Halophilic bacterium

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 phenyl structures . Aniline was further degraded without detection of other metabolites suggesting a complete degradation . Strain DPUZ grows at NaCl concentrations between 0.2 and 2.6 M with an optimum at 0.51 M . It grows at a temperature range between 20 and 40 degrees C with an optimum at 35 degrees C . This is the first study on bacterial metabolism of DPU.

FEMS Microbiol Lett, 2002 Aug 6, 213(2), 149 - 57
The sequence of the major gas vesicle protein, GvpA, influences the width and strength of halobacterial gas vesicles; Beard SJ et al.; Transformation experiments with Haloferax volcanii show that the amino acid sequence of the gas vesicle protein GvpA influences the morphology and strength of gas vesicles produced by halophilic archaea . A modified expression vector containing p-gvpA was used to complement a Vac(-) strain of Hfx . volcanii that harboured the entire p-vac region (from Halobacterium salinarum PHH1) except for p-gvpA . Replacement of p-gvpA with mc-gvpA (from Haloferax mediterranei) led to the synthesis of gas vesicles that were narrower and stronger . Other gene replacements (using c-gvpA from Hbt . salinarum or mutated p-gvpA sequences) led to a significant but smaller increase in gas vesicle strength, and less marked effects on gas vesicle morphology.

J Mol Biol, 2002 Aug 16, 321(3), 383 - 95
The structure of the archeabacterial flagellar filament of the extreme halophile Halobacterium salinarum R1M1 and its relation to eubacterial flagellar filaments and type IV pili; Cohen-Krausz S et al.; Although the phenomenology and mechanics of swimming are very similar in eubacteria and archaeabacteria (e.g . reversible rotation, helical polymorphism of the filament and formation of bundles), the dynamic flagellar filaments seem completely unrelated in terms of morphogenesis, structure and amino acid composition . Archeabacterial flagellar filaments share important features with type IV pili, which are components of retractable linear motors involved in twitching motility and cell adhesion . The archeabacterial filament is unique in: (1) having a relatively smooth surface and a small diameter of approximately 100A as compared to approximately 240A of eubacterial filaments and approximately 50A of type IV pili; (2) being glycosylated and sulfated in a pattern similar to the S-layer; (3) being synthesized as pre-flagellin with a signal-peptide cleavable by membrane peptidases upon transport; and (4) having an N terminus highly hydrophobic and homologous with that of the olygomerization domain of pilin.The synthesis of archeabacterial flagellin monomers as pre-flagellin and their post-translational, extracellular glycosylation suggest a different mode of monomer transport and polymerization at the cell-proximal end of the filament, similar to pili rather than to eubacterial flagellar filaments . The polymerization mode and small diameter may indicate the absence of a central channel in the filament.Using low-electron-dose images of cryo-negative-stained filaments, we determined the unique symmetry of the flagellar filament of the extreme halophile Halobacterium salinarum strain R1M1 and calculated a three-dimensional density map to a resolution of 19A . The map is based on layer-lines of order n=0, +10, -7, +3, -4, +6, and -1 . The cross-section of the density map has a triskelion shape and is dominated by seven outer densities clustered into three groups, which are connected by lower-density arms to a dense central core surrounded by a lower-density shell . There is no evidence for a central channel . On the basis of the homology with the oligomerization domain of type IV pilin and the density distribution of the filament map, we propose a structure for the central core.

Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1291 - 7
Psychrobacter submarinus sp . nov . and Psychrobacter marincola sp . nov., psychrophilic halophiles from marine environments; Romanenko LA et al.; Two novel psychrophilic, halophilic, Psychrobacter-like bacteria, strains KMM 225T and KMM 277T, were isolated from sea water and the internal tissues of an ascidian Polysyncraton sp . specimen, respectively, and characterized using a polyphasic approach, which included phenotypic, genotypic, chemotaxonomic and phylogenetic analyses . The novel marine isolates were Gram-negative, aerobic, coccoid, oxidase- and catalase-positive, non-pigmented, non-motile, psychrophilic and halophilic and they utilized a restricted spectrum of carbon sources . Strains KMM 225T and KMM 277T required sea water or sodium ions for growth and were tolerant of up to 12-15% (w/v) NaCl . Growth of strains KMM 225T and KMM 277T was observed at 4-35 and 7-35 degrees C, respectively . The DNA G+C contents of KMM 225T and KMM 277T were respectively 46-8 and 50.7 mol % . Comparison of almost complete 16S rDNA sequences of strains KMM 225T and KMM 277T revealed that both strains were phylogenetically most closely related to each other (99.9% sequence similarity) and slightly less related to Psychrobacter glacincola, with 97.2 and 97.8% similarity, respectively . DNA-DNA reassociation between KMM 225T and KMM 277T revealed 15% similarity, whereas similarity to other Psychrobacter species was 14-25% . Strains KMM 225T and KMM 277T differed from one another in their growth temperature, organic substrate utilization, antibiotic sensitivity and DNA G+C content . Both strains examined could be distinguished from all previously described Psychrobacter species by their physiological, genotypic and phylogenetic characteristics . On the basis of the physiological and molecular properties of the novel isolates, the names Psychrobacter submarinus sp . nov . (type strain KMM 225T = DSM 14161T) and Psychrobacter marincola sp . nov . (type strain KMM 277T = DSM 14160T) are proposed.

Biochemistry, 2002 Aug 6, 41(31), 10026 - 37
Kinetics of proton uptake and dye binding by photoactive yellow protein in wild type and in the E46Q and E46A mutants; Borucki B et al.; We studied the kinetics of proton uptake and release by photoactive yellow protein (PYP) from Ectothiorhodospira halophila in wild type and the E46Q and E46A mutants by transient absorption spectroscopy with the pH-indicator dyes bromocresol purple or cresol red in unbuffered solution . In parallel, we investigated the kinetics of chromophore protonation as monitored by the rise and decay of the blue-shifted state I(2) (lambda(max) = 355 nm) . For wild type the proton uptake kinetics is synchronized with the fast phase of I(2) formation (tau = 500 micros at pH 6.2) . The transient absorption signal from the dye also contains a slower component which is not due to dye deprotonation but is caused by dye binding to a hydrophobic patch that is transiently exposed in the structurally changed and partially unfolded I(2) intermediate . This conclusion is based on the wavelength, pH, and concentration dependence of the dye signal and on dye measurements in the presence of buffer . SVD analysis, moreover, indicates the presence of two components in the dye signal: protonation and dye binding . The dye binding has a rise time of about 4 ms and is coupled kinetically with a transition between two I(2) intermediates . In the mutant E46Q, which lacks the putative internal proton donor E46, the formation of I(2) is accelerated, but the proton uptake kinetics remains kinetically coupled to the fast phase of I(2) formation (tau = 100 micros at pH 6.3) . For this mutant the protein conformational change, as monitored by the dye binding, occurs with about the same time constant as in wild type but with reduced amplitude . In the alkaline form of the mutant E46A the formation of the I(2)-like intermediate is even faster as is the proton uptake (tau = 20 micros at pH 8.3) . No dye binding occurred in E46A, suggesting the absence of a conformational change . In all of the systems proton release is synchronized with the decay of I(2) . Our results support mechanisms in which the chromophore of PYP is protonated directly from the external medium rather than by the internal donor E46.

Arch Biochem Biophys, 2002 Jul 15, 403(2), 284 - 91
Biochemical and enzymological properties of the polyhydroxybutyrate synthase from the extremely halophilic archaeon strain 56; Hezayen FF et al.; Some members of the archaebacterial family Halobacteriaceae have been determined to accumulate polyhydroxyalkanoate (PHA) and poly(3-hydroxybutyrate) (PHB) . The extremely halophilic archaebacterium strain 56 is capable of accumulating large amounts of PHB . Since measurements of enzyme activities related to archaebacterial PHB biosynthesis have never been achieved, we investigated the enzymology of PHB biosynthesis in strain 56 . Crude extracts of strain 56 cultivated under accumulating conditions showed PHB synthase activity, whereas neither beta-ketothiolase nor NADH/NADPH-dependent acetoacetyl-CoA reductase activity was detectable . An 80-kDa protein, cross-reacting with the anti-PHB synthase antibodies raised against the PHB synthase from Ralstonia eutropha, was identified in the crude extract and was strongly enriched by purification of PHB granules . The granule-associated PHB synthase was enzymologically characterized . Enzyme kinetics showed a specific activity of about 4.6U/mg and Hill plot analysis revealed a K(0.5) of 56 microM with (R)-3-hydroxybutyryl-CoA employed as substrate . A Hill coefficient of 1.75 indicated that the PHB synthase exhibited positive cooperativity . The thioesters 3-hydroxyvaleryl-CoA, 4-hydroxybutyryl-CoA, and 3-hydroxydecanoyl-CoA were not accepted as substrates . Moreover, the PHB synthase was found to be competitively inhibited by CoA, showing an IC(50) of 160 microM . The PHB synthase was stable up to 60 degrees C and still exhibited about 90% of the maximum enzyme activity, which was obtained at 40 degrees C . In contrast to the soluble PHB synthase, the granule-bound PHB synthase was almost independent of the salt concentration . The PHB synthase could not be released from the PHB granules, indicating a covalent attachment to the PHB core . This is the first description of an archaebacterial PHA synthase.

Mol Microbiol, 2002 Aug, 45(3), 665 - 72
Cellular responses to environmental salinity in the halophilic black yeast Hortaea werneckii; Petrovic U et al.; The development of crop plants with increased salt tolerance necessitates the study of naturally salt-tolerant eukaryotic species . We studied the bio-synthesis of glycerol as a compatible solute in the halophilic eukaryotic microorganism, black yeast Hortaea werneckii . A restriction fragment-differential display technique was used to investigate the transcriptome of the organism . Eight differentially expressed genes were identified in response to growth at different salinities . Although the putative functions of their products, P-type ATPase, ubiquinone reductase, aconitase, RNA helicase, Asn-tRNA ligase, isoamyl alcohol oxidase, and phosphatidylinositol-3-kinase, are not intimately related within the cellular machinery, the results presented here are sufficient to propose a model which describes how H . werneckii adapts to extremely high salinities . Some of these mechanisms of adaptation to raised environmental salinity are similar to those in other salt-sensitive species, e.g . glycerol accumulation, there also appear to be novel mechanisms present such as the use of different energy production mechanisms and post-transcriptional regulation of gene expression . Our results have also provided new data on two genes from two other fungal species, the Neurospora crassa B1D1.130 gene and the Aspergillus ustus amdS-A gene.

Mol Microbiol, 2002 Jul, 45(2), 511 - 20
A bZIP protein from halophilic archaea: structural features and dimer formation of cGvpE from Halobacterium salinarum; Plosser P et al.; The cGvpE protein of Halobacterium salinarum PHH4 has been identified as transcriptional activator for the promoter of the c-gvpA gene encoding the major gas vesicle structural protein cGvpA . Molecular modelling of the carboxy-terminal region of cGvpE suggests that this protein resembles a basic leucine-zipper protein, and mutations in the putative DNA binding domain DNAB completely abolish the activator function in Haloferax volcanii transformants . Mutations in the key residues of the putative leucine-zipper region AH6 of cGvpE confirmed that the three residues V159, L166 and L173 were essential for the activator function of cGvpE at the c-gvpA promoter, whereas the cysteine residue C180 could be altered to a leucine or an aspartate residue without the loss of this function . Mutations in basic residues of helix AH4 demonstrated the importance of the lysine K104 for the activator function of cGvpE . A cGvpE protein containing a his-tag at the C-terminus was still able to activate the expression of c-gvpA in vivo . The cGvpE his-purified from Hf . volcanii formed a dimer in Blue-native polyacrylamide gels that could be resolved into monomers by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) . Dimers of cGvpE were already seen using SDS-PAGE, but not with cGvpE mutant proteins containing the alterations L166E or L173E/C180L in the leucine zipper . These results imply that the hydrophobic surface of helix AH6 is indeed required for the establishment of cGvpE dimers.

Lipids, 2002 Jun, 37(6), 541 - 8
Regiospecific enzymatic oxygenation of cis-vaccenic acid during aerobic senescence of the halophilic purple sulfur bacterium Thiohalocapsa halophila; Marchand D et al.; A regiospecific oxygenation of the allylic carbon 10 of cis-vaccenic acid has been observed in senescent cells of the halophilic purple sulfur bacterium Thiohalocapsa halophila incubated under aerobic conditions in darkness . The results obtained strongly suggest that these enzymatic processes involve the initial dioxygenase-mediated formation of 1 O-hydroperoxyoctadec-cis-11-enoic acid, which is not accumulated in the cells of T . halophila owing to its high cytotoxic properties . Deuterium labeling and GC-MS analyses enabled us to demonstrate that subsequent enzymatic conversions of this allylic hydroperoxide involved reduction, cleavage, isomerization, and saturation reactions . Some of the specific oxidation products thus formed could constitute potential T . halophila biomarkers.

Biotechnol Bioeng, 2002 Jun 5, 78(5), 497 - 502
Mechanism of adaptation of an atypical alkaline p-nitrophenyl phosphatase from the archaeon Halobacterium salinarum at low-water environments; Marhuenda-Egea FC et al.; Enzymes suspended in organic solvents represent a versatile system for studying the involvement of water in catalytic properties and their flexibility in adapting to different environmental conditions . The extremely halophilic alkaline p-nitrophenylphosphate phosphatase from the archaeon Halobacterium salinarum was solubilized in an organic medium consisting of reversed micelles of hexadecyltrimethylammoniumbromide in cyclohexane, with 1-butanol as cosurfactant . Hydrolysis of p-nitrophenylphosphate was nonlinear with time when the enzyme was microinjected into reversed micelles that contained substrate . These data are consistent with a kinetic model in which the enzyme is irreversibly converted from an initial form to a final stable form during the first seconds of the encapsulation process . The model features a rate constant (k) for that transition and separate hydrolysis rates, v(1) and v(2), for the two forms of the enzyme . The enzyme conversion may be governed by the encapsulation process .

Proteins, 2002 Aug 1, 48(2), 212 - 9
Signal transduction in the photoactive yellow protein . II . Proton transfer initiates conformational changes; Groenhof G et al.; Molecular dynamics simulation techniques, together with semiempirical PM3 calculations, have been used to investigate the effect of photoisomerization of the 4-hydroxy-cinnamic acid chromophore on the structural properties of the photoactive yellow protein (PYP) from Ectothiorodospira halophila . In this bacteria, exposure to blue light leads to a negative photoactic response . The calculations suggest that the isomerization does not directly destabilize the protein . However, because of the isomerization, a proton transfer from a glutamic acid residue (Glu46) to the phenolate oxygen atom of the chromophore becomes energetically favorable . The proton transfer initiates conformational changes within the protein, which are in turn believed to lead to signaling .

Proteins, 2002 Aug 1, 48(2), 202 - 11
Signal transduction in the photoactive yellow protein . I . Photon absorption and the isomerization of the chromophore; Groenhof G et al.; Molecular dynamics simulation techniques together with time-dependent density functional theory calculations have been used to investigate the effect of photon absorption by a 4-hydroxy-cinnamic acid chromophore on the structural properties of the photoactive yellow protein (PYP) from Ectothiorodospira halophila . The calculations suggest that the protein not only modifies the absorption spectrum of the chromophore but also regulates the subsequent isomerization of the chromophore by stabilizing the isomerization transition state . Although signaling from PYP is thought to involve partial unfolding of the protein, the mechanical effects accompanying isomerization do not appear to directly destabilize the protein .

Can J Microbiol, 2002 May, 48(5), 468 - 72
PCR and blot hybridization for rapid identification of Haloferax species; Asker D et al.; Based on the amplification of a 16S rDNA, a PCR assay for the identification of species of Haloferax to genus level was performed . Two variable regions of the 16S rDNA in Haloferax spp . were selected as genus-specific primers for the PCR assay and hybridization probe . Five genera of halophilic Archaea and Escherichia coli were examined as outside groups . Using this approach, all strains of Haloferax spp . were positive . In contrast, all species belonging to the most closely related genera, including Natrinema, Halorubrum, Halobacterium, and Haloarcula, were negative . In addition, the mass bloom of halophilic Archaea that develops in the El-Mallahet saltern of Alexandria City was positive using the same approach . This assay, which does not require pure cultures of microorganisms, is a specific and rapid method for identifying Haloferax spp . in hypersaline environments.

Int Microbiol, 2002 Mar, 5(1), 3 - 9
Photobacterium damselae subsp . piscicida: an integrated view of a bacterial fish pathogen; Romalde JL; Pasteurellosis, or pseudotuberculosis, is a bacterial septicaemia caused by the halophilic bacterium Photobacterium damselae subsp . piscicida (formerly Pasteurella piscicida) . Although this disease was first described in wild populations of white perch and striped bass, currently the natural hosts of the pathogen are a wide variety of marine fish . The disease has great economic impact both in Japan, where it affects mainly yellowtail cultures, and in the Mediterranean area, due to the losses it causes in seabream and seabass farms . This microorganism serves as a perfect model to study a bacterial fish pathogen, either at an applied level, to resolve or to mitigate the high economic losses of fish farmers, or at a basic level, for a better understanding of P . damselae subsp . piscicida biology . This article discusses the methods employed in our laboratory to study the causative agent of pasteurellosis . It reviews important aspects, from the diverse procedures for the detection and isolation of the pathogen to the latest molecular studies that have allowed its correct taxonomic allocation . Characterization of some virulence mechanisms and the available methods to prevent the disease are also presented.

Evolution Int J Org Evolution, 2002 May, 56(5), 909 - 26
Accelerated molecular evolution in halophilic crustaceans; Hebert PD et al.; In contrast to the stable ionic composition of the oceans, inland waters show striking diversity, possessing salt concentrations varying from I mM to 5 M . Although species diversity is highest in fresh water, some lineages have colonized hypersaline environments where they encounter elevated levels of both ultraviolet (UV) radiation and osmotic stress . This study compares rates of evolution in halophilic and freshwater taxa for two groups of microcrustaceans, anostracans and daphniids, from Australia and North America . The results establish that halophilic species show consistent rate acceleration, involving elevated levels of both insertion/deletion events and of nucleotide substitutions . The elevated pace of molecular evolution does not appear to be linked to selection or to other agents that are known to influence the supply rate of mutations, such as UV exposure, generation length, or shifts in metabolic rate . However, variance in ionic strength, which is known to have potent effects on DNA-protein interactions as well as on the structural properties of DNA and proteins, might account for the lowered fidelity of DNA replication in life from hypersaline settings . Regardless of its cause, the consistent rate acceleration in halophiles suggests that past efforts to employ sequence divergences to date events, such as the age of asexual lineages in Artemia, have resulted in serious overestimates . More generally, the results indicate that coordinated shifts in rates of molecular evolution may occur in lineages exposed to extreme environmental conditions.

Biosci Biotechnol Biochem, 2002 May, 66(5), 1176 - 80
The groESL operon of the halophilic lactic acid bacterium Tetragenococcus halophila; Fukuda D et al.; The groESL operon of the halophilic lactic acid bacterium Tetragenococcus halophila was cloned by a PCR-based method . The molecular masses of GroES and GroEL proteins were calculated to be 10,153 and 56,893 Da, respectively . The amount of groESL mRNA was increased 3.8-fold by heat shock (45 degrees C), and 4-fold by high NaCl (3-4 M) . The Bacillus subtilis sigmaA-like constitutive promoter existed in front of groES, and was used under both normal and stress (heat shock and high salinity) conditions.

Syst Appl Microbiol, 2002 Apr, 25(1), 3 - 12
Primary structure of selected archaeal mesophilic and extremely thermophilic outer surface layer proteins; Claus H et al.; The archaea are recognized as a separate third domain of life together with the bacteria and eucarya . The archaea include the methanogens, extreme halophiles, thermoplasmas, sulfate reducers and sulfur metabolizing thermophiles, which thrive in different habitats such as anaerobic niches, salt lakes, and marine hydrothermals systems and continental solfataras . Many of these habitats represent extreme environments in respect to temperature, osmotic pressure and pH-values and remind on the conditions of the early earth . The cell envelope structures were one of the first biochemical characteristics of archaea studied in detail . The most common archaeal cell envelope is composed of a single crystalline protein or glycoprotein surface layer (S-layer), which is associated with the outside of the cytoplasmic membrane . The S-layers are directly exposed to the extreme environment and can not be stabilized by cellular components . Therefore, from comparative studies of mesophilic and extremely thermophilic S-layer proteins hints can be obtained about the molecular mechanisms of protein stabilization at high temperatures . First crystallization experiments of surface layer proteins under microgravity conditions were successful . Here, we report on the biochemical features of selected mesophilic and extremely archaeal S-layer (glyco-) proteins.

FEMS Microbiol Lett, 2002 Jun 4, 211(2), 213 - 8
The substrate-binding protein TeaA of the osmoregulated ectoine transporter TeaABC from Halomonas elongata: purification and characterization of recombinant TeaA; Tetsch L et al.; TeaABC is a novel, osmoregulated secondary transport system for compatible solutes found in the halophilic proteobacterium Halomonas elongata DSM 2581(T) . Sequence comparisons have confirmed that TeaABC belongs to the family of TRAP transporters and as such it consists of two putative transmembrane proteins (TeaB, TeaC) and a putative periplasmic substrate-binding protein (TeaA) . TeaABC is the only osmoregulated transporter for ectoines found in H . elongata . By overexpressing TeaA-(6)His tag in Escherichia coli we demonstrated that TeaA is processed as predicted and exported to the periplasm . Furthermore, it was proven that TeaA is indeed a periplasmic ectoine-binding protein . The functionality of the purified protein as an ectoine-binding protein was tested employing a modified binding assay.

