reestablished the lateral diffusion rate of the protein was returned to that prior to activation . A similar decrease in lateral mobility as observed upon photoactivation was obtained when bacteriorhodopsin was reconstituted at high protein-to-lipid ratios (>1:10 w/w) . We interpret the shifts in mobility during light adaptation as being caused by transient photoinduced oligomerization of bacteriorhodopsin . These observations are fully supported by freeze-fracture electron microscopy, and the size of the clusters during photoactivation was estimated to consist of two or three trimers. FEMS Microbiol Lett, 2002 Aug 6, 213(2), 199 - 204 Metabolism of diphenylurea by a Marinobacter sp . isolated from a contaminated ephemeral stream bed in the Negev Desert; Sorensen SR et al.; A moderate halophilic Marinobacter sp . (designated strain DPUZ) able to metabolize 1,3-diphenylurea (DPU) was isolated from a contaminated ephemeral desert stream bed near an industrial complex in the northern part of the Negev Desert (Israel) . Metabolism of DPU was accompanied by a transient accumulation of a metabolite identified as aniline using gas chromatography-mass spectrometry, thus indicating a metabolic pathway involving cleavage of the urea bridge between the phe