Extremophiles, 2002 Jun, 6(3), 217 - 23 Epub 2002 Feb 01.
Amino acid composition of bulk protein and salt relationships of selected enzymes of Salinibacter ruber, an extremely halophilic bacterium; Oren A et al.; The extremely halophilic bacterium Salinibacter ruber was previously shown to have a high intracellular potassium content, comparable to that of halophilic Archaea of the family Halobacteriaceae . The amino acid composition of its bulk protein showed a high content of acidic amino acids, a low abundance of basic amino acids, a low content of hydrophobic amino acids, and a high abundance of serine . We tested the level of four cytoplasmic enzymatic activities at different KCl and NaCl concentrations . Nicotinamide adenine dinucleotide (NAD)-dependent isocitrate dehydrogenase functioned optimally at 0.5-2 M KCl, with rates of 60% of the optimum value at 3.3 M . NaCl provided less activation: 70% of the optimum rates in KCl were found at 0.2-1.2 M NaCl, and above 3 M NaCl, activity was low . We also detected nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate activity, which remained approximately constant between 0-3.2 M NaCl and increased with increasing KCl concentration . NAD-dependent malate dehydrogenase functioned best in the absence of salt, but rates as high as 25% of the optimal values were measured in 3-3.5 M KCl or NaCl . NAD-dependent glutamate dehydrogenase, assayed by the reductive amination of 2-oxoglutarate, showed low activity in the absence of salt . NaCl was stimulatory with optimum activity at 3-3.5 M . However, no activity was found above 2.5 M KCl . Although the four activities examined all function at high salt concentrations, the behavior of individual enzymes toward salt varied considerably . The results presented show that Salinibacter enzymes are adapted to function in the presence of high salt concentrations.

Environ Microbiol, 2002 Jun, 4(6), 349 - 60
Prokaryotic genetic diversity throughout the salinity gradient of a coastal solar saltern; Benlloch S et al.; Bacterial and archaeal assemblages have been studied in a multipond solar saltern using a range of microbial ecology techniques by four laboratories simultaneously . These include 16S rDNA sequencing from both denaturing gradient gel electrophoresis (DGGE) and clone libraries, and culturing methods . Water samples from eight ponds were analysed, covering a salinity range from near sea water (4% salt) to saturated sodium chloride (37% salt; ponds called crystallizers) . Clone libraries focused on ponds with salinity of 8%, 22% and 32% . Although different cloning strategies were able to retrieve the same type of dominant sequences, there were differing degrees of success with less abundant sequences . Thus, the use of two sets of primers recovered a higher number of phylotypes . Bacterial and archaeal isolates were, however, different from any of the retrieved environmental sequences . For Bacteria, most sequences in the 8% salt pond were related to organisms of marine origin . Thus, representatives of the alpha-, beta-, gamma- and epsilon-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides group (CFB), high-G+C Gram-positive bacteria and cyanobacteria were found . In the 22% salt pond, alpha- and gamma-Proteobacteria, cyanobacteria and CFB were the only groups found, and most of them were related to specialized halophilic bacteria . From the 32% salt pond, only CFB were found, and most of the sequences retrieved clustered with Salinibacter ruber, an extremely halophilic bacterium . A decrease in the richness of bacterial genera was therefore apparent along the gradient . Archaea behaved quite similarly . In the lowest salinity ponds, sequences were related to environmental clones of Marine Archaea Group III (Thermoplasmales relatives) and to unclassified branches of Euryarchaeaota . In the 8%, 22% and 32% ponds, most of the clones were related to different cultured strains of Halobacteriaceae . Finally, most sequences from the crystallizers clustered with the uncultured square archaeon SPhT . Crenarchaeaota were not detected . Despite the fact that higher prokaryotic richness was apparent in the lower salinity ponds than in the crystallizers, the diversity index from clone libraries calculated according to Shannon and Weaver did not show this trend . This was because diversity in the crystallizers can be considered as 'microdiversity', the co-existence of several closely related clones of Bacteria (the S . ruber cluster) and Archaea (the SPhT cluster) . Regardless of the changes in abundance, both Bacteria and Archaea showed the same pattern; as salinity increased, the number of different clusters decreased, and only one cluster became dominant . Both clusters, however, showed a considerable degree of microdiversity . The meaning of such microdiversity remains to be determined.

Biochem J, 2002 Sep 15, 366(Pt 3), 959 - 64
Lipid modification of proteins in Archaea: attachment of a mevalonic acid-based lipid moiety to the surface-layer glycoprotein of Haloferax volcanii follows protein translocation; Konrad Z et al.; Once the newly synthesized surface (S)-layer glycoprotein of the halophilic archaeaon Haloferax volcanii has traversed the plasma membrane, the protein undergoes a membrane-related, Mg(2+)-dependent maturation event, revealed as an increase in the apparent molecular mass and hydrophobicity of the protein . To test whether lipid modification of the S-layer glycoprotein could explain these observations, H . volcanii cells were incubated with a radiolabelled precursor of isoprene, {(3)H}mevalonic acid . In Archaea, isoprenoids serve as the major hydrophobic component of archaeal membrane lipids and have been shown to modify other haloarchaeal S-layer glycoproteins, although little is known of the mechanism, site or purpose of such modification . In the present study we report that the H . volcanii S-layer glycoprotein is modified by a derivative of mevalonic acid and that maturation of the protein was prevented upon treatment with mevinolin (lovastatin), an inhibitor of mevalonic acid biosynthesis . These findings suggest that lipid modification of S-layer glycoproteins is a general property of halophilic archaea and, like S-layer glycoprotein glycosylation, lipid-modification of the S-layer glycoproteins takes place on the external cell surface, i.e . following protein translocation across the membrane.

Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 789 - 93
Salinicoccus alkaliphilus sp . nov., a novel alkaliphile and moderate halophile from Baer Soda Lake in Inner Mongolia Autonomous Region, China; Zhang W et al.; A novel alkaliphilic and moderately halophilic gram-positive coccus, designated strain T8T, was isolated from Baer Soda Lake in Inner Mongolia Autonomous Region, China . Strain T8T grew in the presence of 0-25% (w/v) NaCl and at pH 6.5-11.5, with optimum growth at 10% (w/v) NaCl and pH 9.0 . It grew at 10.0-46.0 degrees C, with an optimum growth temperature of 32.0 degrees C . The organism was strictly aerobic, non-motile, non-sporulating and catalase- and oxidase-positive . The DNA G+C content was 49.6 mol% . The cell wall contained Lys and Gly . The major isoprenoid quinone was menaquinone 6 (MK-6) . Phylogenetic analyses based on 16S rDNA sequence comparisons indicate that strain T8T is a member of the genus Salinicoccus . DNA-DNA relatedness of less than 50% with the described species of Salinicoccus supported the view that this organism represents a novel species of the genus Salinicoccus . The name Salinicoccus alkaliphilus sp . nov . is proposed for this novel species . The type strain is T8T (= AS 1.2691T = JCM 11311T).

Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 729 - 38
Haloferax alexandrinus sp . nov., an extremely halophilic canthaxanthin-producing archaeon from a solar saltern in Alexandria (Egypt); Asker D et al.; An extremely halophilic red micro-organism designated strain TM(T) was isolated from a solar saltern in Alexandria, Egypt . The micro-organism stains gram-negative, is very pleomorphic, non-motile and strictly aerobic and requires at least 10 g NaCl l(-1) for growth . The growth optimum is 250 g NaCl l(-1) . Growth is also observed over a wide range of MgSO4 concentrations (10-40 g l(-1)) . Aerobic reduction of nitrate without gas production was detected . Cells grew aerobically in a minimal salts medium containing ammonium chloride and glucose . Strain TM(T) produced acid from fructose, glucose, rhamnose, maltose and glycerol . The G+C content of the DNA was 59.5+/-0.3 mol % . On the basis of polar lipid analysis, the isolate belonged to the genus Haloferax . Analysis of the 16S rDNA sequence showed the highest similarity (>99%) to be to the type strain Haloferax volcanii . Although the spectrum of antibiotic susceptibility was similar to that of validly described species of the genus Haloferax, the strain could be distinguished from them by its different response to josamycin and rifampicin . Strain TM(T) is unique within the genus Haloferax in producing canthaxanthin . Comparative analysis of phenotypic properties and DNA-DNA hybridization between strain TM(T) and Haloferax species supported the conclusion that TM(T) is a novel species within this genus, for which the name Haloferax alexandrinus sp . nov . is proposed . The type strain is TM(T) (= JCM 10717T = IFO 16590T).

Mikrobiologiia, 2002 Mar-Apr, 71(2), 160 - 5
{Antagonistic interactions between stress factors during the growth of microorganisms under conditions simulating the parameters of their natural ecotopes}; Arzumanian VG et al.; Two stress factors, hypoxia (microaerobic conditions) and a high salt concentration, if applied simultaneously to aerobic microorganisms, display an antagonistic mode of interaction . As a result, the NaCl level that is usually optimal for moderate halophiles (5-6%) becomes optimal for the growth of weak halophiles (Rhodococcus erythropolis and Shewanella sp . CN32); the halotolerant yeast Yarrowia lypolytica acquires halophilic properties (with a growth optimum at a NaCl concentration of 10%), and the growth rate of the extremely halophilic Halobacterium salinarum increases at supraoptimal salt concentrations (25-34%) . This phenomenon is apparently due to multiple changes in metabolic reactions . In particular, high salt concentrations suppress respiration and the formation of enzymes (superoxide dismutase and catalase) that protect the cell from toxic oxygen species . Therefore, establishment of microaerobic conditions compensates for the loss of these protective mechanisms and enables cell growth at higher salt concentrations than under aerobic conditions . Of some importance can also be the increase in the intracellular concentrations of osmoprotectants caused by the suppression of their intracellular oxidation . The implications of this phenomenon for the ecophysiology of microorganisms (including oiloxidizing species) and for the classification of weak and moderate halophiles are discussed.

Mikrobiologiia, 2002 Mar-Apr, 71(2), 149 - 59
{The biology and osmoadaptation of haloalkaliphilic methanotrophs}; Trotsenko IuA et al.; There is increasing evidence for the presence and activity of methanotrophic bacteria in saline and alkaline aquatic environments located in different ecogeographical regions . Alkalitolerant halophilic and alkaliphilic halotolerant methanotrophs of type I were found to be able to utilize methane and methanol, to oxidize ammonium ions, and to transform various organic compounds in a wide range of water salinities (up to 12% NaCl) and pH values (from 5 to 11) . The ecophysiological importance of methanotrophs in microbial communities inhabiting saline and alkaline aquatic environments is due to their involvement in the global cycles of methane and major bioelements (C, N, and S) . Specific cyto- and biochemical properties of haloalkaliphilic methanotrophs--the synthesis of osmoprotectants (ectoine, 5-hydroxyproline, and sucrose), the accumulation of potassium ions, the formation of glycoprotein S layers on the outer surface of their cell walls, and the modification of the chemical composition of their membranes--allow them to adapt to highly saline and alkaline habitats . Due to their specific properties, haloalkaliphilic methanotrophs may become of use in modern biotechnology.

Structure (Camb), 2002 May, 10(5), 629 - 37
Reversal of halophilicity in a protein-DNA interaction by limited mutation strategy; Bergqvist S et al.; Comparison of the genes of functionally homologous proteins in organisms existing in different environments shows that adaptation is most often accomplished by mutation of an existing protein . However, from such comparisons, the significance of individual residues to the particular environmental adaptation is not generally discernable among the mass of changes that occur over evolutionary time . This can be exemplified by the general transcription factor found in eukaryotes and archaea, the TATA binding protein (TBP) . TBP from Pyrococcus woesei is adapted for optimal binding to DNA at high salt and high temperature, with 34% of the amino acids altered in comparison to its nearest known mesophilic counterpart . We demonstrate that the halophilic nature of this protein can be attributed to only three mutations, revealing that the important phenotype of halophilicity could be rapidly acquired in evolutionary time.

Extremophiles, 2002 Apr, 6(2), 97 - 102
Purification and characterization of an extremely halophilic acetoacetyl-CoA thiolase from a newly isolated Halobacterium strain ZP-6; Liu T et al.; The extremely halophilic archaeon ZP-6 was isolated from Ai-Ding salt lake in Xinjiang Uighur Autonomous Region of the People's Republic of China . Based on its physiological properties, 16S rDNA sequence, and DNA-DNA homology with known haloarchaea, the isolate was tentatively identified as a Halobacterium sp . An acetoacetyl-CoA thiolase was purified and characterized from this organism . The native enzyme has a molecular mass of 80 +/- 8 kDa and consists of two identical subunits of 43 +/- 2 kDa each . The N-terminus 14 amino acid residues were sequenced and showed identity with the respective part of a putative thiolase (AcaB1) of Halobacterium sp . NRC-1 . The purified enzyme has an optimal pH of 7.9 for acetoacetyl-CoA thiolysis . The thiolytic activity was inhibited by the presence of Mg'- and was stimulated by KCl or NaCl . The thiolysis reaction of Halobacterium sp . ZP-6 thiolase can be inhibited by either substrate when present in excess . The distinct kinetic profile indicates that the thiolase from Halobacterium sp . ZP-6 may have a different catalytic mechanism from the so-called ping-pong mechanism employed by other thiolases . To our knowledge, this is the first report of the purification and characterization of a halophilic thiolase from an archaeal species.

Extremophiles, 2002 Apr, 6(2), 135 - 42
Stability of Natrialba magadii NDP kinase: comparisons with other halophilic proteins; Polosina YY et al.; Nucleoside diphosphate kinase from the haloalkaliphilic archaeon Natrialba magadii (Nm NDPK) is a homooligomeric hexamer with a monomer molecular weight of 23 kDa . Its main function is to exchange gamma-phosphates between nucleoside triphosphates and diphosphates . Previously it was shown that Nm NDPK is active over a wide range of NaCl concentrations, which is not typical of extremely halophilic proteins . In this paper more detailed investigations of kinase function and stability were carried out using circular dichroism, differential scanning calorimetry, size-exclusion chromatography, and biochemical methods . A possible mechanism for stabilization of halophilic proteins that allows them to function in a wide range of NaCl concentrations is proposed.

Extremophiles, 2002 Apr, 6(2), 131 - 4
Evidence of interspecies hydrogen transfer from glycerol in saline environments; Cayol JL et al.; Two halanaerobic bacteria--Halanaerobium saccharolytica subsp . senegalense and Halanaerobium sp . strain FR1H--produced acetate, H2, and CO2 from glycerol fermentation, but the glycerol consumption rate was low . In contrast, in the presence of the moderately halophilic hydrogenotrophic sulfate-reducing bacterium, Desulfohalobium retbaense, used as H2 scavenger in the coculture, glycerol oxidation by both halanaerobes significantly increased . Cocultures of both halanaerobes with D . retbaense on glycerol led to acetate, hydrogen sulfide, and CO2 production, whereas glycerol fermentation by the two strains led to the production of acetate, hydrogen, and CO2 . The increased glycerol oxidation by H . saccharolytica and strain FRI H in coculture with D . retbaense resulted from low H2 partial pressure caused by the hydrogen-oxidizing activity of D . retbaense . These results provide the first evidence of interspecies hydrogen transfer in saline environments and indicate that this mechanism may play an important role in organic matter mineralization in hypersaline ecosystems.

J Appl Microbiol, 2002, 92(6), 1136 - 42
Purification and properties of a histidine decarboxylase from Tetragenococcus muriaticus, a halophilic lactic acid bacterium; Konagaya Y et al.; AIMS: A histidine decarboxylase from Tetragenococcus muriaticus, a halophilic histamine-producing bacterium isolated from Japanese fermented squid liver sauce, was purified to homogeneity, for the first time . METHODS AND RESULTS: The enzyme was purified 16-fold from cell-free extract by ammonium sulphate precipitation, anion exchange chromatography and hydroxyapatite chromatography . The pure enzyme consisted of two polypeptide chains with molecular mass of 28.8 and 13.4 kDa . The N-terminal amino acid sequences of these polypeptides highly correlated with those of the alpha- and beta-chains of other Gram-positive bacterial histidine decarboxylases . The optimum and stable pH for the enzyme was 4.5-7.0 and 4.0-7.0, respectively . This enzyme did not decarboxylate lysine, arginine, tyrosine, tryptophan and ornithine . The enzyme activity decreased with the addition of NaCl . At pH 4.8, the Vmax and Km values were 16.8 micromol histamine min-1 mg-1 and 0.74 mmol l-1, respectively . CONCLUSIONS: The very similar physiological properties of this enzyme and almost identical N-terminal amino acid sequences to those from other Gram-positive bacteria indicated that this enzyme may be evolutionally highly conserved among Gram-positive bacteria . SIGNIFICANCE AND IMPACT OF THE STUDY: Information on this enzyme could be useful for studying the mechanism of histamine accumulation in salted foods . In addition, the N-terminal amino acid sequence can be utilized to design oligonucleotide probes, which may prove valuable in the rapid monitoring of halophilic histamine producers in salted products.

FEMS Microbiol Lett, 2002 Apr 9, 209(2), 155 - 60
NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning, sequence determination and overexpression in Escherichia coli; Camacho M et al.; A gene encoding NADP-dependent Ds-threo-isocitrate dehydrogenase was isolated from Haloferax volcanii genomic DNA by using a combination of polymerase chain reaction and screening of a lambda EMBL3 library . Analysis of the nucleotide sequence revealed an open reading frame of 1260 bp encoding a protein of 419 amino acids with 45837 Da molecular mass . This sequence is highly similar to previously sequenced isocitrate dehydrogenases . In the alignment of the amino acid sequences with those from several archaeal and mesophilic NADP-dependent isocitrate dehydrogenases, the residues involved in dinucleotide binding and isocitrate binding are well conserved . We have developed methods for the expression in Escherichia coli and purification of the enzyme from H . volcanii . This expression was carried out in E . coli as inclusion bodies using the cytoplasmic expression vector pET3a . The enzyme was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing EDTA, MgCl(2) and 3 M NaCl . Maximal activity was obtained after several hours incubation at room temperature.

J Bacteriol, 2002 Jun, 184(11), 3114 - 21
Functional role for a 2-oxo acid dehydrogenase in the halophilic archaeon Haloferax volcanii; Wanner C et al.; The archaeon Haloferax volcanii was previously shown to contain and transcribe the genes for a 2-oxo acid dehydrogenase (OADH) complex, but their presence remained a mystery because no enzymatic activity with any of the known OADH substrates could be found, and an inactivation of one of the genes did not lead to any phenotype . Here we report the identification of an additional oadh gene cluster in the genome of H . volcanii . In contrast to previously known oadh loci, it contains three genes, oadh2A1, oadh2A2, and oadh2ld, with coding capacity for the E1alpha and E1beta subunits and an unattached lipoyl domain, but it is devoid of the genes for a complete E2 and an E3 . The genes were isolated by complementation of a nitrate respiration-deficient mutant of H . volcanii and therefore were shown to be functional in vivo . Phylogenetic analyses revealed that the deduced E1alpha and E1beta subunits of OADH2 group with bacterial acetoin dehydrogenases but not with the OADH1 subunits, and thus, H . volcanii has obtained the two gene groups independently . Comparison of the wild type and the mutant allowed us to exclude a function of OADH2 in the aerobic or anaerobic degradation of acetoin or glucose . Instead, it could be shown that OADH2 is important during nitrate-respirative growth on Casamino Acids . Many physiological and biochemical experiments failed to indicate that OADH2 uses any of the previously known OADH substrates . Growth potentials of the mutant were markedly different in media with a single carbon source versus media with mixed carbon sources.

J Bacteriol, 2002 Jun, 184(11), 3078 - 85
New type of osmoregulated solute transporter identified in halophilic members of the bacteria domain: TRAP transporter TeaABC mediates uptake of ectoine and hydroxyectoine in Halomonas elongata DSM 2581(T); Grammann K et al.; The halophilic bacterium Halomonas elongata synthesizes as its main compatible solute the aspartate derivative ectoine . We constructed a deletion mutant of H . elongata, KB1, defective in ectoine synthesis and tolerating elevated salt concentrations only in the presence of external compatible solutes . The dependency of KB1 on solute uptake for growth in high-salt medium was exploited to select insertion mutants unable to accumulate external solutes via osmoregulated transporters . One insertion mutant out of 7,200 failed to accumulate the osmoprotectants ectoine and hydroxyectoine . Genetic analysis of the insertion site proved that the mutation affected an open reading frame (ORF) of 1,281 bp (teaC) . The nucleotide sequence upstream of teaC was determined, and two further ORFs of 603 bp (teaB) and 1,023 bp (teaA) were identified . Deletion of teaA and teaB proved that all three genes are mandatory for ectoine uptake . Sequence comparison showed significant identity of TeaA, TeaB, and TeaC to the transport proteins of the recently identified tripartite ATP-independent periplasmic transporter family (TRAP-T) . The affinity of the cells for ectoines was determined (K(s) = 21.7 microM), suggesting that the transporter TeaABC exhibits high affinity for ectoines . An elevation of the external osmolarity resulted in a strong increase in ectoine uptake via TeaABC, demonstrating that this transporter is osmoregulated . Deletion of teaC and teaBC in the wild-type strain led to mutants which excreted significant amounts of ectoine into the medium when cultivated at high salt concentrations . Therefore, the physiological role of TeaABC may be primarily to recover ectoine leaking through the cytoplasmic membrane.

Annu Rev Biophys Biomol Struct, 2002, 31, 257 - 73 Epub 2001 Oct 25.
The search and its outcome: high-resolution structures of ribosomal particles from mesophilic, thermophilic, and halophilic bacteria at various functional states; Yonath A; We determined the high-resolution structures of large and small ribosomal subunits from mesophilic and thermophilic bacteria and compared them with those of the thermophilic ribosome and the halophilic large subunit . We confirmed that the elements involved in intersubunit contacts and in substrate binding are inherently flexible and that a common ribosomal strategy is to utilize this conformational variability for optimizing its functional efficiency and minimizing nonproductive interactions . Under close-to-physiological conditions, these elements maintain well-ordered characteristic conformations . In unbound subunits, the features creating intersubunit bridges within associated ribosomes lie on the interface surface, and the features that bind factors and substrates reach toward the binding site only when conditions are ripe.

Arch Microbiol, 2002 May, 177(5), 361 - 70 Epub 2002 Feb 13.
Polyphasic characterization of benthic, moderately halophilic, moderately thermophilic cyanobacteria with very thin trichomes and the proposal of Halomicronema excentricum gen . nov., sp . nov; Abed RM et al.; A new genus of moderately halophilic, moderately halotolerant and moderately thermophilic cyanobacteria with very thin trichomes is described . The four strains included in this genus were isolated from benthic microbial mats in a man-made hypersaline pond . Trichomes were around 1 microm thick, with small constrictions at the cross-walls and diffluent colorless sheaths . Thylakoids were parallel to the cell wall, but thylakoids and nucleoid were often excentrically arranged within the cytoplasm with respect to the main trichome axis . Strains grew at between 3.2 and 12-15% (w/v) salinity with optima between 3.2 and 12% . They showed lower temperature limits around 20 degrees C and upper limits between 45 and 50 degrees C, with optima between 28 and 45-50 degrees C . Carotenoid and mycosporine amino-acid complements were identical among strains . Phylogenetic analyses based on 16S rRNA gene sequence showed that all strains were closely related (99% or higher similarity) and distantly related to other cyanobacteria (91% or lower similarity) . We propose the new genus and species Halomicronema excentricum for these strains . The type strain is TFEP1.

Life Sci Space Res, 1980, 18, 109 - 21
Lipids as a principle for the identification of archaebacteria; Tornabene TG et al.; The 'Archaebacteria' consist of several distinct subgroups including methanogens, extreme halophiles and specific thermoacidophiles . These bacteria are distinct from other bacteria with respect to their characteristic RNA compositions, the absence of muramic acid in the cell walls and the predominance of nonsaponifiable {correction of nonsaponifable} lipids . The lipid composition of the archaebacteria consists of isoprenoid and hydroisoprenoid hydrocarbons and isopranyl glycerol ether lipids . The pathways for the biosynthesis of the lipid components are those shared by most microorganisms and demonstrate a close relationship; however, an independent line of descent is indicated by the formation of the isopranyl glycerol ether lipids . This discontinuity formulates a point for delineating the early stages of biological evolution and for dividing bacteria into two subgroups.

J Appl Microbiol, 2002, 92(4), 695 - 705
Widespread distribution of the bacteriocin-producing lactic acid cocci in Miso-paste products; Onda T et al.; AIMS: A survey was conducted on the ecological distribution of bacteriocin-producing lactic acid bacteria in Miso-pastes, a typical fermented food in Japan . METHODS AND RESULTS Nine Miso-pastes were sampled for isolation of bacteriocin-producers . Almost all isolated enterococcal strains produced bacteriocins but no isolated tetragenococci did so . The bacteriocin-producing isolates were divided into nine groups by phenotypic tests . As the phenotypic characters were highly diverse, these strains could not be identified to species level on the basis of their phenotypes . The nine representative strains from each group were identified by 16S rRNA analysis . These bacteriocin-producers with one exception (Lactococcus sp.) were identified as strains of the Enterococcus faecium 'species group' . The bacteriocins of the nine strains were classified into five types according to their antibacterial spectral patterns and their SDS-PAGE profiles . The bacteriocins inhibited undesirable bacteria in Miso-pastes, such as Bacillus subtilis, but did not inhibit the useful Tetragenococcus halophila . CONCLUSIONS: The bacteriocin-producing lactic acid cocci were widespread at high frequencies in Miso-pastes . They were considered to play an important role in preventing the growth of undesirable bacteria . SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that bacteriocin-producers act effectively as safe biopreservatives and may contribute to the biopreservation in Miso-pastes.

J Ind Microbiol Biotechnol, 2002 Jan, 28(1), 48 - 55
Microbial diversity and complexity in hypersaline environments: a preliminary assessment; Litchfield CD et al.; The microbial communities in solar salterns and a soda lake have been characterized using two techniques: BIOLOG, to estimate the metabolic potential, and amplicon length heterogeneity analysis, to estimate the molecular diversity of these communities . Both techniques demonstrated that the halophilic Bacteria and halophilic Archaea populations in the Eilat, Israel saltern are dynamic communities with extensive metabolic potentials and changing community structures . Halophilic Bacteria were detected in Mono Lake and the lower salinity ponds at the Shark Bay saltern in Western Australia, except when the crystallizer samples were stressed by exposure to Acid Green Dye #9899 . At Shark Bay, halophilic Archaea were found only in the crystallizer samples . These data confirm both the metabolic diversity and the phylogenetic complexity of the microbial communities and assert the need to develop more versatile media for the cultivation of the diversity of bacteria in hypersaline environments.

J Ind Microbiol Biotechnol, 2002 Jan, 28(1), 23 - 31
Halocins and sulfolobicins: the emerging story of archaeal protein and peptide antibiotics; O'Connor EM et al.; Production of antibiotic peptides and proteins is a near-universal feature of living organisms regardless of phylogenetic classification . Bacteriocins (proteinaceous antimicrobials from the domain Bacteria) have been studied for over 75 years, and the eucaryocins (proteinaceous antimicrobials from the domain Eucarya) since the early 1960s . However, one domain of organisms, the Archaea, containing hyperthermophiles, extreme halophiles and the methanogens, is just beginning to be scrutinized for the production of peptide antibiotics . Production of archaeal proteinaceous antimicrobials (archaeocins) from extreme halophiles (halocins) is a nearly universal feature of the rod-shaped haloarchaea . Halocin activity is first detectable in culture supernatants at the beginning of the transition into stationary phase, concomitant with an induction of transcription of the structural gene . Halocins are diverse in size, consisting of proteins as large as 35 kDa and peptide "microhalocins" as small as 3.6 kDa . The 36 amino acids of microhalocin HalS8 are located in the interior of a 311-residue pro-protein from which they are liberated by an unknown mechanism . Microhalocins are hydrophobic and robust, withstanding heat, desalting and exposure to organic solvents . Unlike the peptide bacteriocins and the eucaryocins, microhalocins possess a large number of neutral residues and are not cationic, leaving their mechanism(s) of action mostly a mystery . While microhalocins affect a variety of haloarchaeal genera (kingdom Euryarchaeota), they also exhibit cross-kingdom toxicity, inhibiting or killing Sulfolobus species (kingdom Crenarchaeota) . Finally, archaeocins also are produced by the hyperthermophile "Sulfolobus islandicus" . These 20-kDa protein antibiotics are not excreted into the environment, but are associated with small particles apparently derived from the cell's S-layer.

Appl Microbiol Biotechnol, 2002 Mar, 58(3), 358 - 63 Epub 2001 Dec 11.
Yield production, chemical composition, and functional properties of emulsifier H28 synthesized by Halomonas eurihalina strain H-28 in media containing various hydrocarbons; Martinez-Checa F et al.; Halomonas eurihalina strain H-28 is a moderately halophilic bacterium that produces an extracellular polysaccharide not only in media with glucose but also in media supplemented with hydrocarbons (n-tetradecane, n-hexadecane, n-octane, xylene, mineral light oil, mineral heavy oil, petrol, or crude oil) . In this study we investigated yield production, chemical composition, viscosity, and emulsifying activity of exopolysaccharides (EPS) extracted from the different media used . The largest amounts of biopolymer were synthesized in media with glucose and n-hexadecane . Chemical composition varied with culture conditions; thus EPS from cultures grown in the presence of hydrocarbons had lower contents of carbohydrates and proteins than EPS from media with glucose . However, the percentages of uronic acids, acetyls, and sulfates were always higher than glucose EPS . Crude oil was the substrate most effectively emulsified . All EPS were capable of emulsifying crude oil more efficiently than the three control surfactants tested (Tween 20, Tween 80, and Triton X-100) . All polymers gave low viscosity solutions . EPS H28 could be attractive for application in the oil industry and/or in bioremediation processes, bearing in mind not only its functional properties, but also the capacity of producer strain H-28 to grow in the presence of high salt concentrations and oil substrates.

J Mol Microbiol Biotechnol, 2002 May, 4(3), 175 - 81
Regulation of gas vesicle formation in halophilic archaea; Pfeifer F et al.; The halophilic archaea Halobacterium salinarum and Haloferax mediterranei produce gas vesicles depending on the growth phase and on environmental factors such as light, salt, or oxygen . Fourteen different gvp genes (gvpACNO and gvpDEFGHIJKLM) are involved in their formation, and the regulation of gvp gene expression occurs at the transcriptional and translational level . Haloferax volcanii offers a clean genetic background for the functional analysis of gas vesicle genes by transformation experiments . Such experiments show that the promoter of the gvpA gene encoding the major gas vesicle structural protein is activated by the endogenous basic leucine-zipper protein GvpE . On the other hand, the GvpD protein, which contains a p-loop motif, is involved either directly or indirectly in the repression of the gvpA promoter activity . Eight of the fourteen p-gvp genes (p-gvpAO and p-gvpFGJKLM) enable gas vesicle formation in Hf . volcanii transformants and thus constitute the minimal p-vac region.

Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 555 - 8
Saccharomonospora halophila sp . nov., a novel halophilic actinomycete isolated from marsh soil in Kuwait; Al-Zarban SS et al.; An actinomycete, strain 8T, was isolated from marsh soil in Kuwait . The strain was aerobic, gram-positive, halophilic and produced light blue to greyish aerial mycelium . The warty spores were sessile, occurring singly or in pairs on aerial mycelium . The mycelium was stable and did not fragment during ageing . Chemotaxonomic markers of the isolate were consistent with its classification as Saccharomonospora . The strain possessed meso-diaminopimelic acid as the diagnostic amino acid in the peptidoglycan . The diagnostic sugars were arabinose and galactose; polar lipids were phosphatidyl inositol, phosphatidyl ethanolamine, hydroxy-phosphatidyl ethanolamine, lyso-phosphatidyl ethanolamine and diphosphatidyl glycerol; the principal menaquinone was MK-9(H4); and the iso/anteiso-branched fatty acid pattern was combined with 10-methyl-branched and 2-hydroxy-branched fatty acids . Saccharomonospora cyanea DSM 44106T was the closest phylogenetic neighbour of strain 8T, showing 96.8% 16S rDNA sequence similarity . These data, together with distinct physiological traits, led to the conclusion that the novel isolate represents a new species within the genus Saccharomonospora for which the name Saccharomonospora halophila sp . nov . is proposed . The type strain is strain 8T (= DSM 44411T =NRRL B-24125T).

Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 549 - 53
Vibrio calviensis sp . nov., a halophilic, facultatively oligotrophic 0.2 microm-fiIterabIe marine bacterium; Denner EB et al.; A gram-negative, facultatively anaerobic, straight to slightly curved rod-shaped bacterium (RE35F/12T) sensitive to vibriostatic agent O/129 was previously isolated from sea water (Western Mediterranean Sea, Bay of Calvi, Corsica, France) by 0.2 microm-membrane filtration . Strain RE35/F12T (= CIP 107077T = DSM 14347T) was facultatively oligotrophic, halophilic, required Na+ for growth and produced acid but no gas from D-glucose under anaerobic conditions . Comparative 165 rRNA gene-sequence analyses demonstrated that the bacterium is most closely related (94.3%) to Vibrio scophthalmi . Similarities to the sequences of all other established Vibrio species ranged from 93.6% (with Vibrio aestuarianus) to 90.7% (with Vibrio rumoiensis) . Strain RE35/F12T occupies a distinct phylogenetic position; this is similar to the case of Vibrio hollisae, because RE35F/12T represents a relatively long subline of descent sharing a branching point with the outskirts species V . hollisae . The G+C content of the DNA was 49.5 mol% . Ubiquinone Q-8 was the main respiratory lipoquinone, and 16:1omega9cis, 16:0 and 18:1trans9, cis11 were the major cellular fatty acids, 16:1omega9cis being predominant . The polyamine pattern was characterized by the presence of the triamine sym-norspermidine . On the basis of the polyphasic information summarized above, a new Vibrio species is described for which the name Vibrio calviensis sp . nov . is proposed.

Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 485 - 91
Salinibacter ruber gen . nov., sp . nov., a novel, extremely halophilic member of the Bacteria from saltern crystallizer ponds; Anton J et al.; Five brightly red-pigmented, motile, rod-shaped, extremely halophilic bacteria were isolated from saltern crystallizer ponds in Alicante (two strains) and Mallorca (three strains), Spain . They grew optimally at salt concentrations between 20 and 30% and did not grow below 15% salts . Thus, these isolates are among the most halophilic organisms known within the domain Bacteria . The temperature optimum was 37-47 degrees C . A single, yet to be identified pigment was present, with an absorption maximum at 482 nm and a shoulder at 506-510 nm . The G+C content of the DNA was 66.3-67.7 mol% and, together, they formed a homogeneous genomic group with DNA-DNA similarities above 70% . The 16S rRNA gene sequences were almost identical to sequences recovered earlier from the saltern biomass by amplification of bacterial small-subunit rRNA genes from DNA extracted from the environment . This phylotype, earlier described as 'Candidatus Salinibacter', was shown by fluorescence in situ hybridization to contribute between 5 and 25% of the prokaryote community of the saltern crystallizers . We have therefore succeeded in isolating a bacterium from the natural environment that, although being a major component of the community, was previously known by its phylotype only . Isolation of the organism now allows formal description of a novel genus and species, for which we propose the name Salinibacter ruber gen . nov., sp . nov . The type strain is strain M31T (= DSM 13855T = CECT 5946T).

DNA Seq, 2001 Dec, 12(5-6), 337 - 44
The gas vesicle gene (gvp) cluster of the cyanobacterium Pseudanabaena sp . strain PCC 6901; Albouy D et al.; A gene cluster located downstream from gvpA in the cyanobacterium Pseudanabaena sp . strain PCC 6901 has been cloned and sequenced . The three genes, orf1, gvpN and gvpJ, are consecutive with no intergenic region . In contrast to GvpN and GvpJ, which share high similarity at the amino acid level with their counterparts in other cyanobacteria and halophilic archaea, Orf1 is only 29% identical to the C-terminal part of GvpC from Anabaena flos-aquae and its sequence organization is reminiscent of the halophilic archaeal GvpC.

J Biotechnol, 2002 May 9, 95(2), 171 - 9
Screening and characterization of aldehyde dehydrogenase gene from Halomonas salina strain AS11; Sripo T et al.; A population survey was made of moderately halophilic bacteria in prawn pond sediment in the Songkla region of Thailand . Twenty-two isolated halophilic bacteria capable of growing on modified ATCC culture medium 1270 for halobacterium were then assayed for aldehyde dehydrogenase (ALDH) activity which might be involved in the metabolism of xenobiotic compounds . One isolate, designated AS11, was selected based on its high amount of ALDH activity . This organism can grow at sodium chloride concentrations ranging from 2.5 to 25%, although optimum growth occurs at 5% NaCl . Phenotypic and phylogenetic studies indicated that AS11 was an isolate of Halomonas salina . The aldh gene coding for this enzyme was then cloned . The open reading frame of the aldh gene was 1521-bp long and coded for a protein of 506 amino acid residues with a calculated molecular mass of 55 kDa . The aldh gene product proved to be 76% identical to the NAD-dependent acetaldehyde dehydrogenase gene from Pseudomonase aeruginosa.

Mar Pollut Bull, 2002 Jan, 44(1), 32 - 8
Biological pollution in the Mediterranean Sea: invasive versus introduced macrophytes; Boudouresque CF et al.; The authors have listed 85 species of macrophytes that have probably been introduced to the Mediterranean . Among them, nine species can be considered as invasive, i.e., playing a conspicuous role in the recipient ecosystems, taking the place of keystone species and/or being economically harmful: Acrothamnion preissii, Asparagopsis armata, Lophocladia lallemandii, Womersleyella setacea (Rhodophyta), Sargassum muticum, Stypopodium schimperi (Fucophyceae), Caulerpa racemosa, Caulerpa taxifolia and Halophila stipulacea (Plantae) . These data fit well the Williamson and Fitter's "tens rule", which states that, on average, 1 out of 10 introduced species becomes invasive . Though some features (e.g . life traits, geographical origin) can increase the likelihood of a successful invasion, the success of invaders is far from being predictable . Since the beginning of the 20th century, the number of introduced species to the Mediterranean has nearly doubled every 20 years . Should these kinetics continue, and according to the tens rule, it can be expected that 5-10 newly introduced macrophytes shall become invasive in the next 20 years.

Zh Mikrobiol Epidemiol Immunobiol, 2001 Nov-Dec, (6), 3 - 7
{Studies on halophilic vibrios causing a food poisoning outbreak in the city of Vladivostok}; Smolikova LM et al.; V . parahaemolyticus and V . alginolyticus strains isolated from patients during an outbreak of an acute enteric disease in Vladivostok in 1997 were studied . All strains were found to possess typical taxonomic signs . V . parahaemolyticus isolated from humans had direct heat stable haemolysin exotoxin . The overwhelming majority of these strains belonged to serovar O3K6 . Among the cultures under study 7 phage types were determined: phage types 1, 2, 7, 10 in 8 V . parahaemolyticus strains and phage types 2, 4 . 5 . 7 in 5 V . alginolyticus strains . The diagnostic halophilic phage lyzed vibrios in 30.2% of strains . The cultures under study were found to be highly sensitive to chloramphenicol, cefotaxime, nalidixic acid and cyprofloxacin . The study proved that the outbreak of alimentary toxicoinfection was caused by vibrios of serogroup O3:K6.

Extremophiles, 2002 Feb, 6(1), 15 - 20
Investigation into a stress-inducible promoter region from Marinococcus halophilus using green fluorescent protein; Bestvater T et al.; A 480-bp Marinococcus halophilus DNA-fragment upstream of the ectoine genes ectABC was linked to the reporter gene gfp(uv) in the stress probe plasmid pBRGFP(uv) . On the basis of green fluorescence emitted after application of different types of stress to recombinant Escherichia coli, the DNA sequence was characterized as a stress-responding promoter region . In E . coli, osmotic stress resulted in an increasing fluorescence intensity, whereas the effects of heat or cold were insignificant, suggesting that transcription and/or translation is controlled mainly by an osmotic stress/low-water stress response . Additionally, the stress-counteracting effects of the compatible solutes ectoine and betaine were demonstrated.

Huan Jing Ke Xue, 2001 Nov, 22(6), 44 - 8
{Treatment of ion-exchange monosodium glutamate wastewater by yeast}; Yang Q et al.; Two strains of yeasts, Candida halophila and Rhodotorula glutinis, were acquired through screening from ion-exchange monosodium glutamate wastewater (IEMGW) which was characterized by its high contents of COD (40,690 mg/L), NH(4+)-N (16,914 mg/L) and SO(4)2- (18,000 mg/L) . The mixture of the two species had a good COD removal performance for treating IEMGW in a pH rang of 4-9 . The yeast technology was superior to the conventional activated sludge in terms of COD removal and COD decomposition rate, In a dilution rate range of wastewater from 1:1 to 1:9, a COD removal of 84.5% and an average COD decomposition rate of over 1.0 kg/(kg.d) were obtained under an initial pH of 4.

Plant Cell Environ, 2002 Feb, 25(2), 131 - 139
Molecular and genetic aspects of plant responses to osmotic stress; Xiong L et al.; Drought, high salinity and freezing impose osmotic stress on plants . Plants respond to the stress in part by modulating gene expression, which eventually leads to the restoration of cellular homeostasis, detoxification of toxins and recovery of growth . The signal transduction pathways mediating these adaptations can be dissected by combining forward and reverse genetic approaches with molecular, biochemical and physiological studies . Arabidopsis is a useful genetic model system for this purpose and its relatives including the halophyte Thellungiella halophila, can serve as valuable complementary genetic model systems.

New Microbiol, 2002 Jan, 25(1), 93 - 6
Virulence genes in halophilic Vibrio spp . isolated in common mussels; Deriu A et al.; Twenty-five Vibrio strains belonging to nine different species, isolated in common mussels, were examined for the presence of different virulence genes: ctxA, tcpA, toxR, toxS, ace, zot and vpi previously found in pathogenic Vibrio cholerae strains . Our results suggest that there is a wide dissemination of Vibrio cholerae virulence genes among the various Vibrio species tested . This finding raises the question of whether a different approach should be taken to study "environmental" Vibrio strains.

Int J Syst Evol Microbiol, 2002 Jan, 52(Pt 1), 7 - 76
The neomuran origin of archaebacteria, the negibacterial root of the universal tree and bacterial megaclassification; Cavalier-Smith T; Prokaryotes constitute a single kingdom, Bacteria, here divided into two new subkingdoms: Negibacteria, with a cell envelope of two distinct genetic membranes, and Unibacteria, comprising the new phyla Archaebacteria and Posibacteria, with only one . Other new bacterial taxa are established in a revised higher-level classification that recognizes only eight phyla and 29 classes . Morphological, palaeontological and molecular data are integrated into a unified picture of large-scale bacterial cell evolution despite occasional lateral gene transfers . Archaebacteria and eukaryotes comprise the clade neomura, with many common characters, notably obligately co-translational secretion of N-linked glycoproteins, signal recognition particle with 7S RNA and translation-arrest domain, protein-spliced tRNA introns, eight-subunit chaperonin, prefoldin, core histones, small nucleolar ribonucleoproteins (snoRNPs), exosomes and similar replication, repair, transcription and translation machinery . Eubacteria (posibacteria and negibacteria) are paraphyletic, neomura having arisen from Posibacteria within the new subphylum Actinobacteria (possibly from the new class Arabobacteria, from which eukaryotic cholesterol biosynthesis probably came) . Replacement of eubacterial peptidoglycan by glycoproteins and adaptation to thermophily are the keys to neomuran origins . All 19 common neomuran character suites probably arose essentially simultaneously during the radical modification of an actinobacterium . At least 11 were arguably adaptations to thermophily . Most unique archaebacterial characters (prenyl ether lipids; flagellar shaft of glycoprotein, not flagellin; DNA-binding protein lob; specially modified tRNA; absence of Hsp90) were subsequent secondary adaptations to hyperthermophily and/or hyperacidity . The insertional origin of protein-spliced tRNA introns and an insertion in proton-pumping ATPase also support the origin of neomura from eubacteria . Molecular co-evolution between histones and DNA-handling proteins, and in novel protein initiation and secretion machineries, caused quantum evolutionary shifts in their properties in stem neomura . Proteasomes probably arose in the immediate common ancestor of neomura and Actinobacteria . Major gene losses (e.g . peptidoglycan synthesis, hsp90, secA) and genomic reduction were central to the origin of archaebacteria . Ancestral archaebacteria were probably heterotrophic, anaerobic, sulphur-dependent hyperthermoacidophiles; methanogenesis and halophily are secondarily derived . Multiple lateral gene transfers from eubacteria helped secondary archaebacterial adaptations to mesophily and genome re-expansion . The origin from a drastically altered actinobacterium of neomura, and the immediately subsequent simultaneous origins of archaebacteria and eukaryotes, are the most extreme and important cases of quantum evolution since cells began . All three strikingly exemplify De Beer's principle of mosaic evolution: the fact that, during major evolutionary transformations, some organismal characters are highly innovative and change remarkably swiftly, whereas others are largely static, remaining conservatively ancestral in nature . This phenotypic mosaicism creates character distributions among taxa that are puzzling to those mistakenly expecting uniform evolutionary rates among characters and lineages . The mixture of novel (neomuran or archaebacterial) and ancestral eubacteria-like characters in archaebacteria primarily reflects such vertical mosaic evolution, not chimaeric evolution by lateral gene transfer . No symbiogenesis occurred . Quantum evolution of the basic neomuran characters, and between sister paralogues in gene duplication trees, makes many sequence trees exaggerate greatly the apparent age of archaebacteria . Fossil evidence is compelling for the extreme antiquity of eubacteria {over 3500 million years (My)} but, like their eukaryote sisters, archaebacteria probably arose only 850 My ago . Negibacteria are the most ancient, radiating rapidly into six phyla . Evidence from molecular sequences, ultrastructure, evolution of photosynthesis, envelope structure and chemistry and motility mechanisms fits the view that the cenancestral cell was a photosynthetic negibacterium, specifically an anaerobic green non-sulphur bacterium, and that the universal tree is rooted at the divergence between sulphur and non-sulphur green bacteria . The negibacterial outer membrane was lost once only in the history of life, when Posibacteria arose about 2800 My ago after their ancestors diverged from Cyanobacteria.

Int J Syst Evol Microbiol, 2002 Jan, 52(Pt 1), 229 - 34
Pseudoalteromonas issachenkonii sp . nov., a bacterium that degrades the thallus of the brown alga Fucus evanescens; Ivanova EP et al.; Eleven non-pigmented strains of gram-negative, aerobic, marine bacteria with polar flagella were isolated from the thallus of the brown alga Fucus evanescens collected in the Kraternaya Bight of the Kurile Islands in the Pacific Ocean . These organisms were conspecific and exhibited high levels of genetic relatedness (up to 91%) . The G+C contents of the DNAs of these strains were 42.9-43.3 mol% . These halophilic bacteria had bacteriolytic, proteolytic and haemolytic activities and degraded algal polysaccharides, synthesizing a number of glycoside hydrolases (fucoidanases, laminaranases, alginases, agarases, pullulanases, beta-glucosidases, beta-galactosidases, beta-N-acetylglucosaminidases and beta-xylosidases) . By 16S rDNA analysis, the bacteria were shown to belong to the genus Pseudoalteromonas, a member of the gamma-subclass of the Proteobacteria . DNA from the strains isolated from the brown alga showed 27-54% genetic relatedness with respect to DNAs of other type strains of the genus Pseudoalteromonas . The phenotypic characteristics, together with the genetic evidence, indicate that this group of epiphytic bacteria represents a distinct species, Pseudoalteromonas issachenkonii sp . nov., for which the type strain is KMM 3549T (= LMG 19697T = CIP 106858T).

Int J Syst Evol Microbiol, 2002 Jan, 52(Pt 1), 149 - 55
Halorubrum tebenquichense sp . nov., a novel halophilic archaeon isolated from the Atacama Saltern, Chile; Lizama C et al.; A novel extremely halophilic archaeon was isolated from Lake Tebenquiche, situated in the northern part of the Atacama Saltern, Chile . The cells of these micro-organisms were mostly irregularly disc-shaped . They grew in medium containing saturated concentrations of NaCl and did not require magnesium for optimal growth . The polar lipid composition revealed the presence of mannosyl-2-sulfate-(1-4)-glycosyl-archaeol, the main glycolipid of the genus Halorubrum, and two new glycolipids . The G+C content of the DNA was 63.2 mol% . Phylogenetic analysis of the 16S rRNA gene placed strain ALT6-92T within the Halorubrum cluster . The low DNA-DNA hybridization value justified classification in a new species for which the name Halorubrum tebenquichense sp . nov . is proposed . The type strain is ALT6-92T (= CECT 5317T = DSM 14210T).

Int J Syst Evol Microbiol, 2002 Jan, 52(Pt 1), 123 - 30
Halomonas alimentaria sp . nov., isolated from jeotgal, a traditional Korean fermented seafood; Yoon JH et al.; A gram-negative, moderately halophilic bacterial strain, YKJ-16T, which was isolated from jeotgal, a traditional Korean food, was considered to be a member of the genus Halomonas . Cells of strain YKJ-16T are non-motile and cocci or short rods, unlike most Halomonas species . However, chemotaxonomic and phylogenetic analyses demonstrated that strain YKJ-16T belongs to the genus Halomonas . The predominant isoprenoid quinone is ubiquinone-9 . The major fatty acids are C18.1omega7c, C16:0, C19:0 cyclo omega8c and C16:1omega7c and/or iso C15:0 20H . The phylogenetic tree showed that strain YKJ-16T forms a distinct evolutionary lineage within the radiation comprising Halomonas species and forms a coherent cluster with Halomonas halodenitrificans, Halomonas cupida and Halomonas pacifica . Levels of 16S rDNA similarity between strain YKJ-16T and the type strains of other Halomonas species are 93.0-96.3% . Levels of DNA-DNA relatedness indicate a taxonomic status of strain YKJ-16T as a species different from the three species that form the coherent cluster mentioned above . Morphologically, strain YKJ-16T is also clearly differentiated from the type strains of H . cupida and H . pacifica . Accordingly, on the basis of the phenotypic characteristics, 16S rDNA sequence analysis and DNA relatedness data, strain YKJ-16T should be placed in the genus Halomonas as a novel species . The name Halomonas alimentaria sp . nov . is proposed with strain YKJ-16T (= KCCM 41042T = JCM 10888T) as the type strain.

Folia Microbiol (Praha), 2001, 46(4), 297 - 302
Growth of moderately halophilic bacteria isolated from sea water using phenol as the sole carbon source; Munoz JA et al.; Moderately halophilic bacteria utilizing phenol as the sole carbon source were isolated by selective enrichment from sea water . The isolate (Gram-negative motile rods) was identified as Deleya venusta . It grew well in the presence of up to 1600 mg/L of phenol and 8% NaCl under aerobic conditions . When the cells were treated with chloramphenicol prior to the addition of phenol they did not utilize added phenol, even after prolonged incubation . Thus, the enzymes necessary for phenol metabolism appeared to be inducible.

Biochemistry, 2002 Feb 12, 41(6), 1915 - 22
Light-induced unfolding of photoactive yellow protein mutant M100L; Sasaki J et al.; Light-activation of the PAS domain protein photoactive yellow protein (PYP) is believed to trigger a negative phototactic response in the phototropic bacterium Halorhodospira halophila . To investigate transient conformational changes of the PYP photocycle, we utilized the PYP mutant M100L that displays an increased lifetime of the putative signaling-state photointermediate PYP(M) by 3 orders of magnitude, as previously reported for the M100A mutant {Devanathan, S., Genick, U . K., Canestrelli, I . L., Meyer, T . E., Cusanovich, M . A., Getzoff, E . D., and Tollin, G . Biochemistry (1998) 37, 11563-11568} . The FTIR difference spectrum of PYP(M) and the ground state of M100L demonstrated extensive peptide-backbone structural changes as observed in the FTIR difference spectrum of the wild-type protein and PYP(M) . The conformational change investigated by CD spectroscopy in the far-UV region showed reduction of the alpha-helical content by approximately 40%, indicating a considerable amount of changes in the secondary structure . The optical activity of the p-coumaric acid chromophore completely vanished upon PYP(M) in contrast to the dark state, indicating deformation of the binding pocket structure in PYP(M) . The tertiary structural changes were further monitored by small-angle X-ray scattering measurements, which demonstrated a significant increase of the radius of gyration of the molecule by approximately 5% in PYP(M) . These structural changes were reversed concomitantly with the chromophore anionization upon the dark state recovery . The observed changes of the quantities provided a more vivid view of the structural changes of the mutant PYP in going from PYP(M) to PYP(dark), which can be regarded as a process of folding of the secondary and the tertiary structures of the "PAS" domain structure, coupled with the p-coumaric acid chromophore deprotonation and isomerization.

Syst Appl Microbiol, 2001 Nov, 24(3), 464 - 74
Taxonomic study of extreme halophilic archaea isolated from the "Salar de Atacama", Chile; Lizama C et al.; A large number of halophilic bacteria were isolated in 1984-1992 from the Atacama Saltern (North of Chile) . For this study 82 strains of extreme halophilic archaea were selected . The characterization was performed by using the phenotypic characters including morphological, physiological, biochemical, nutritional and antimicrobial susceptibility test . The results, together with those from reference strains, were subjected to numerical analysis, using the Simple Matching (S(SM)) coefficient and clustered by the unweighted pair group method of association (UPGMA) . Fifteen phena were obtained at an 70% similarity level . The results obtained reveal a high diversity among the halophilic archaea isolated . Representative strains from the phena were chosen to determine their DNA base composition and the percentage of DNA-DNA similarity compared to reference strains . The 16S rRNA studies showed that some of these strains constitutes a new taxa of extreme halophilic archaea.

J Bacteriol, 2002 Feb, 184(3), 840 - 5
Cytochrome aa(3) in Haloferax volcanii; Tanaka M et al.; A cytochrome in an extremely halophilic archaeon, Haloferax volcanii, was purified to homogeneity . This protein displayed a redox difference spectrum that is characteristic of a-type cytochromes and a CN(-) complex spectrum that indicates the presence of heme a and heme a(3) . This cytochrome aa(3) consisted of 44- and 35-kDa subunits . The amino acid sequence of the 44-kDa subunit was similar to that of the heme-copper oxidase subunit I, and critical amino acid residues for metal binding, such as histidines, were highly conserved . The reduced cytochrome c partially purified from the bacterial membrane fraction was oxidized by the cytochrome aa(3), providing physiological evidence for electron transfer from cytochrome c to cytochrome aa(3) in archaea.

Extremophiles, 2001 Dec, 5(6), 385 - 92
Archaeology of Archaea: geomicrobiological record of Pleistocene thermal events concealed in a deep-sea subseafloor environment; Inagaki F et al.; A record of the history of the Earth is hidden in the Earth's crust, like the annual rings of an old tree . From very limited records retrieved from deep underground, one can infer the geographical, geological, and biological events that occurred throughout Earth's history . Here we report the discovery of vertically shifted community structures of Archaea in a typical oceanic subseafloor core sample (1410 cm long) recovered from the West Philippine Basin at a depth of 5719 m . Beneath a surface community of ubiquitous deep-sea archaea (marine crenarchaeotic group I; MGI), an unusual archaeal community consisting of extremophilic archaea, such as extreme halophiles and hyperthermophiles, was present . These organisms could not be cultivated, and may be microbial relicts more than 2 million years old . Our discovery of archaeal rDNA in this core sample, probably associated with the past terrestrial volcanic and submarine hydrothermal activities surrounding the West Philippine Basin, serves as potential geomicrobiological evidence reflecting novel records of geologic thermal events in the Pleistocene period concealed in the deep-sea subseafloor.

Extremophiles, 2001 Dec, 5(6), 375 - 84
Expression of the 2,4-D degradative pathway of pJP4 in an alkaliphilic, moderately halophilic soda lake isolate, Halomonas sp . EF43; Kleinsteuber S et al.; The broad host range plasmid pJP4, which carries genes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid, and 3-chlorobenzoic acid, was used in conjugation experiments with mixed cultures enriched from water and sediment samples from an alkaline pond in the area of Szegedi Feherto, a soda lake in south Hungary . pJP4-encoded mercury resistance was used as a selection marker . One of the transconjugants, the alkaliphilic, moderately halophilic strain EF43, stably maintained the plasmid and was able to degrade 2,4-D and 3-chlorobenzoate under alkaline conditions in the presence of an additional carbon source such as pyruvate, benzoate, or alpha-ketoglutarate, indicating that the degradative genes of pJP4 were expressed in this strain . However, it was unable to grow on these chloroaromatic substrates when the substrate was the sole source of carbon and energy . Chemostat cultivation experiments revealed that the 2,4-D degradation rate during growth on benzoate or pyruvate was limited by the low activity of chlorocatechol-degrading enzymes, particularly chloromuconate cycloisomerase . Strain EF43 was identified as Halomonas sp . on the basis of 16S rRNA sequencing and additional taxonomic studies . 16S rRNA sequence analysis revealed that strain EF43 is closely related to typical soda lake isolates belonging to the genus Halomonas.

J Microbiol Methods, 2002 Feb, 48(2-3), 161 - 70
Biomarker techniques to screen for bacteria that produce polyunsaturated fatty acids; Nichols DS et al.; The production of polyunsaturated fatty acids (PUFA) by bacteria has been firmly established for over two decades although it is still commonly ignored . Investigations of Antarctic sea ice have revealed a high diversity of novel bacterial taxa with the ability to produce PUFA . The majority are psychrophilic (requiring low temperatures for growth) and halophilic (requiring the presence of salts for growth), in contrast to the bacterial community present in the underlying water column . Specific fatty acids may be used as indicators of PUFA-producing bacteria in environmental samples . Structural studies of bacterial phospholipids have been particularly revealing in suggesting biomarkers specific for prokaryotic PUFA input . The use of negative ion fast atom bombardment tandem mass spectrometry for the analysis of bacterial phospholipids has identified species specific for certain groups of bacterial PUFA producers . The phylogeny of PUFA production in the gamma-Proteobacteria also suggests the future use of PUFA genes for the assessment of marine bacterial biodiversity.

Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 2133 - 43
Alcalilimnicola halodurans gen . nov., sp . nov., an alkaliphilic, moderately halophilic and extremely halotolerant bacterium, isolated from sediments of soda-depositing Lake Natron, East Africa Rift Valley; Yakimov MM et al.; An alkaliphilic, halotolerant, Gram-negative, heterotrophic, aerobic and rod-shaped organism was isolated from drying soda and at a water-covered site of Lake Natron, Tanzania, by means of the most-probable-number technique developed for anoxygenic, phototrophic sulfur bacteria . It had an absolute requirement for alkalinity, but not for salinity; growth occurred at salt concentrations of 0-28% (w/v), with optimal growth at 3-8% (w/v) NaCl . The bacterium preferentially metabolized volatile fatty acids and required vitamins for growth . The name Alcalilimnicola halodurans gen . nov., sp . nov . is proposed for the novel isolate, placed in the gamma-Proteobacteria within the family Ectothiorhodospiraceae on the basis of analysis of the 16S rDNA sequence, polar lipids, fatty acids and DNA base composition . Although Alcalilimnicola halodurans is closely related to the extreme anoxygenic, phototrophic sulfur bacteria of the genus Halorhodospira, it is not phototrophic.

Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 2087 - 93
Jeotgalibacillus alimentarius gen . nov., sp . nov., a novel bacterium isolated from jeotgal with L-lysine in the cell wall, and reclassification of Bacillus marinus Rüger 1983 . as mMrinibacillus marinus gen nov., comb . nov; Yoon JH et al.; A moderately halophilic, round-endospore-forming bacterium (strain YKJ-13T) was isolated from jeotgal, a traditional Korean fermented seafood, and studied by a polyphasic taxonomic approach . This organism was related to the phylogenetic clade comprising members of Bacillus rRNA group 2 and formed a cluster with Bacillus marinus with a bootstrap fidelity value of 93.6% . The peptidoglycan type was A1alpha linked directly through L-Lys . Based on cell morphology, peptidoglycan type and phylogeny, strain YKJ-13T, together with B . marinus, is considered to be a member of Bacillus rRNA group 2 . Strain YKJ-13T was also characterized by having MK-7 and MK-8 as the predominant menaquinones and iso-C15:0 as the major fatty acid . The DNA G+C content was 44 mol% . Strain YKJ-13T exhibited a 16S rDNA similarity value of 95.7% with B . marinus DSM 1297T, its closest phylogenetic relative . Levels of 16S rDNA similarity between strain YKJ-13T and other Bacillus spp . were less than 94.2% . Therefore, on the basis of the data presented, the name Jeotgalibacillus alimentarius gen . nov., sp . nov . is proposed for strain YKJ-13T (= KCCM 80002T = JCM 10872T) . It is also proposed that B . marinus be reclassified in Marinibacillus gen . nov . as Marinibacillus marinus comb . nov.

Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 2031 - 6
Vibrio agarivorans sp . nov., a novel agarolytic marine bacterium; Macian MC et al.; It is proposed that the new Vibrio species Vibrio agarivorans accommodates two agarolytic, halophilic, fermentative bacterial strains isolated from Mediterranean sea water . The cells were gram-negative, oxidase-positive, polarly flagellated bacilli that fermented glucose without gas production and that produced no decarboxylases . They used a wide range of compounds as sole carbon and energy sources . The DNA G+C content was 44.8 mol% . Phylogenetic analysis based on complete 16S and 23S rDNA sequences revealed that the strains belong to the gamma-Proteobacteria, and are specifically related to Vibrio species . Their nearest relatives were species of the Vibrio fischeri group, sharing 16S rDNA sequence similarities below 97% with the agarolytic strains . The type strain is 289T (= CECT 5085T = DSM 13756T).

Int J Food Microbiol, 2001 Oct 22, 70(1-2), 71 - 7
Histamine formation by Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce; Kimura B et al.; We examined histamine formation in cultures of Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce . T . muriaticus formed histamine in low acidity (pH 5.8), O2 limiting conditions with optimal NaCl and glucose concentrations of 5-7% (w/v) and above 1%, respectively . Histamine formation could not be prevented even at 20% (w/v) NaCl, indicating that NaCl could not prevent histamine formation by this bacterium . A conspicuous amount of histamine accumulated only during the late stationary phase regardless of the growth conditions . Studies of cell suspension experiments confirmed the results obtained from cultured cells.

Biosci Biotechnol Biochem, 2001 Oct, 65(10), 2343 - 6
Characterization of nucleoside diphosphate kinase from moderately halophilic eubacteria; Yonezawa Y et al.; Nucleoside diphosphate kinase was purified to apparent homogeneity from naturally isolated moderately halophilic eubacteria by ATP-agarose and phenyl-5PW column chromatographies . The molecular mass of this enzyme was 15 kDa by time-of-flight mass-spectrometry . This protein showed anomalous mobility on SDS-PAGE which is typical of a halophilic protein . It was stable and active over a wide range of salt concentrations, from 0 to 4.0 M NaCl.

Biologist (London), 2001 Dec, 48(6), 278 - 82
Microbial survivors: thermophiles, halophiles, and other prodigies; Howland JL; When biologists encounter microbes flourishing in boiling water and other extreme habitats, they often consider such creatures as merely odd, and only search for possible protective mechanisms . But it may also be that extreme habitats resemble those where life first occurred, and that such organisms provide links with earlier evolution.

J Biotechnol, 2002 Feb 14, 93(2), 159 - 64
An extreme halophilic enzyme active at low salt in reversed micelles; Marhuenda-Egea FC et al.; Possible biotechnological applications of extreme halophilic enzymes are strongly determined by their high salt requirement of around 4 M NaCl . Consequently, the use of these in organic media seemed to be unlikely . However, we have succeeded in dissolving a halophilic enzyme, p-nitrophenylphosphate phosphatase from the archaeon Halobacterium salinarum, in an organic medium by creating a reverse micellar system with very low salt concentration . The enzyme retained its catalytic properties in reversed micelles made with an anionic surfactant (dioctyl sodium sulphosuccinate) or with a cationic surfactant (hexadecyltrimethylammonium bromide) in cyclohexane plus 1-butanol as co-surfactant . The dependence of the rate of hydrolysis of p-nitrophenylphosphate phosphate on the molar water/surfactant ratio (w(0) value) showed a bell-shaped curve for each surfactant system . Kinetic parameters were determined in each system . The enzymatic reaction appeared to follow Michaelis-Menten kinetics with the anionic surfactant only . The kinetic behaviour was determined at different concentrations of Mn(2+) in reversed micelles of dioctyl sodium sulphosuccinate as surfactant.

J Mol Evol, 2002 Jan, 54(1), 134 - 7
Curiously modern DNA for a "250 million-year-old" bacterium; Nickle DC et al.; Studies of ancient DNA have attracted considerable attention in scientific journals and the popular press . Several of the more extreme claims for ancient DNA have been questioned on biochemical grounds (i.e., DNA surviving longer than expected) and evolutionary grounds (i.e., nucleotide substitution patterns not matching theoretical expectations for ancient DNA) . A recent letter to Nature from Vreeland et al . (2000), however, tops all others with respect to age and condition of the specimen . These researchers extracted and cultured a bacterium from an inclusion body from what they claim is a 250 million-year (Myr)-old salt crystal . If substantiated, this observation could fundamentally alter views about bacterial physiology, ecology and evolution . Here we report on molecular evolutionary analyses of the 16S rDNA from this specimen . We find that 2-9-3 differs from a modern halophile, Salibacillus marismortui, by just 3 unambiguous bp in 16S rDNA, versus the approximately 59 bp that would be expected if these bacteria evolved at the same rate as other bacteria . We show, using a Poisson distribution, that unless it can be shown that S . marismortui evolves 5 to 10 times more slowly than other bacteria for which 16S rDNA substitution rates have been established, Vreeland et al.'s claim would be rejected at the 0.05 level . Also, a molecular clock test and a relative rates test fail to substantiate Vreeland et al.'s claim that strain 2-9-3 is a 250-Myr-old bacterium . The report of Vreeland et al . thus falls into a long series of suspect ancient DNA studies.

Proc Natl Acad Sci U S A, 2001 Dec 4, 98(25), 14356 - 61
Fast dynamics of halophilic malate dehydrogenase and BSA measured by neutron scattering under various solvent conditions influencing protein stability; Tehei M et al.; Protein thermal dynamics was evaluated by neutron scattering for halophilic malate dehydrogenase from Haloarcula marismortui (HmMalDH) and BSA under different solvent conditions . As a measure of thermal stability in each case, loss of secondary structure temperatures were determined by CD . HmMalDH requires molar salt and has different stability behavior in H(2)O, D(2)O, and in NaCl and KCl solvents . BSA remains soluble in molar NaCl . The neutron experiments provided values of mean-squared atomic fluctuations at the 0.1 ns time scale . Effective force constants, characterizing the mean resilience of the protein structure, were calculated from the variation of the mean-squared fluctuation with temperature . For HmMalDH, resilience increased progressively with increasing stability, from molar NaCl in H(2)O, via molar KCl in D(2)O, to molar NaCl in D(2)O . Surprisingly, however, the opposite was observed for BSA; its resilience is higher in H(2)O where it is less stable than in D(2)O . These results confirmed the complexity of dynamics-stability relationships in different proteins . Softer dynamics for BSA in D(2)O showed that the higher thermostability is associated with entropic fluctuations . In the halophilic protein, higher stability is associated with increased resilience showing the dominance of enthalpic terms arising from bonded interactions . From previous data, it is suggested that these are associated with hydrated ion binding stabilizing the protein in the high-salt solvent.

J Food Prot, 2001 Nov, 64(11), 1716 - 21
Levels of Vibrio vulnificus and organoleptic quality of raw shellstock oysters (Crassostrea virginica) maintained at different storage temperatures; Lorca TA et al.; Temperature abuse during raw oyster harvesting and storage may allow for the multiplication of natural spoilage flora as well as microbial pathogens, thus posing a potential health threat to susceptible consumers and compromising product quality . The objective of this study was to provide a scientific basis for determining whether different refrigeration and abuse temperatures for raw oysters would result in a spoiled product before it became unsafe . Raw shellstock oysters (Crassostrea virginica) purchased from a commercial Virginia processor were subjected to different temperature abuse conditions (7, 13, and 21 degrees C) over a 10-day storage period . Salinity, pH, halophilic plate count (HPC), total culturable Vibrio counts, and culturable Vibrio vulnificus counts were determined at each abuse condition . V . vulnificus isolates were confirmed by a specific enzyme-linked immunosorbent assay . Olfactory analysis was performed to determine consumer acceptability of the oysters at each abuse stage . The pH of the oysters decreased over time in each storage condition . The HPC increased 2 to 4 logs for all storage conditions, while olfactory acceptance decreased over time . V . vulnificus levels increased over time, reaching 10(5) to 10(6) CFU/g by day 6 . The length of storage had a greater effect on the bacterial counts and olfactory acceptance of the oysters (P < 0.05) over time than did the storage temperature (P < 0.05).

J Clin Microbiol, 2001 Dec, 39(12), 4233 - 40
Evaluation of typing of vibrio parahaemolyticus by three PCR methods using specific primers; Wong HC et al.; Vibrio parahaemolyticus is a halophilic bacterium frequently involved in human outbreaks of seafood-associated gastroenteritis . For epidemiological purposes, different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE) or ribotyping, have been developed for this pathogen; however, these methods are mostly labor-intensive and time-consuming . In this work, we designed and evaluated three rapid PCR typing methods for this pathogen using primers designed on the basis of the following specific sequences: conserved ribosomal gene spacer sequence (RS), repetitive extragenic palindromic sequence (REP), and enterobacterial repetitive intergenic consensus sequence (ERIC) . Typing patterns and clustering analysis indicated that these methods apparently differentiated V . parahaemolyticus strains from reference strains of interspecific Escherichia coli, V . cholerae, and V . vulnificus and were also valuable in subspecies typing of this pathogen . Forty domestic strains of V . parahaemolyticus, representing a wide range of PFGE patterns, were grouped into 15, 27, and 27 patterns, with discrimination indexes of 0.91, 0.97, and 0.98, by RS-, REP-, and ERIC-PCR, respectively . The discriminative abilities of these PCR methods closely approached or even exceeded those of PFGE and ribotyping . REP-PCR is preferable to ERIC-PCR because of the greater reproducibility of its fingerprints, while RS-PCR may be a practical method because it generates fewer amplification bands and patterns than the alternatives.

Nucleic Acids Res, 2001 Nov 15, 29(22), 4518 - 29
Box C/D RNA guides for the ribose methylation of archaeal tRNAs . The tRNATrp intron guides the formation of two ribose-methylated nucleosides in the mature tRNATrp; Clouet d'Orval B et al.; Following a search of the Pyrococcus genomes for homologs of eukaryotic methylation guide small nucleolar RNAs, we have experimentally identified in Pyrococcus abyssi four novel box C/D small RNAs predicted to direct 2'-O-ribose methylations onto the first position of the anticodon in tRNALeu(CAA), tRNALeu(UAA), elongator tRNAMet and tRNATrp, respectively . Remarkably, one of them corresponds to the intron of its presumptive target, pre-tRNATrp . This intron is predicted to direct in cis two distinct ribose methylations within the unspliced tRNA precursor, not only onto the first position of the anticodon in the 5' exon but also onto position 39 (universal tRNA numbering) in the 3' exon . The two intramolecular RNA duplexes expected to direct methylation, which both span an exon-intron junction in pre-tRNATrp, are phylogenetically conserved in euryarchaeotes . We have experimentally confirmed the predicted guide function of the box C/D intron in halophile Haloferax volcanii by mutagenesis analysis, using an in vitro splicing/RNA modification assay in which the two cognate ribose methylations of pre-tRNATrp are faithfully reproduced . Euryarchaeal pre-tRNATrp should provide a unique system to further investigate the molecular mechanisms of RNA-guided ribose methylation and gain new insights into the origin and evolution of the complex family of archaeal and eukaryotic box C/D small RNAs.

Extremophiles, 2001 Oct, 5(5), 285 - 94
Bioenergetics and solute uptake under extreme conditions; Albers SV et al.; The ion and particularly the proton and sodium ion permeabilities of cytoplasmic membranes play crucial roles in the bioenergetics of microorganisms . The proton and sodium permeabilities of membranes increase with 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 constant (homeoproton permeability) . Thermophilic bacteria are an exception . They rely on the less permeable sodium ions to generate a sodium motive force, which is subsequently used to drive energy-requiring membrane-bound processes . Transport of solutes across bacterial and archaeal membranes 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 uptake systems . Several high-affinity ATP-binding cassette (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.

J Membr Biol, 2001 Oct 1, 183(3), 195 - 204
Characterization of inverted membrane vesicles from the halophilic archaeon Haloferax volcanii; Ring G et al.; Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood . One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model membrane systems . In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic archaeon Haloferax volcanii, is presented . The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells . Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles . Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the membrane preparation . The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform for the study of various membrane-related phenomena in archaea.

Appl Microbiol Biotechnol, 2001 Oct, 57(1-2), 200 - 4
High-pressure adaptation by salt stress in a moderately halophilic bacterium obtained from open seawater; Tanaka T et al.; High-pressure adaptation was examined using a moderately halophilic bacterium (Micrococcus roseus), which was isolated from open seawater and capable of growing in 15% w/v NaCl (optimum NaCl concentration: 3% w/v) . After treatment at 207 MPa, colony-forming units (CFUs) significantly decreased; however, the loss of integral cells after pressure was only 30% when direct cell count was performed microscopically . In order to investigate the piezotolerance of M . roseus under high pressure without morphological change, the survival of cells was examined under pressure at 138 MPa for 2 h . M . roseus in 3% NaCl was still sensitive to pressure at 138 MPa . However, the cells in the third generations showed remarkably increased pressure resistance, and no significant loss of viability was confirmed . Furthermore, when M . roseus was cultured in 1, 3, 5, 10 and 15% NaCl, the survival ratio proportionally increased at increased NaCl concentration . M . roseus cultured in 15% NaCl was remarkably resistant (94.7% viability) to pressure at 138 MPa, even when suspended in lower concentration of NaCl . This suggests that NaCl concentrations in growth culture affect the piezotolerance of M . roseus and that this species has an ability to adapt to high pressure.

Arch Microbiol, 2001 Oct, 176(4), 294 - 300
Actinopolyspora halophila has two separate pathways for betaine synthesis; Nyyssola A et al.; The extremely halophilic actinomycete Actinopolyspora halophila is a rare example of a heterotrophic eubacterium producing betaine from simple carbon sources . A . halophila synthesized remarkably high intracellular concentrations of betaine . The highest betaine concentration, determined at 24% (w/v) NaCl, was 33% of the cellular dry weight . Trehalose was synthesized as a compatible solute, accounting for up to 9.7% of the cellular dry weight . The betaine concentration was shown to increase with increasing NaCl concentration, whereas the trehalose concentration was highest at the lowest NaCl concentration used (15% w/v) . A . halophila was capable of accumulating betaine from the medium, while at the same time betaine was also excreted back into the medium by the cells . Along with the de novo synthesis of betaine, A . halophila was able to take up choline from the medium and oxidize it to betaine . Some basic characteristics of the choline oxidation system are described . Choline was oxidized to betaine aldehyde in a reaction in which H2O2 generation and oxygen consumption were coupled . Betaine aldehyde was also oxidized, but with lesser efficiency . In addition, betaine aldehyde was oxidized further to betaine in a reaction in which NAD(P)+ was reduced.

Arch Microbiol, 2001 Oct, 176(4), 243 - 54
True marine and halophilic anoxygenic phototrophic bacteria; Imhoff JF; Anoxygenic phototrophic bacteria are widely distributed in marine sediments and shallow waters of the coastal zone, where they often form intensely colored mass developments . The phototrophic bacteria have adapted to the whole spectrum of salt concentrations, from freshwater to saturated brines, and it is apparent that individual species have adapted well to particular habitats and mineral salts compositions, both qualitatively and quantitatively . This adaptation is reflected not only in the demand for defined ranges of salt concentrations, but also in the phylogenetic relationships of these bacteria, as established by 16S rDNA sequences . Major phylogenetic branches of purple sulfur bacteria are represented by: (1) marine and extremely halophilic Ectothiorhodospiraceae, (2) truly marine and halophilic Chromatiaceae and (3) freshwater Chromatiaceae, some of which are tolerant to low salt concentrations and are successful competitors in brackish and marine habitats . Quite similarly, salt-dependent green sulfur bacteria form distinct phylogenetic lines . In addition, also among the phototrophic alpha-Proteobacteria (purple nonsulfur bacteria), distinct phylogenetic lines of salt-dependent species are recognized . Available data give rise to the assumption that salt concentrations of natural habitats are an important selective factor that determines the development of a selected range of phototrophic bacteria in an exclusive way . As a consequence, the salt responses of these bacteria are reflected in their phylogenetic relationships.

J Org Chem, 1997 Nov 14, 62(23), 8049 - 8057
Intramolecular Geminal and Vicinal Element Effects in Substitution of Simple Bromo(chloro)alkenes by Methoxide and Thiolate Ions . An Example of a Single Step Substitution?
Beit-Yannai M, Rappoport Z, Shainyan BA, Danilevich YS.
Intramolecular element effects k(Br)/k(Cl) for substitution of geminal bromochloroalkenes BrC(Cl)=C(Br)Cl (1), BrC(Cl)=CCl(2) (2), Me(2)C=C(Br)Cl (3), and XCH=C(Br)Cl (X = Cl, 4; X = Br, 5), with MeO(-) and RS(-) nucleophiles were investigated . 3 did not give substitution, and 4 and 5 gave substitution with MeO(-) via an initial elimination (to acetylene)-addition route, followed by further reactions . In reactions of 4 with thiolates, geminal element effects of 2-10 were obtained . Formation of RSC(Cl)=C(Cl)Y, Y = SR, Br, is ascribed to an initial halophilic reaction, followed by addition of RSCl to the formed acetylene . Reaction of 2 with MeO(-) gave a high vicinal element effect, and RS(-) gave a high geminal element effect . Reaction of 1 with both MeO(-) and RS(-) ions gave high (2 orders of magnitude) geminal element effects, which were interpreted as indicating a rate-determining C-X bond cleavage . This is supported by the high k(Br)/k(Cl) intermolecular element effects (k(1)/k(Cl(2)C=CCl(2)) with MeO(-) and PhCH(2)S(-) ions . Mechanistic alternatives based on these observations are discussed.

Inorg Chem, 1997 Oct 8, 36(21), 4798 - 4803
1H and (13)C NMR Studies of an Oxidized HiPIP; Bertini I et al.; 1H-(13)C HETCOR NMR spectra have been recorded for the oxidized HiPIP I from Ectothiorhodospira halophila for which an extended (1)H assignment was available . The hyperfine shifts of the alpha and beta carbons of the coordinated cysteines, as well as those of their attached protons, have been discussed in terms of the current magnetic coupling models and of the mechanisms of spin density delocalization . Through HSQC spectra preceded by a proton 180 degrees pulse, the nonselective T(1) values of the protons have been accurately obtained . It is shown how the nuclear T(1) values can be used as constraints, together with NOEs, for solution structure determination even when the present magnetic coupling scheme occurs . The oxidized cluster is shown to have an effective relaxation time much shorter than that in the reduced state.

Int J Syst Evol Microbiol, 2001 Sep, 51(Pt 5), 1625 - 32
Halomonas maura sp . nov., a novel moderately halophilic, exopolysaccharide-producing bacterium; Bouchotroch S et al.; Four moderately halophilic, exopolysaccharide-producing bacterial strains isolated from soil samples collected from a saltern at Asilah (Morocco) are reported . These four strains were initially considered to belong to the genus Halomonas . Their DNA G+C contents varied between 62.2 and 64.1 mol % . DNA-DNA hybridization revealed a considerable degree of DNA-DNA similarity amongst all four strains (75.5-80.8%) . Nevertheless, similarity with the reference strains of phylogenetically close relatives was lower than 40% . 16S rRNA gene sequences were compared with those of other species of Halomonas and other gram-negative bacteria and they were sufficiently distinct phylogenetically from other recognized Halomonas species to warrant their designation as a novel species . The name Halomonas maura sp . nov . is therefore proposed, with strain S-31T (= CECT 5298T= DSM 13445T) as the type strain . The fatty acid composition of strain S-31T revealed the presence of 18:1omega7c, 16:1omega7c/2-OH i15:0 and 16:0 as the major components . Growth rate analysis showed that strain S-31T had specific cationic requirements for Na+ and Mg2+.

J Bacteriol, 2001 Nov, 183(21), 6365 - 71
An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli; Jung KH et al.; Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis, alter their swimming behavior by phototaxis responses to changes in light intensity and color using visual pigment-like sensory rhodopsins (SRs) . In N . pharaonis, SRII (NpSRII) mediates photorepellent responses through its transducer protein, NpHtrII . Here we report the expression of fusions of NpSRII and NpHtrII and fusion hybrids with eubacterial cytoplasmic domains and analyze their function in vivo in haloarchaea and in eubacteria . A fusion in which the C terminus of NpSRII is connected by a short flexible linker to NpHtrII is active in phototaxis signaling for H . salinarum, showing that the fusion does not inhibit functional receptor-transducer interactions . We replaced the cytoplasmic portions of this fusion protein with the cytoplasmic domains of Tar and Tsr, chemotaxis transducers from enteric eubacteria . Purification of the fusion protein from H . salinarum and Tar fusion chimera from Escherichia coli membranes shows that the proteins are not cleaved and exhibit absorption spectra characteristic of wild-type membranes . Their photochemical reaction cycles in H . salinarum and E . coli membranes, respectively, are similar to those of native NpSRII in N . pharaonis . These fusion chimeras mediate retinal-dependent phototaxis responses by Escherichia coli, establishing that the nine-helix membrane portion of the receptor-transducer complex is a modular functional unit able to signal in heterologous membranes . This result confirms a current model for SR-Htr signal transduction in which the Htr transducers are proposed to interact physically and functionally with their cognate sensory rhodopsins via helix-helix contacts between their transmembrane segments.

Genome Res, 2001 Oct, 11(10), 1641 - 50
Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence; Kennedy SP et al.; The genome of the halophilic archaeon Halobacterium sp . NRC-1 and predicted proteome have been analyzed by computational methods and reveal characteristics relevant to life in an extreme environment distinguished by hypersalinity and high solar radiation: (1) The proteome is highly acidic, with a median pI of 4.9 and mostly lacking basic proteins . This characteristic correlates with high surface negative charge, determined through homology modeling, as the major adaptive mechanism of halophilic proteins to function in nearly saturating salinity . (2) Codon usage displays the expected GC bias in the wobble position and is consistent with a highly acidic proteome . (3) Distinct genomic domains of NRC-1 with bacterial character are apparent by whole proteome BLAST analysis, including two gene clusters coding for a bacterial-type aerobic respiratory chain . This result indicates that the capacity of halophiles for aerobic respiration may have been acquired through lateral gene transfer . (4) Two regions of the large chromosome were found with relatively lower GC composition and overrepresentation of IS elements, similar to the minichromosomes . These IS-element-rich regions of the genome may serve to exchange DNA between the three replicons and promote genome evolution . (5) GC-skew analysis showed evidence for the existence of two replication origins in the large chromosome . This finding and the occurrence of multiple chromosomes indicate a dynamic genome organization with eukaryotic character.

Biol Sci Space, 2000 Dec, 14(4), 323 - 31
{Microbes on the edge of global biosphere}; Naganuma T; The search for life on the edge of global biosphere is a frontier to bridge conventional bio/ecology and exo/astrobiology . This communication reviews the foci of microbiological studies on the inhabitants of the selected "edges", i.e., deep-sea, deep subsurface and Antarctic habitats . The deep-sea is characterized as the no-light (non-photosynthetic) habitat, and the primary production is mostly due to the chemosynthetic autotrophy at the hydrothermal vents and methane-rich seeps . Formation of the chemosynthesis-dependent animal communities in the deep leads to the idea that such communities may be found in "ocean" of the Jovian satellite, Europa . The oxygen minimal layer (OML) in mid-water provides another field of deep-sea research . Modern OML is a relatively thin layer, found between the water depth of 200 and 1000 m, but was much thicker during the periods of oceanic anoxia events (OAEs) in the past . The history of oceanic biosphere is regarded as the cycle of OAE and non-OAE periods, and the remnants of the past OAEs may be seen in the modem OML . Anoxic (no-O2) condition is also characteristic of deep subsurface biosphere . Microorganisms in deep subsurface biosphere exploit every available oxidant, or terminal electron acceptor (TEA), for anaerobic respiration . Sulfate, nitrate, iron (III) and CO2 are the representative TEAs in the deep subsurface . Subsurface of hydrothermal vents, or sub-vent biosphere, may house brine (high salt) habitats and halophilic microorganisms . Some sub-vent halophiles were phylogenetically closely similar to the ones found in the Antarctic habitats which are extremely dry by the liophilizing climate . Below the 3000-4000 m-thick glacier on Antarctica, there have been >70 lakes with liquid water located . One of such sub-glacial lakes, Lake Vostok, has been a target of "life in extreme environments" and is about to be drill-penetrated for microbiological studies . These 'microbiological platforms' will provide new knowledge about the diversity and potential of the Earth's life and facilitate the capability of astrobiologial exploration.

FEMS Microbiol Lett, 2001 Sep 25, 203(2), 235 - 9
Na+-driven multidrug efflux pump VcmA from Vibrio cholerae non-O1, a non-halophilic bacterium; Huda MN et al.; A chromosomal DNA fragment from Vibrio cholerae non-O1 containing a drug resistance determinant was cloned and sequenced . The deduced amino acid sequence suggested that the determinant gene encodes a multidrug efflux pump . We designated the pump VcmA . Escherichia coli cells transformed with a plasmid carrying the vcmA gene showed increased resistance against norfloxacin, ciprofloxacin, ofloxacin, daunomycin, doxorubicin, streptomycin, kanamycin, ethidium bromide, 4',6-diamidino-2-phenylindole dihydrochloride, Hoechst 33342 and acriflavine . Na+ (or Li+)-dependent efflux of ethidium bromide was detected in transformant cells . Efflux of Na+, elicited by ethidium bromide, was observed from transformant cells . Thus, we concluded that the VcmA is a Na+/drug antiporter.

Appl Environ Microbiol, 2001 Oct, 67(10), 4891 - 5
Detection of indigenous Halobacillus populations in damaged ancient wall paintings and building materials: molecular monitoring and cultivation; Pinar G et al.; Several moderately halophilic gram-positive, spore-forming bacteria have been isolated by conventional enrichment cultures from damaged medieval wall paintings and building materials . Enrichment and isolation were monitored by denaturing gradient gel electrophoresis and fluorescent in situ hybridization . 16S ribosomal DNA analysis showed that the bacteria are most closely related to Halobacillus litoralis . DNA-DNA reassociation experiments identified the isolates as a population of hitherto unknown Halobacillus species.

Biophys J, 2001 Oct, 81(4), 1868 - 80
Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents; Solovyova A et al.; We have investigated the potential of sedimentation velocity analytical ultracentrifugation for the measurement of the second virial coefficients of proteins, with the goal of developing a method that allows efficient screening of different solvent conditions . This may be useful for the study of protein crystallization . Macromolecular concentration distributions were modeled using the Lamm equation with the approximation of linear concentration dependencies of the diffusion constant, D = D(o) (1 + k(D)c), and the reciprocal sedimentation coefficient s = s(o)/(1 + k(s)c) . We have studied model distributions for their information content with respect to the particle and its non-ideal behavior, developed a strategy for their analysis by direct boundary modeling, and applied it to data from sedimentation velocity experiments on halophilic malate dehydrogenase in complex aqueous solvents containing sodium chloride and 2-methyl-2,4-pentanediol, including conditions near phase separation . Using global modeling for three sets of data obtained at three different protein concentrations, very good estimates for k(s) and s degrees and also for D degrees and the buoyant molar mass were obtained . It was also possible to obtain good estimates for k(D) and the second virial coefficients . Modeling of sedimentation velocity profiles with the non-ideal Lamm equation appears as a good technique to investigate weak inter-particle interactions in complex solvents and also to extrapolate the ideal behavior of the particle.

Meteorit Planet Sci, 1998 Jul, 33(4), 813 - 9
Survival strategies for microorganisms in hypersaline environments and their relevance to life on early Mars; Litchfield CD; There are two groups of microorganisms that live and grow in hypersaline (>10-15% NaCl) environments: the halophilic Archaea and the halotolerant Bacteria and algae . In order to grow and reproduce in such high-salt, low-water activity environments, these organisms have made basic biochemical adaptations in their proteins, osmoregulation mechanisms, nucleic acids, and lipids . The environment of the halophiles and especially how the halophilic Archaea have adapted to that environment are reviewed in this paper . Along with this review is a brief description of how these adaptations could be important in the detection of life on early Mars assuming similar types of salts and a carbon-based life.

Syst Appl Microbiol, 1988, 10, 211 - 4
The ribosomal gene spacer region in archaebacteria; Achenbach-Richter L et al.; Sequences for the spacer regions that separate the 16S and 23S ribosomal RNA genes have been determined for four more (strategically placed) archaebacteria . These confirm the general rule that methanogens and extreme halophiles have spacers that contain a single tRNAala gene, while tRNA genes are not found in the spacer region of the true extreme thermophiles . The present study also shows that the spacer regions from the sulfate reducing Archaeglobus and the extreme thermophile Thermococcus (both of which cluster phylogenetically with the methanogens and extreme halophiles) contain each a tRNAala gene . Thus, not only all methanogens and extreme halophiles show this characteristic, but all organisms on the "methanogen branch" of the archaebacterial tree appear to do so . The finding of a tRNA gene in the spacer region of the extreme thermophile Thermococcus celer is the first known phenotypic property that links this organism with its phylogenetic counterparts, the methanogens, rather than with its phenotypic counterparts, the sulfur-dependent extreme thermophiles.

Syst Appl Microbiol, 1987, 9, 6 - 8
Polar lipid composition of a new halobacterium; Tindall BJ et al.; Investigations of the polar lipid composition of a new aerobic, extremely halophilic aracheabacterium capable of nitrate reduction have shown that this organism contains two previously unknown phospholycolipids derived from diphytanyl glycerol diethers . Comparison of the lipid pattern from this new isolate with other known strains indicate that this organism is novel . On the basis of the unique polar lipid pattern it can be concluded that this organism represents a new taxon, at least at the species level.

Syst Appl Microbiol, 1986, 7, 194 - 7
Characteristic archaebacterial 16S rRNA oligonucleotides; McGill TJ et al.; A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides . This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures . This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified . Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures . Signatures that would establish between group relationships were also sought and found . In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae . This inclusion is in fact supported by six strong signature oligonucleotides . However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae . Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply . The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria . This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

Syst Appl Microbiol, 1986, 7, 161 - 77
Archaebacterial phylogeny: perspectives on the urkingdoms; Woese CR et al.; Comparisons of complete 16S ribosomal RNA sequences have been used to confirm, refine and extend earlier concepts of archaebacterial phylogeny . The archaebacteria fall naturally into two major branches or divisions, I--the sulfur-dependent thermophilic archaebacteria, and II--the methanogenic archaebacteria and their relatives . Division I comprises a relatively closely related and phenotypically homogeneous collection of thermophilic sulfur-dependent species--encompassing the genera Sulfolobus, Thermoproteus, Pyrodictium and Desulfurococcus . The organisms of Division II, however, form a less compact grouping phylogenetically, and are also more diverse in phenotype . All three of the (major) methanogen groups are found in Division II, as are the extreme halophiles and two types of thermoacidophiles, Thermoplasma acidophilum and Thermococcus celer . This last species branches sufficiently deeply in the Division II line that it might be considered to represent a separate, third Division . However, both the extreme halophiles and Tp . acidophilum branch within the cluster of methanogens . The extreme halophiles are specifically related to the Methanomicrobiales, to the exclusion of both the Methanococcales and the Methanobacteriales . Tp . acidophilum is peripherally related to the halophile-Methanomicrobiales group . By 16S rRNA sequence measure the archaebacteria constitute a phylogenetically coherent grouping (clade), which excludes both the eubacteria and the eukaryotes--a conclusion that is supported by other sequence evidence as well . Alternative proposals for archaebacterial phylogeny, not based upon sequence evidence, are discussed and evaluated . In particular, proposals to rename (reclassify) various subgroups of the archaebacteria as new kingdoms are found wanting, for both their lack of proper experimental support and the taxonomic confusion they introduce.

Syst Appl Microbiol, 1984, 5, 97 - 105
The phylogenetic relationships of three sulfur dependent archaebacteria; Woese CR et al.; Oligonucleotide catalogs have been determined for the 16S ribosomal RNAs of three sulfur dependent (i.e . "thermoacidophilic") archaebacteria--Sulfolobus acidocaldarius, S . solfataricus, and Thermoproteus tenax . The three form a group specifically related to one another, but are only distantly related to the other archaebacteria--i.e . the group comprising the methanogens, extreme halophiles, and (peripherially) the genus Thermoplasma . The three catalogs exhibit two features unique among bacteria: (1) an unusually high number of long pyrimidine runs, and (2) a remarkably high number of (post-transcriptionally) modified nucleotides.

Nature, 1987 May 28, 327(6120), 348 - 9
A possible biochemical missing link among archaebacteria; Achenbach-Richter L et al.; Until recently all archaebacteria isolated conformed to one of three basic phenotypes: they were either methanogens, extreme halophiles, or ('sulphur-dependent') extreme thermophiles . However, a novel phenotype, that fits none of these categories, has recently been described . The organism, strain VC-16 (tentatively called "Archaeoglobus fulgidus") reduces sulphate--the only archaebacterium so far known to do so--and makes very small quantities of methane, although it lacks some of the cofactors normally associated with methanogenesis . These characteristics suggest that strain VC-16 might represent a transition form between an anaerobic thermophilic sulfur-based type of metabolism (which seems to be the ancestral metabolism for archaebacteria and methanogenesis (which somehow then derives from it) . We here show that the lineage represented by strain VC-16 arises from the archaebacterial tree precisely where such an interpretation would predict that it would, between the Methanococcus lineage (which is the deepest of the methanogen branchings) and that of Thermococcus (the deepest of all branchings on the methanogen side of the tree).

Curr Microbiol, 1985, 12, 69 - 72
Widespread distribution of a 7S RNA in archaebacteria; Luehrsen KR et al.; An RNA of nonribosomal origin was found in the extreme halophilic bacteria . This novel small RNA was found to be a homogeneous species by RNA fingerprinting . Analysis of the ribonuclease T1 oligonucleotides gave no evidence of the presence of posttranscriptional modifications . Comparisons of electrophoretic mobility with other RNAs of known size suggest that this is a 7S RNA containing 325-375 nucleotides . An RNA of similar mobility was found in all major divisions of the archaebacteria . Insufficient sequence information is available to determine whether these RNAs are homologs of any other known small RNA.

Geochim Cosmochim Acta, 1992 Sep, 56(9), 3469 - 79
Diphytanyl glycerol ether distributions in sediments of the Orca Basin; Pease TK et al.; Archaebacterially produced diphytanyl glycerol ether (DPGE) was examined in core sediments from the Orca Basin, an anoxic hypersaline basin in the northwestern Gulf of Mexico, to observe its spatial variability and potential origin . A differential extraction protocol was employed to quantify the isopranyl glycerol ethers associated with unbound, intermediate-bound, and kerogen-bound lipid fractions . Archaebacterial lipids were evident at all depths for the unbound and intermediate-bound fractions . Concentrations of DPGE ranged from 0.51 to 2.91 micrograms/g dry sediment at the surface and showed secondary maxima deeper in basin sediments . Intermediate-bound DPGE concentrations exhibited an inverse relationship to unbound DPGE concentrations . Kerogen-bound DPGE concentrations were normally below detection limits . Earlier studies describing the general homogeneity of lipid components within the overlying brine and at the brine/seawater interface suggest that the large-scale sedimentary DPGE variations observed in this study result from spatial and temporal variations in in situ production by methanogenic or extremely halophilic archaebacteria.

Arch Microbiol, 1988, 148, 365 - 71
An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria; Jensen RA et al.; The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase . Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns . A new extreme-halophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria . In addition to the conventional sensitivity to feedback inhibition by L-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors . Thus, L-tryptophan inhibited activity while L-tyrosine, L-methionine, L-leucine and L-isoleucine activated the enzyme . L-Isoleucine and L-phenylalanine were effective at micromolar levels; other effectors operated at mM levels . A regulatory mutant selected for resistance to growth inhibition caused by beta-2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished . Inhibition by L-tryptophan was also lost, and activation by allosteric activators was diminished . Not only was sensitivity to feedback inhibition by L-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis) . It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster.

Int J Syst Bacteriol, 1988 Apr, 38(2), 139 - 42
Methanohalophilus zhilinae sp . nov., an alkaliphilic, halophilic, methylotrophic methanogen; Mathrani IM et al.; Methanohalophilus zhilinae, a new alkaliphilic, halophilic, methylotrophic species of methanogenic bacteria, is described . Strain WeN5T (T = type strain) from Bosa Lake of the Wadi el Natrun in Egypt was designated the type strain and was further characterized . This strain was nonmotile, able to catabolize dimethylsulfide, and able to grow in medium with a methyl group-containing substrate (such as methanol or trimethylamine) as the sole organic compound added . Sulfide (21 mM) inhibited cultures growing on trimethylamine . The antibiotic susceptibility pattern of strain WeN5T was typical of the pattern for archaeobacteria, and the guanine-plus-cytosine content of the deoxyribonucleic acid was 38 mol% . Characterization of the 16S ribosomal ribonucleic acid sequence indicated that strain WeN5T is phylogenetically distinct from members of previously described genera other than Methanohalophilus and supported the partition of halophilic methanogens into their own genus.

Syst Appl Microbiol, 1991, 14, 358 - 63
The sequence of Methanospirillum hungatei 23S rRNA confirms the specific relationship between the extreme halophiles and the Methanomicrobiales; Burggraf S et al.; We have determined the sequence of the 23S rRNA from the methanogenic archaeon Methanospirillum hungatei . This is the first such sequence from a member of the Methanomicrobiales . Moreover, it brings additional evidence to bear on the possible specific relationship between this particular group of methanogens and the extreme halophiles . Such evidence is critical in that several new (and relatively untested) methods of phylogenetic inference have lead to the controversial conclusion that the extreme halophiles are either not related to the archaea, or are only peripherally so . Analysis of the Methanospirillum hungatei 23S rRNA sequence shows the Methanomicrobiales are indeed a sister group of the extreme halophiles, further strengthening the conclusions reached from analysis of 16S rRNA sequences.

Syst Appl Microbiol, 1991, 14(4), 364 - 71
Archaeal phylogeny: reexamination of the phylogenetic position of Archaeoglobus fulgidus in light of certain composition-induced artifacts; Woese CR et al.; A major and too little recognized source of artifact in phylogenetic analysis of molecular sequence data is compositional difference among sequences . The problem becomes particularly acute when alignments contain ribosomal RNAs from both mesophilic and thermophilic species . Among prokaryotes the latter are considerably higher in G + C content than the former, which often results in artificial clustering of thermophilic lineages and their being placed artificially deep in phylogenetic trees . In this communication we review archaeal phylogeny in the light of this consideration, focusing in particular on the phylogenetic position of the sulfate reducing species Archaeoglobus fulgidus, using both 16S rRNA and 23S rRNA sequences . The analysis shows clearly that the previously reported deep branching of the A . fulgidus lineage (very near the base of the euryarchaeal side of the archaeal tree) is incorrect, and that the lineage actually groups with a previously recognized unit that comprises the Methanomicrobiales and extreme halophiles.

Adv Space Res, 1986, 6(12), 299 - 306
Survival strategies of microorganisms in extreme saline environments; Imhoff JF; Halophilic representatives are found in all main lines of evolutionary descendence of microbes: in archaebacteria, Gram-negative and Gram-positive eubacteria, and also in eucaryotes . In principle all halophilic microorganisms have to adapt their surface and membrane structures to their highly ionic environments . Concerning their intracellular compartment two different strategies have been developed: Inorganic ions are largely excluded in some microorganisms while such ions are actively accumulated in others . In particular the second group of organisms has to adapt the whole metabolic machinery to the highly ionic conditions of several molar salts, whereas in the first group only the outer surface of the cytoplasmic membrane and the extracytoplasmic structures are in contact with high concentrations of inorgainic ions . In this latter group, a variety of organic solutes is accumulated in response to increases of the salinity of the environment.

Sci Am, 1997 Apr, 276(4), 82 - 7
Extremophiles; Madigan MT et al.; NASA: The authors examine the presence of bacteria in extreme climates and their role in biotechnology . Within the past 30 years, scientists have discovered bacteria in areas long thought to be sterile due to extremes in heat, cold, or pH . Enzymes from these bacteria are used in many areas of industry . Examples discussed include the use of enzymes from thermophilic bacteria for polymerase chain reactions, use of enzymes in detergents, and the use of halophiles to enhance extraction of crude oil . Methods of harvesting extremozymes are discussed .

Orig Life Evol Biosph, 1988, 18(1-2), 59 - 64
Archaebacterial class I and class II aldolases from extreme halophiles; Altekar W et al.; Both, class I (Schiff-base forming) and class II (metal requiring) fructose biphosphate aldolases were found to be distributed among halophilic archaebacteria . The aldolase activity from Halobacteriium halobium, H . salinarium, H . cutirubrum, H . mediterranei and H . volcanii exhibited properties of a bacterial class II aldolase as it was metal-dependent for activity and therefore inhibited by EDTA . In contrast, aldolase from H . saccharovorum, Halobacterium R-113, H . vallismortis and Halobacterium CH-1 formed a Schiff-base intermediate with the substrate and therefore resembled to eukaryotic class I type . The type of aldolase did not vary by changes in the growth medium.

Orig Life Evol Biosph, 1993 Feb, 23(1), 53 - 64
Comparison of membrane ATPases from extreme halophiles isolated from ancient salt deposits; Stan-Lotter H et al.; Halophilic microorganisms were isolated from Triassic and Permian salt deposits . Two were rods and grew as red colonies; another was a coccus and produced pink colonies . The rods lysed in solutions that lacked added sodium chloride . Growth of all isolates was inhibited by aphidicolin and their bulk proteins were acidic as judged from isoelectric focusing . Therefore, these organisms were tentatively identified as extreme halophiles . Whole cell proteins patterns of the isolates following gel electrophoresis were distinct and differed from those of representative type strains of halophilic bacteria . The membrane ATPases from the rods were similar to the enzyme from Halobacterium saccharovorum with respect to subunit composition, enzymatic properties and immunological cross-reaction, but differed slightly in amino acid composition . If the age of the microbial isolated is similar to that of the salt deposits, they can be considered repositories of molecular information of great evolutionary interest.

Int J Syst Bacteriol, 1990 Apr, 40(2), 209 - 10
Haloarcula marismortui (Volcani) sp . nov., nom . rev., an extremely halophilic bacterium from the Dead Sea; Oren A et al.; An extremely halophilic red archaebacterium isolated from the Dead Sea (Ginzburg et al., J . Gen . Physiol . 55: 187-207, 1970) belongs to the genus Haloarcula and differs sufficiently from the previously described species of the genus to be designated a new species; we propose the name Haloarcula marismortui (Volcani) sp . nov., nom . rev . because of the close resemblance of this organism to "Halobacterium marismortui," which was first described by Volcani in 1940 . The type strain is strain ATCC 43049.

Lakartidningen, 2001 Aug 8, 98(32-33), 3400 - 2
{Vibrio vulnificus . A marine bacterium with lethal potential}; Melhus A et al.; Vibrio vulnificus is a halophilic gram-negative rod widespread in the aquatic environment and associated with primary septicemia and severe wound infections . The first Swedish case was reported in 1994 . Ever since, sporadic cases have occurred in the south of Sweden whenever the coastal water temperature has exceeded 20 degrees C . Critical for a successful outcome in these infections has been early diagnosis with appropriate antibiotic and surgical treatment . A review of this subject was prompted by two cases of fulminant septicemia, which both presented themselves as atypical erysipelas.

Int J Antimicrob Agents, 2001 Aug, 18(2), 135 - 40
Antimicrobial susceptibility of potentially pathogenic halophilic vibrios isolated from seafood; Ottaviani D et al.; Susceptibility patterns to 27 antimicrobial agents and beta-lactamase production were investigated in potentially pathogenic halophilic vibrios from seafood . The effect of salinity on the response to the drugs in vitro was also studied . All isolates were uniformly sensitive to choramphenicol, imipenem, meropenem but resistant to lincomycin . All were highly sensitive to oxolinic acid, trimethoprim-sulphamethoxazole, doxycycline, flumequine, cefotaxime, nalidixic acid and ciprofloxacin . Some strains of V . harveyi, V . alginolyticus and V . parahaemolyticus apparently had mechanisms of resistance to several beta-lactam antibiotics other than by the production of beta-lactamases . Sixty-nine strains produced penicillinase but a low correlation between beta-lactamase activity and resistance to beta-lactam antibiotics was noted . The salt concentration affected the in vitro susceptibility of halophilic vibrios and the effect of salinity depended on both the individual strains and the antimicrobial tested.

Biochim Biophys Acta, 2001 Aug 30, 1520(2), 154 - 62
Sequencing, phylogenetic and transcriptional analysis of the glyoxylate bypass operon (ace) in the halophilic archaeon Haloferax volcanii; Serrano JA et al.; The glyoxylate cycle occurs in the three domains of living organisms: Eukarya, Bacteria and Archaea . We have isolated and sequenced the ace (acetate assimilation) gene operon, comprising the glyoxylate cycle key enzymes isocitrate lyase and malate synthase genes (icl or aceA and ms or aceB), from the halophilic archaeon Haloferax volcanii . This is the first time that these genes are sequenced in an organism from the domain Archaea . Phylogenetic analysis of the sequenced genes revealed that isocitrate lyase shows a significant identity with isocitrate lyases from Eukarya and Bacteria, but it is not more closely related to eukaryal or bacterial enzymes, and that malate synthase from H . volcanii has very little identity with any other known protein . This enzyme forms a new class of malate synthases . Transcriptional analysis indicated that both genes are cotranscribed in a single 2.7 kb mRNA molecule . The genes were transcribed only when acetate was the carbon source, indicating transcriptional regulation . Two sets of palindromic sequences were found in the promoter region, possibly involved in binding of transcriptional regulators (repressors and/or activators).

Chemosphere, 2001 Aug, 44(5), 1145 - 51
Photo-oxidation of biodegraded crude oil and toxicity of the photo-oxidized products; Maki H et al.; We investigated the physicochemical changes resulting from irradiation by sunlight of biodegraded crude oil . An Arabian light crude oil sample was first subjected to microbial degradation . n-Alkanes and aromatic compounds such as naphthalenes, fluorenes, dibenzothiophenes and phenanthrenes possessing short, alkyl side chain(s) were almost completely degraded, while the contents of the saturated and aromatic fractions were reduced by 70% and 40%, respectively . This biodegraded oil was then suspended in seawater and exposed to sunlight irradiation for several weeks . The most remarkable change caused by the irradiation was a substantial decline in the aromatic fraction with a concomitant increase in the resin and asphaltene fractions . A 13C-nuclear magnetic resonance (NMR) spectroscopic analysis showed that the aromaticity of the biodegraded oil was significantly lower in the irradiated sample . A field desorption-mass spectrometric (FD-MS) analysis showed that sunlight irradiation reduced the average molecular weight of the oil components and formed oxygenated compounds . Consistent with this observation is that the oxygen content in the oil increased as the irradiation was prolonged . The bioavailability of the biodegraded oil was increased by the photo-oxidation: the growth of seawater microbes was minimal when the non-irradiated biodegraded oil was used as the source of carbon and energy; however, growth was significant when irradiated biodegraded oil was used . The concentration of dissolved organic carbon (DOC) increased linearly during the sunlight irradiation of the biodegraded oil, and this increase was matched by an increase in ultraviolet-absorptive materials in the seawater . The photochemically formed, water-soluble fraction (WSF) showed acute toxicity against the halophilic crustacean, Artemia.

Biometals, 2001 Jun, 14(2), 135 - 42
Cloning and expression of the ferredoxin gene from extremely halophilic archaeon Haloarculajaponica strain TR-1; Matsuo T et al.; The gene encoding a ferredoxin (Fd) from Haloarculajaponica strain TR-1 was cloned and sequenced . Sequence analysis of the cloned Ha . japonica Fd gene revealed that the structural gene consisted of an open reading frame of 387 nucleotides encoding 129 amino acids . The deduced amino acid sequence of Ha . japonica Fd showed 84 to 98% identity with corresponding sequences in other extremely halophilic archaea . The Ha . japonica Fd gene was inserted into the shuttle vector pWL102 and used to transform Ha . japonica . Ha . japonica Fd could then be produced as a fusion with His-Tag (6xHis) in Ha . japonica host cells . The absorption and ESR spectra of the Fd/His Tag fusion protein revealed the presence of a {2Fe-2S} cluster which is characteristic of native Ha . japonica Fd.

Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1457 - 62
Chromohalobacter salexigens sp . nov., a moderately halophilic species that includes Halomonas elongata DSM 3043 and ATCC 33174; Arahal DR et al.; Two strains that were originally isolated and characterized as members of the moderately halophilic species Halomonas elongata, strains DSM 3043 (= 1H11) and ATCC 33174 (= 1H15), were studied in detail . Their complete 16S rRNA sequences were determined and, when compared to sequences available from the databases, they showed a close phylogenetic relationship to Chromohalobacter marismortui . In addition, DNA-DNA hybridization experiments showed that both strains are members of the same species, but their DNA relatedness to the type strains of Halomonas elongata, ATCC 33173T, and Chromohalobacter marismortui, ATCC 17056T, is very low . Phenotypically, the two strains showed very similar features, related to those of Chromohalobacter, but clear differences were found between these two strains and Chromohalobacter marismortui . On the basis of these data, it is proposed that Halomonas elongata DSM 3043 and ATCC 33174 should be included in a new species of the genus Chromohalobacter, Chromohalobacter salexigens sp . nov . The type strain is DSM 3043T (= ATCC BAA-138T = CECT 5384T = CCM4921T = CIP106854T = NCIMB 13768T).

Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1443 - 8
Transfer of Halomonas canadensis and Halomonas israelensis to the genus Chromohalobacter as Chromohalobacter canadensis comb . nov . and Chromohalobacter israelensis comb . nov.; Arahal DR et al.; 16S rRNA gene sequence comparisons and DNA-DNA hybridization data support the conclusion that two species previously described as members of the genus Halomonas, Halomonas israelensis and Halomonas canadensis, should be placed in the genus Chromohalobacter . Both H . israelensis ATCC 43985T (= Ba1T) and H . canadensis ATCC 43984T (= NRCC 41227T) have been used extensively for physiological studies for many years; nevertheless, they were not named and classified taxonomically until quite recently . Their phenotypic resemblance (at least 65% Jaccard similarity) to some members of the genus Halomonas and the degree of DNA-DNA relatedness (lower than 60%) to other described species of this genus permitted the conclusion that they were distinct species belonging to the genus Halomonas . In this study, the 16S rDNA of both species has been sequenced completely and found to share higher similarity to the available sequences of the moderately halophilic bacterium Chromohalobacter marismortui than to sequences of members of the genus Halomonas . C . marismortui is the sole species of the genus Chromohalobacter, also included in the family Halomonadaceae, and shares many phenotypic features with H . canadensis and H . israelensis . It is proposed that the two species should be renamed as Chromohalobacter canadensis comb . nov . and Chromohalobacter israelensis comb . nov . An emended description of the genus Chromohalobacter is given in order to include the features of these two species.

Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1283 - 9
Thalassomonas viridans gen . nov., sp . nov., a novel marine gamma-proteobacterium; Macian MC et al.; A new genus and species are proposed for two halophilic, strictly aerobic, chemo-organotrophic, marine bacterial strains . These bacteria are gram-negative, motile rods isolated from oysters cultivated off the Mediterranean coast at Valencia (Spain) . They produce green/blue-green diffusible pigment . The G+C content of the DNA of the proposed type strain (XOM25T) is 48.4 mol % . A 16S rRNA gene sequence analysis of the two strains has shown that the new isolates represent a branch within the gamma-Proteobacteria, close to the genus Colwellia . The type species of the new genus is Thalassomonas viridans gen . nov., sp . nov., with the type strain XOM25T (= CECT 5083T = DSM 13754T).

Eur J Biochem, 2001 Aug, 268(15), 4366 - 73
Post-translational modification of the S-layer glycoprotein occurs following translocation across the plasma membrane of the haloarchaeon Haloferax volcanii; Eichler J; The halophilic archaeon Haloferax volcanii is surrounded by a protein shell solely comprised of the S-layer glycoprotein . While the gene sequence and glycosylation pattern of the protein and indeed the three-dimensional structure of the surface layer formed by the protein have been described, little is known of the biosynthesis of the S-layer glycoprotein . In the following, pulse-chase radiolabeling and cell-fractionation studies were employed to reveal that newly synthesized S-layer glycoprotein undergoes a maturation step following translocation of the protein across the plasma membrane . The processing step, detected as an increase in the apparent molecular mass of the S-layer glycoprotein, is unaffected by inhibition of protein synthesis and is apparently unrelated to glycosylation of the protein . Maturation requires the presence of magnesium ions, involved in membrane association of the S-layer glycoprotein, and results in increased hydrophobicity of the protein as revealed by enhanced detergent binding . Thus, along with protein glycosylation, additional post-translational modifications apparently occur on the external face of the haloarchaeal plasma membrane, the proposed topological homologue of the lumenal face of the eukaryal endoplasmic reticulum membrane.

Appl Environ Microbiol, 2001 Aug, 67(8), 3618 - 29
Distribution of archaea in a black smoker chimney structure; Takai K et al.; Archaeal community structures in microhabitats in a deep-sea hydrothermal vent chimney structure were evaluated through the combined use of culture-independent molecular analyses and enrichment culture methods . A black smoker chimney was obtained from the PACMANUS site in the Manus Basin near Papua New Guinea, and subsamples were obtained from vertical and horizontal sections . The elemental composition of the chimney was analyzed in different subsamples by scanning electron microscopy and energy-dispersive X-ray spectroscopy, indicating that zinc and sulfur were major components while an increased amount of elemental oxygen in exterior materials represented the presence of oxidized materials on the outer surface of the chimney . Terminal restriction fragment length polymorphism analysis revealed that a shift in archaeal ribotype structure occurred in the chimney structure . Through sequencing of ribosomal DNA (rDNA) clones from archaeal rDNA clone libraries, it was demonstrated that the archaeal communities in the chimney structure consisted for the most part of hyperthermophilic members and extreme halophiles and that the distribution of such extremophiles in different microhabitats of the chimney varied . The results of the culture-dependent analysis supported in part the view that changes in archaeal community structures in these microhabitats are associated with the geochemical and physical dynamics in the black smoker chimney.

FEMS Microbiol Lett, 2001 Jul 24, 201(2), 221 - 7
Use of a green fluorescent protein gene as a reporter in Zymomonas mobilis and Halomonas elongata; Douka E et al.; We investigated the applicability of the green fluorescent protein of Aequorea victoria as a reporter for gene expression in the strictly fermentative Gram-negative ethanologenic bacterium Zymomonas mobilis and in the moderately halophilic bacterium Halomonas elongata . We have succeeded to express a mutated gene of green fluorescent protein under the control of different promoters in Z . mobilis and H . elongata grown under various glucose or salt concentrations, respectively . Our results demonstrate that gfp can serve as an easily assayable reporter gene in both organisms . Maximum fluorescence was obtained in Z . mobilis grown aerobically and in H . elongata grown under elevated salt concentration in solid medium . For both bacteria the fluorescence obtained was higher when the gfp gene was placed under the control of a native promoter.

Acta Crystallogr D Biol Crystallogr, 2001 Aug, 57(Pt 8), 1157 - 8 Epub 2001 Jul 23.
Crystallization and preliminary X-ray analysis of catalase-peroxidase from the halophilic archaeon Haloarcula marismortui; Yamada Y et al.; Catalase-peroxidases are bifunctional enzymes found in many microorganisms . Crystals of catalase-peroxidase from the halophilic archaeon Haloarcula marismortui were obtained using the hanging-drop vapour-diffusion method . The rhombic plate-shaped crystals were grown from purified protein solution using (NH(4))(2)SO(4) as precipitant at 293 K . The crystal belongs to the monoclinic system, space group C2, and diffracted beyond 2.0 A resolution.

Extremophiles, 2001 Jun, 5(3), 169 - 75
Isolation of the fibrocrystalline body, a structure present in haloarchaeal species, from Halobacterium salinarum; Alba I et al.; An organized structure, the fibrocrystalline body (FB), has been isolated from the archaeon Halobacterium salinarum . The structure is also present in, and can be isolated from, other extreme halophilic archaea . FB is present in the cytoplasm during the exponential growth and early stationary phases . This structure is affected by vincristine, an antitumoral drug, which targets tubulin . The drug causes fragmentation of the FB, changes in the cell shape, and growth inhibition . Taken together, these results point toward an important role in the life of the cell for this highly organized structure.

FEMS Microbiol Ecol, 2001 Jul, 36(2-3), 123 - 130
The contribution of halophilic Bacteria to the red coloration of saltern crystallizer ponds(1); Oren A et al.; Analysis of the pigments extracted from solar saltern crystallizer ponds in Santa Pola near Alicante and on the Balearic island of Mallorca, Spain, showed that 5-7.5% of the total prokaryotic pigment absorbance could be attributed to a novel carotenoid or carotenoid-like compound . This unidentified pigment was identical to the sole pigment present in Salinibacter ruber, the only described member of a newly discovered genus of red halophilic Bacteria related to the Cytophaga-Flavobacterium-Bacteroides group . On the basis of fluorescence in situ hybridization experiments it has been shown that Salinibacter is an important component of the microbial community of Spanish saltern ponds . The red color of saltern crystallizer ponds may thus not only be due to red halophilic Archaea and to beta-carotene-rich Dunaliella cells as previously assumed, but may contain a bacterial contribution as well . The Salinibacter pigment was not detected in samples collected from crystallizer ponds of the salterns of Eilat, Israel, and only traces of it may have been present in the Newark, CA, USA, salterns . The community structure of the prokaryote community inhabiting saltern crystallizers thus shows significant geographic variations . Polar lipid analyses of the biomass collected from the Santa Pola salterns showed that the total contribution of Salinibacter and other Bacteria to the total biomass was minor, the most important component of the community being halophilic Archaea.

Lett Appl Microbiol, 2001 Jul, 33(1), 61 - 4
Correlation between medium acidification and pathogenicity in environmental halophilic non-cholera vibrios; Ottaviani D et al.; AIMS: The metabolic characterization and pathogenicity of vibrios isolated from seafood were studied . METHODS AND RESULTS: Strains of halophilic vibrios, grown in the presence of 0.5% glucose, induced high medium acidification and were non-culturable after 24 h, while moderately acidifying strains were culturable, produced cytotoxins, and remained lethal when inoculated intraperitoneally in mice . Highly acidifying strains failed to elicit pathogenicity in vivo and in vitro . CONCLUSION: The high acidification of the medium and the self-killing activity of NCVs might be considered a significant phenotypic marker of virulence and/or cytotoxicity . SIGNIFICANCE AND IMPACT OF THE STUDY: We suggest the medium acidification test as possible screening method for pathogenic NCVs in food microbiology.

J Basic Microbiol, 2001, 41(2), 97 - 104
Adenine toxicity and transport in the moderately halophilic eubacterium Halomonas elongata; Perlman AM et al.; The presence of adenine in the L-alanine defined medium substantially inhibited the growth of the moderately halophilic eubacterium Halomonas elongata . Extensive attempts to reverse the adenine toxicity for growth were made using a variety of purine and pyrimidine compounds, vitamins, and amino acids . Of the compounds tested, only cytosine was found to reverse the adenine growth inhibition . This indicates a mechanism similar to that found for some strains of Escherichia coli in which the presence of exogenous purines (e.g . adenine) was found to stop purine de novo synthesis and repress the synthesis of the pyrimidine salvage enzyme cytosine deaminase . H . elongata was found to possess an active adenine uptake system that was sodium dependent with only lithium having a considerable capacity to replace the sodium . A competition study indicated that the adenine transport system was quite specific . This paper represents the initial study of purine and pyrimidine salvage pathways and adenine uptake for the moderately halophilic eubacteria.

FEMS Microbiol Lett, 2001 May 1, 198(2), 111 - 5
Kinetic regulation of an alkaline p-nitrophenylphosphate phosphatase from Halobacterium salinarum in low water system by Mn2+ and monovalent cations; Marhuenda-Egea FC et al.; Reversed micelles were used as a cytoplasmic model to study the effect of the multi-ionic equilibria on kinetics of extreme halophilic enzymes . The enzymatic system used was an alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium) . This enzyme was solubilised in reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant . The p-nitrophenylphosphate phosphatase is a good system to study the regulation of the enzymatic activity, because it utilises manganese, water and potassium or sodium as cofactors and reacts with p-nitrophenylphosphate . Kinetic behaviour was determined by the ratio between {Mn2+} and {Na+} or {K+} . When the {Mn2+} increased and {Na+} or {K+} decreased, the kinetics showed cooperative behaviour . Rabin's model describes the kinetic behaviour of the p-nitrophenylphosphate phosphatase in reversed micelles.

J Nat Prod, 2001 Feb, 64(2), 256 - 9
Characterization of novel methyl-branched chain fatty acids from a halophilic Bacillus species; Carballeira NM et al.; The 4-methylated fatty acids 4,9-dimethyldecanoic, 4,11-dimethyldodecanoic, 4,10-dimethyldodecanoic, and 4,13-dimethyltetradecanoic acid as well as the 2-methylated fatty acids 2,13-dimethyltetradecanoic and 2,12-dimethyltetradecanoic acid were identified for the first time in nature in the halophilic bacterium Bacillus sp . isolated from the salt pans of Burgas in Bulgaria . The principal fatty acids in this bacterium were a series of iso-anteiso fatty acids with chain lengths between C11 and C19, but an interesting series of linear alkylbenzene fatty acids with chain lengths between C10 and C14, such as 12-phenyldodecanoic acid, were also identified . The novel 4-methylated fatty acids were characterized using a combination of GC-MS and chemical transformations such as N-acylpyrrolidide derivatization . The 2-methylated fatty acids were also identified by GC-MS and gas chromatographic coelution with synthetic samples . The novel methyl-branched fatty acids probably originated from the selective incorporation of methylmalonyl-CoA by one of the fatty acid-synthesizing enzymes of the bacterium.

Microbiology, 2001 Jul, 147(Pt 7), 1745 - 54
Use of a halobacterial bgaH reporter gene to analyse the regulation of gene expression in halophilic archaea; Gregor D et al.; The bgaH reading frame encoding a beta-galactosidase of 'Haloferax alicantei' was used as a reporter gene to investigate three different promoter regions derived from gvpA genes of Haloferax mediterranei (mc-gvpA) and Halobacterium salinarum (c-gvpA and p-gvpA) in Haloferax volcanii transformants . The fusion of bgaH at the start codon of each gvpA reading frame (A1-bgaH fusion genes) caused translational problems in some cases . Transformants containing constructs with fusions further downstream in the gvpA reading frame (A-bgaH) produced beta-galactosidase, and colonies on agar plates turned blue when sprayed with X-Gal . The beta-galactosidase activities quantified by standard ONPG assays correlated well with the mRNA data determined with transformants containing the respective gvpA genes: the cA-bgaH fusion gene was completely inactive, the mcA-bgaH transformants showed low amounts of products, whereas the pA-bgaH fusion gene was constitutively expressed in the respective transformants . The transcription of each A-bgaH gene was activated by the homologous transcriptional activator protein GvpE . The cGvpE, pGvpE and mcGvpE proteins were able to activate the promoter of pA-bgaH and mcA-bgaH, whereas the promoter of cA-bgaH was only activated by cGvpE . Among the three GvpE proteins tested, cGvpE appeared to be the strongest transcriptional activator.

Ann Dermatol Venereol, 2001 May, 128(5), 653 - 5
{Necrotizing dermatitis due to Vibrio Vulnificus}; Dieng MT et al.; BACKGROUND: Vibrio vulnificus is a Gram-negative halophilic pathogen for man that can cause septicemia in patients with chronic liver disease . In healthy subjects, infections are generally local or regional . We report two cases of necrotizing dermo-hypodermitis due to Vibrio vulnificus . CASE REPORTS: The first patient was a 21-year-old fisherman who developed extensive necrotizing dermo-hypodermitis 24 hours after a penetrating scratch with a fish bone . The second patient was also a fisherman . This 35-year-old man developed a massive area of necrotic tissue on the left leg, also 48 hours after a penetrating fish bone scratch . Both patients exhibited neutrophil hyperleukocytosis and thrombocytopenia . Bacteriological study of the pus and damaged tissue identified Vibrio vulnificus . Blood cultures were negative . Liver tests and liver ultrasound were normal . The course was favorable in both cases with antibiotic treatment and excision of the necrotic tissue . DISCUSSION: For these two cases of Vibrio vulnificus necrotizing dermo-hypodermitis, the diagnosis was based on epidemiological, clinical and bacteriological arguments . Neither of these patients had liver disease, explaining the limited locoregional nature of the infection . The severity of the skin necrosis and its rapid course are related to the toxins produced by V . vulnificus . Curative medical and surgical care is generally effective in cases without septicemia and prognosis is good . Education of exposed subjects, fishermen and patients with chronic liver disease, should be helpful for prevention.

Appl Environ Microbiol, 2001 Jul, 67(7), 3077 - 85
Microbial diversity of the brine-seawater interface of the Kebrit Deep, Red Sea, studied via 16S rRNA gene sequences and cultivation methods; Eder W et al.; The brine-seawater interface of the Kebrit Deep, northern Red Sea, was investigated for the presence of microorganisms using phylogenetic analysis combined with cultivation methods . Under strictly anaerobic culture conditions, novel halophiles were isolated . The new rod-shaped isolates belong to the halophilic genus Halanaerobium and are the first representatives of the genus obtained from deep-sea, anaerobic brine pools . Within the genus Halanaerobium, they represent new species which grow chemoorganotrophically at NaCl concentrations ranging from 5 to 34% . The cellular fatty acid compositions are consistent with those of other Halanaerobium representatives, showing unusually large amounts of Delta7 and Delta11 16:1 fatty acids . Phylogenetic analysis of the brine-seawater interface sample revealed the presence of various bacterial 16S rRNA gene sequences dominated by cultivated members of the bacterial domain, with the majority affiliated with the genus Halanaerobium . The new Halanaerobium 16S rRNA clone sequences showed the highest similarity (99.9%) to the sequence of isolate KT-8-13 from the Kebrit Deep brine . In this initial survey, our polyphasic approach demonstrates that novel halophiles thrive in the anaerobic, deep-sea brine pool of the Kebrit Deep, Red Sea . They may contribute significantly to the anaerobic degradation of organic matter enriched at the brine-seawater interface.

FEMS Microbiol Lett, 2001 Jun 25, 200(2), 221 - 7
Heterologous overexpression of glucose dehydrogenase from the halophilic archaeon Haloferax mediterranei, an enzyme of the medium chain dehydrogenase/reductase family; Pire C et al.; The first gene encoding a glucose dehydrogenase (GDH) from a halophilic organism has been sequenced . Amino acid sequence alignments of GDH from Haloferax mediterranei show a high degree of homology with the thermoacidophilic GDHs and with other enzymes from the medium chain dehydrogenase/reductase family . Heterologous overexpression using the mesophilic organism Escherichia coli as the host has been performed and the expression product was obtained as inclusion bodies . To obtain the halophilic enzyme in its native form refolding and reactivation in a saline environment were required . A pure and highly concentrated sample of the enzyme was obtained using a purification procedure based on the protein's halophilicity . This method may be useful as a general procedure for purifying other halophilic proteins from mesophilic hosts.

Arch Microbiol, 2001 May, 175(5), 369 - 75
Alkalispirillum mobile gen . nov., spec . nov., an alkaliphilic non-phototrophic member of the Ectothiorhodospiraceae; Rijkenberg MJ et al.; From cultures of the anoxygenic phototroph Halorhodospira halophila SL-1, an aerobic, gram-negative spirillum was isolated . This moderately halophilic, alkaliphilic bacterium was motile by means of a single polar flagellum . It is described here as Alkalispirillum mobile gen . nov., spec . nov . Phylogenetic analysis of the Alkalispirillum mobile 16S rRNA gene led to its classification in the gamma-subclass of the Proteobacteria, as it appears closely related to phototrophic purple sulfur bacteria of the genera Ectothiorhodospira and Halorhodospira . Surprisingly, A . mobile is an obligate aerobe . The organism grows optimally with a number of carboxylic acids (such as sodium acetate) as carbon source, at 2% (i.e . approximately 0.34 M) sodium chloride, at pH 9-10, and at temperatures ranging from 35 to 38 degrees C . The dominant cellular fatty acids of Alkalispirillum mobile are C12:0, C16:0, C18:1cis11, and C18:0; its G+C content is 66.2+/-0.5 mol%.

Arch Microbiol, 2001 May, 175(5), 360 - 8
Mechanisms of acetate formation and acetate activation in halophilic archaea; Brasen C et al.; The halophilic archaea Halococcus (Hc.) saccharolyticus, Haloferax (Hf.) volcanii, and Halorubrum (Hr.) saccharovorum were found to generate acetate during growth on glucose and to utilize acetate as a growth substrate . The mechanisms of acetate formation from acetyl-CoA and of acetate activation to acetyl-CoA were studied . Hc . saccharolyticus, exponentially growing on complex medium with glucose, formed acetate and contained ADP-forming acetyl-CoA synthetase (ADP-ACS) rather than acetate kinase and phosphate acetyltransferase or AMP-forming acetyl-CoA synthetase . In the stationary phase, the excreted acetate was completely consumed, and cells contained AMP-forming acetyl-CoA synthetase (AMP-ACS) and a significantly reduced ADP-ACS activity . Hc . saccharolyticus, grown on acetate as carbon and energy source, contained only AMP-ACS rather than ADP-ACS or acetate kinase . Cell suspensions of Hc . saccharolyticus metabolized acetate only when they contained AMP-ACS activity, i.e., when they were obtained after growth on acetate or from the stationary phase after growth on glucose . Suspensions of exponential glucose-grown cells, containing only ADP-ACS but not AMP-ACS, did not consume acetate . Similar results were obtained for the phylogenetic distantly related halophilic archaea Hf . volcanii and Hf . saccharovorum . We conclude that, in halophilic archaea, the formation of acetate from acetyl-CoA is catalyzed by ADP-ACS, whereas the activation of acetate to acetyl-CoA is mediated by an inducible AMP-ACS.

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 1171 - 7
Halomonas marisflavae sp . nov., a halophilic bacterium isolated from the Yellow Sea in Korea; Yoon JH et al.; A halophilic Gram-negative bacterial strain, SW32T, which was isolated from a sample from the Yellow Sea of Korea, was subjected to a polyphasic taxonomic study . This organism grew optimally in the presence of 0.5-12% NaCl . On the basis of phenotypic and phylogenetic data, strain SW32T appeared to be a member of the genus Halomonas . Strain SW32T formed a distinct evolutionary lineage within the phylogenetic clade comprising Halomonas species and the genera Zymobacter and Carnimonas . The 16S rDNA sequence of strain SW32T contains 19 signature characteristics of the genus Halomonas and the family Halomonadaceae . Strain SW32T possessed a single polar flagellum, ubiquinone-9 as the predominant respiratory lipoquinone and C18:1, C16:0 and C16:1 omega7c and/or iso-C15:0 20H as the major fatty acids . The DNA G+C content was 59 mol% . Levels of 16S rDNA similarity between strain SW32T and the type strains of all validly described Halomonas species were 92.0-93.8% . Strain SW32T exhibited 16S rDNA similarity values of 92.7% to Zymobacter palmae IAM 14233T and 91.6% to Carnimonas nigrificans CECT 4437T . These data indicate that strain SW32T was related enough to members of the genus Halomonas to be placed as a new species within that genus . Therefore the name Halomonas marisflavae sp . nov . is proposed for strain SW32T . The type strain of the new species is strain SW32T (= KCCM 80003T = JCM 10873T).

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 1133 - 42
Transfer of Natrialba asiatica B1T to Natrialba taiwanensis sp . nov . and description of Natrialba aegyptiaca sp . nov., a novel extremely halophilic, aerobic, non-pigmented member of the Archaea from Egypt that produces extracellular poly(glutamic acid); Hezayen FF et al.; A novel extremely halophilic member of the Archaea, strain 40T, was isolated from Egypt (Aswan) . This isolate requires at least 1.6 M sodium chloride for growth and exhibits optimal growth between 37 and 42 degrees C . Determination of the entire 16S rRNA gene sequence revealed the highest similarity to the type strain of Natrialba asiatica (> 99%) . Polar lipid analysis indicated that strain 40T and Natrialba asiatica have essentially identical compositions, indicating that the former is a member of genus Natrialba . However, physiological and biochemical data provided evidence that Natrialba asiatica strains B1T and 172P1T, as well as strain 40T, are sufficiently different to be divided in three different species . The G+C content of strain 40T was 61.5+/-0.6 mol% . In addition, DNA-DNA hybridization data supported the placement of the isolate in a new species in the genus Natrialba, Natrialba aegyptiaca sp . nov., and indicated that Natrialba asiatica strain B1T should also be placed in a separate species, Natrialba taiwanensis sp . nov . Morphological studies of strain 40T indicated clearly that this isolate appears in three completely different cell shapes (cocci, rods, tetrads) under different conditions of growth, including different sodium chloride concentrations and different growth temperatures . Another interesting property of strain 40T is the ability to produce an extracellular polymer, which was found to be composed predominantly of glutamic acid (85% w/w), representing poly(glutamic acid), carbohydrates (12.5% w/w) and unidentified compounds (2.5% w/w) . Among the Archaea, production of an extracellular polysaccharide has been described for some members of the genera Haloferax and Haloarcula.

J Biotechnol, 2001 Jun 15, 88(2), 119 - 28
Antigen presentation using novel particulate organelles from halophilic archaea; Stuart ES et al.; A presentation vehicle was developed based on particulate gas vesicles produced by halophilic archaea . Gas vesicle epitope displays were prepared using standard coupling methods or recombinant DNA technology . When presented in the context of gas vesicle preparations, either the hapten, TNP, or a model six amino acid recombinant insert in the outer gas vesicle protein, GvpC was rendered immunogenic . Assays to quantify humoral responses indicated that each preparation elicited strong antibody responses in the absence of exogenous adjuvant . Thus, each preparation elicited a humoral response when injected into mice and this response was long lived and exhibited immunologic memory . Recombinant gas vesicle preparations therefore constitute a new, self-adjuvanting carrier/display vehicle for presentation of an array of peptidyl epitopes.

Syst Appl Microbiol, 2001 Apr, 24(1), 15 - 25
Gene transfer systems and their applications in Archaea; Luo Y et al.; Members of the Archaea domain are extremely diverse in their adaptation to extreme environments, yet also widespread in "normal" habitats . Altogether, among the best characterized archaeal representatives all mechanisms of gene transfer such as transduction, conjugation, and transformation have been discovered, as briefly reviewed here . For some halophiles and mesophilic methanogens, usable genetic tools were developed for in vivo studies . However, on an individual basis no single organism has evolved into the "E . coli of Archaea" as far as genetics is concerned . Currently, and unfortunately, most of the genome sequences available are those of microorganisms which are either not amenable to gene transfer or not among the most promising candidates for genetic studies.

J Biol Chem, 2001 Aug 10, 276(32), 29906 - 14 Epub 2001 Jun 06.
Characterization of a novel complex from halophilic archaebacteria, which displays chaperone-like activities in vitro; Franzetti B et al.; We isolated a protein, P45, from the extreme halophilic archaeon Haloarcula marismortui, which displays molecular chaperone activities in vitro . P45 is a weak ATPase that assembles into a large ring-shaped oligomeric complex comprising about 10 subunits . The protein shows no significant homology to any known protein . P45 forms complexes with halophilic malate dehydrogenase during its salt-dependent denaturation/renaturation and decreases the rate of deactivation of the enzyme in an ATP-dependent manner . Compared with other halophilic proteins, the P45 complex appears to be much less dependent on salt for its various activities or stability . In vivo experiments showed that P45 accumulates when cells are exposed to a low salt environment . We suggest, therefore, that P45 could protect halophilic proteins against denaturation under conditions of cellular hyposaline stress.

Microbiol Res, 2001, 156(1), 49 - 58
Aromatic hydrocarbon degradation patterns and catechol 2,3-dioxygenase genes in microbial cultures from deep anoxic hypersaline lakes in the eastern Mediterranean sea; Brusa T et al.; Several mixed cultures able to grow on different aromatic hydrocarbons were obtained from different depths (between 3500 and 3660 m under the sea surface) of water/brine interfaces (1 to 5 m over the estimated brine surface) of three deep hypersaline anoxic basins (Urania, Discovery and Atalante) in the eastern Mediterranean sea . Eight strains which completely removed toluene from the medium in six to 10 days were isolated from one of the mixed cultures obtained from the Urania basin . The strains grew on toluene and yeast extract in the presence of NaCl concentrations of up to 50 and 100 g l(-1), respectively, indicating that they are halotolerant rather than halophilic . Even though DNA fingerprinting methods showed that the strains were strictly related, two groups could be found on the basis of the plasmid profile . Metabolic profiling and partial sequencing (350 bp) of the 16S rDNA showed that the strains were related to Pseudomonas mendocina . A 320 bp fragment of the catechol 2,3-dioxygenase gene from all the strains was aimplified by PCR . The sequence of the fragment showed 100% identity with xylE from pWW53 of Pseudomonas putida MT53 isolated from soil . Southern hybridisation experiments showed that catechol 2,3-dioxygenase is plasmid encoded.

Arch Microbiol, 2001 Mar, 175(3), 208 - 19
Selenihalanaerobacter shriftii gen . nov., sp . nov., a halophilic anaerobe from Dead Sea sediments that respires selenate; Blum JS et al.; We isolated an obligately anaerobic halophilic bacterium from the Dead Sea that grew by respiration of selenate . The isolate, designated strain DSSe-1, was a gram-negative, non-motile rod . It oxidized glycerol or glucose to acetate + CO2 with concomitant reduction of selenate to selenite plus elemental selenium . Other electron acceptors that supported anaerobic growth on glycerol were nitrate and trimethylamine-N-oxide; nitrite, arsenate, fumarate, dimethylsulfoxide, thiosulfate, elemental sulfur, sulfite or sulfate could not serve as electron acceptors . Growth on glycerol in the presence of nitrate occurred over a salinity range from 100 to 240 g/l, with an optimum at 210 g/l . Analysis of the 16S rRNA gene sequence suggests that strain DSSe-1 belongs to the order Halanaerobiales, an order of halophilic anaerobes with a fermentative or homoacetogenic metabolism, in which anaerobic respiratory metabolism has never been documented . The highest 16S rRNA sequence similarity (90%) was found with Acetohalobium arabaticum (X89077) . On the basis of physiological properties as well as the relatively low homology of 16S rRNA from strain DSSe-1 with known genera, classification in a new genus within the order Halanaerobiales, family Halobacteroidaceae is warranted . We propose the name Selenihalanaerobacter shriftii . Type strain is strain DSSe-1 (ATCC accession number BAA-73).

Extremophiles, 2001 Apr, 5(2), 73 - 83
Potential of halotolerant and halophilic microorganisms for biotechnology; Margesin R et al.; Halotolerant or halophilic microorganisms, able to live in saline environments, offer a multitude of actual or potential applications in various fields of biotechnology . The technical applications of bacteriorhodopsin comprise holography, spatial light modulators, optical computing, and optical memories . Compatible solutes are useful as stabilizers of biomolecules and whole cells, salt antagonists, or stress-protective agents . Biopolymers, such as biosurfactants and exopolysaccharides, are of interest for microbially enhanced oil recovery . Other useful biosubstances are enzymes, such as new isomerases and hydrolases, that are active and stable at high salt contents . Halotolerant microorganisms play an essential role in food biotechnology for the production of fermented food and food supplements . The degradation or transformation of a range of organic pollutants and the production of alternative energy are other fields of applications of these groups of extremophiles.

Mikrobiologiia, 2001 Jan-Feb, 70(1), 77 - 85
{Halonatronum saccharophilum gen . nov . sp . nov--a new haloalkalophilic bacteria from the order Haloanaerobiales from Lake Magadi}; Zhilina TN et al.; A new alkaliphilic and moderately halophilic chemoorganotrophic anaerobic bacterium (strain Z-7986), which is spore-forming, rod-shaped, and has a gram-negative cell wall pattern, was isolated from the coastal lagoon mud of the highly mineralized Lake Magadi (Kenya) . The organism is an obligatorily carbonate- and sodium chloride-dependent . It is a motile peritrichously flagellated rod that has developed within 3-17% NaCl concentration (with an optimum at 7-12% NaCl) and within a pH range of 7.7-10.3 (with an optimum at pH values of 8-8.5) . It is a moderate thermophile with a broad temperature optimum from 36-55 degrees C and a growth maximum at 60 degrees C . The bacterium catabolizes glucose, fructose, sucrose, maltose, starch, glycogen, N-acetyl-D-glucosamine, and, to a slight degree, peptone and yeast extract . Its anabolism requires yeast extract or casamino acids . Glucose fermentation yields formate, acetate, ethanol, H2, and CO2 . The bacterium is sulfidetolerant and capable of the nonspecific reduction of S0 to H2S . The G + C content of the DNA is 34.4 mol % . The analysis of the 16S rRNA sequence revealed that strain Z-7986 belongs to the order Haloanaerobiales and represents a new genus in the family Halobacteroidaceae . We suggest calling the organism Halonatronum saccharophilum gen . nov . sp . nov . The type strain of this species is Z-7986T (= DSM13868, = Uniqem 211).

J Biotechnol, 2001 May 18, 87(3), 255 - 61
Stability of an extreme halophilic alkaline phosphatase from Halobacterium salinarium in non-conventional medium; Marhuenda-Egea FC et al.; Alkaline p-nitrophenylphosphate phosphatase from the halophilic archaeon Halobacterium salinarum (earlier halobium) was solubilised in organic medium using reversed micelles of hexadecyltrimethylammonium bromide in cyclohexane, with 1-butanol as co-surfactant . The stability of alkaline p-nitrophenylphosphate phosphatase in this system was studied at different conditions, w(0) ({H(2)O}/{surfactant}), salt concentration, with and without Mn(+2) . At all the conditions assayed, alkaline p-nitrophenylphosphate phosphatase was more stable in reversed micelles than in bulk aqueous solution (at 25 degrees C) . The stabilisation effect of the reversed micelles was dramatic when the enzyme was dialysed against Mn(+2)-free buffer since the enzyme lost all the activity within 90 min in aqueous medium, but it retained approximately 72% of the initial enzymatic activity for 90 min in reversed micelles.

Appl Microbiol Biotechnol, 2001 Mar, 55(2), 214 - 8
Improved osmotolerance of recombinant Escherichia coli by de novo glycine betaine biosynthesis; von Weymarn N et al.; The genes from the extreme halophile Ecto-thiorhodospira halochloris encoding the biosynthesis of glycine betaine from glycine were cloned into Escherichia coli . The accumulation of glycine betaine and its effect on osmotolerance of the cells were studied . In mineral medium with NaCl concentrations from 0.15 to 0.5 M, the accumulation of both endogenously synthesized and exogenously provided glycine betaine stimulated the growth of E . coli . The intracellular levels of glycine betaine and the cellular yields were clearly higher for cells receiving glycine betaine exogenously than for cells synthesizing it . The lower level of glycine betaine accumulation in cells synthesizing it is most likely a consequence of the limited availability of precursors (e.g . S-adenosylmethionine) rather than the result of a low expression level of the genes . Glycine betaine also stimulated the growth of E . coli and decreased acetate formation in mineral medium with high sucrose concentrations (up to 200 g.l(-1)).

Int J Syst Evol Microbiol, 2001 Mar, 51(Pt 2), 425 - 31
Filobacillus milensis gen . nov., sp . nov., a new halophilic spore-forming bacterium with Orn-D-Glu-type peptidoglycan; Schlesner H et al.; A spore-forming, halophilic bacterium was isolated from surface sediment located on the beach of Palaeochori Bay near to a shallow water hydrothermal vent area, Milos, Greece . The bacterium, designated SH 714T, consisted of motile, strictly aerobic rods which contained an Orn-D-Glu type murein and a G+C content of 35 mol% . Thin sections showed a cell wall typical for Gram-positive bacteria; the peptidoglycan layer, however, was very thin . The Gram-reaction of the organism was negative . Comparative 16S rRNA gene sequencing demonstrated that the isolate represents a new line of descent within the spore-forming rods branching at the periphery of the rRNA group 1 Bacillus (Bacillus sensu stricto) . The nearest phylogenetic neighbours of the unknown bacterium were Bacillus haloalkaliphilus, Marinococcus albus and Halobacillus species . Based on phylogenetic and phenotypic evidence it is proposed that the unknown bacterium be classified as Filobacillus milensis gen . nov., sp . nov . The type strain is SH 714T (= DSM 13259T = ATCC 700960T).

Int J Syst Evol Microbiol, 2001 Mar, 51(Pt 2), 327 - 37
Dethiosulfovibrio russensis sp . nov., Dethosulfovibrio marinus sp . nov . and Dethosulfovibrio acidaminovorans sp . nov., novel anaerobic, thiosulfate- and sulfur-reducing bacteria isolated from 'Thiodendron' sulfur mats in different saline environments; Surkov AV et al.; Four strains of strictly anaerobic, sulfur- and thiosulfate-reducing bacteria, SR12T, SR13, SR15T and WS100T, were isolated from 'Thiodendron' sulfur mats obtained from different saline environments . All isolates were motile, Gram-negative, non-spore-forming curved rods with pointed or rounded ends . The sizes of cells varied from 0.9 x 3-5 microm for strains SR12T, SR13 and SR15T to 0.9 x 4.8 microm for strain WS100T . All strains could form long spiral filamentous cells up to 70-110 microm during the early stage of growth . All strains were motile by a tumbling movement and possessed lateral flagella arranged at the concave side of cells . Incomplete cross-septa were distinctive features of all strains . Growth occurred at temperatures of 10-40 degrees C with an optimum at 28 degrees C . The pH limits for growth were 5.5 to 8.0, with optimal growth at pH 6.5-7.0 . All isolates were obligately anaerobic and slightly halophilic and grew in media containing 0.5-5% NaCl with an optimum at 2% NaCl . All strains were chemoorganoheterotrophic, having a fermentative type of metabolism and utilized proteins, peptides, amino acids and some organic acids, but not sugars, fatty acids or alcohols . Some organic substrates (isoleucine, valine, alanine, glutamate) were utilized only by strain SR12T in the presence of sulfur or thiosulfate . Fermentation of citrate yielded mainly acetate, CO2 and H2 . Sulfur and thiosulfate were reduced to hydrogen sulfide during the fermentation of organic substances, which increased cell yields and growth rates . Sulfate, sulfite, fumarate, nitrate, Fe2O3, MnO2, DMSO and elemental selenium were not used as electron acceptors by these strains . The G+C contents of the DNA were 51 mol% for strains SR12T, SR13 and SR15T and 52 mol% for strain WS100T . Based on morphological, physiological and phylogenetic similarities, all four isolates could be assigned to three new species of the genus Dethiosulfovibrio, named Dethiosulfovibrio russensis (type strain DSM 12538T), Dethiosulfovibrio marinus (type strain DSM 12537T) and Dethiosulfovibrio acidaminovorans (type strain DSM 12590T).

Mikrobiologiia, 2000 Sep-Oct, 69(5), 694 - 9
{Clusterization of halophilic and halotolerant eubacteria using whole-cell protein electrophoresis data}; Bykova SA et al.; Total cell proteins of the nineteen halophilic and halotolerant eubacteria isolated from marine sediments and highly mineralized formation waters of oil fields were investigated by SDS gel electrophoresis . The microorganisms studied, phenotypically identified as belonging to the genera Dietzia, Rhodococcus, Staphylococcus, Cytophaga, Brevibacterium, and Archangium, were found to form clearly distinguishable clusters (20-30% similarity at the generic level) on the dendrogram derived from electrophoretic protein patterns . Protein similarity data confirmed the heterogeneity of Rhodococcus maris and its relatedness to the genus Dietza.

Extremophiles, 2001 Feb, 5(1), 61 - 9
Random sequence analysis of genomic DNA of an anaerobic, thermophilic, halophilic bacterium, Halothermothrix orenii; Mijts BN et al.; A pBluescriptSK+ vector library consisting of 3.360 clones with an average insert size of 3.5 kb was constructed from the genome of Halothermothrix orenii, a halophilic and thermoanaerobic member of the family Haloanaerobiaceae . From both ends, 77 clones were sequenced using T3 and T7 vector primers generating 154 sequence tags, representing approximately 85 kb of the genome . Comparison of sequence tags against the Gen-Bank database using BLASTX identified 66 known proteins and 15 conserved hypothetical proteins . The putative proteins included a V-ATPase, hydrogenases, and enzymes with potential for industrial applications . The overall G + C% of the codons used was 42.9% with a third-position G + C content of 38.6% . High levels of excess acidic amino acids were not detected in the putative proteins of H . orenii as compared to the mesophilic haloanaerobes . This lack may be the result of reduced activity of acidic, halophilic enzymes at high temperatures and intermediate salt concentrations.

FEMS Microbiol Lett, 2001 Apr 1, 197(1), 117 - 21
Plasmid transfer and susceptibility to antibiotics in the halophilic phototrophs Rhodovibrio salinarum and Rhodothalassium salexigens; Borghese R et al.; The present study defines a series of genetic procedures to be used for molecular studies in photosynthetic halophilic species such as Rhodovibrio salinarum and Rhodothalassium salexigens . In both species, the minimal inhibitory concentrations for the antibiotics tetracycline, rifampicin, chloramphenicol, spectinomycin, streptomycin, and kanamycin were determined . In addition, conjugal transfer of IncP and IncQ plasmids from Escherichia coli was demonstrated and the resistance markers expressed in these halophiles were determined . Finally, Rth . salexigens growth dependence on variable salt concentrations was measured: maximal growth rates were seen at 6% and 4% NaCl under phototrophic and chemotrophic conditions, respectively . To the best of our knowledge, this is the first report analyzing the genetic properties of two representative species of halophilic purple non-sulfur phototrophs.

FEBS Lett, 2001 Mar 30, 493(2-3), 134 - 8
NaCl-activated nucleoside diphosphate kinase from extremely halophilic archaeon, Halobacterium salinarum, maintains native conformation without salt; Ishibashi M et al.; Enzymes from extremely halophilic archaea are readily denatured in the absence of a high salt concentration . However, we have observed here that a nucleoside diphosphate kinase prepared from Halobacterium salinarum was active and stable in the absence of salt, though it has the amino acid composition characteristic of halophilic enzymes . Recombinant nucleoside diphosphate kinase expressed in Escherichia coli requires salt for activation in vitro, but once it acquires the proper folding, it no longer requires the presence of salts for its activity and stability.

Arch Microbiol, 2001 Jan, 175(1), 52 - 61
Different glycolytic pathways for glucose and fructose in the halophilic archaeon Halococcus saccharolyticus; Johnsen U et al.; The glucose and fructose degradation pathways were analyzed in the halophilic archaeon Halococcus saccharolyticus by 13C-NMR labeling studies in growing cultures, comparative enzyme measurements and cell suspension experiments . H . saccharolyticus grown on complex media containing glucose or fructose specifically 13C-labeled at C1 and C3, formed acetate and small amounts of lactate . The 13C-labeling patterns, analyzed by 1H- and 13C-NMR, indicated that glucose was degraded via an Entner-Doudoroff (ED) type pathway (100%), whereas fructose was degraded almost completely via an Embden-Meyerhof (EM) type pathway (96%) and only to a small extent (4%) via an ED pathway . Glucose-grown and fructose-grown cells contained all the enzyme activities of the modified versions of the ED and EM pathways recently proposed for halophilic archaea . Glucose-grown cells showed increased activities of the ED enzymes gluconate dehydratase and 2-keto-3-deoxy-gluconate kinase, whereas fructose-grown cells contained higher activities of the key enzymes of a modified EM pathway, ketohexokinase and fructose-1-phosphate kinase . During growth of H . saccharolyticus on media containing both glucose and fructose, diauxic growth kinetics were observed . After complete consumption of glucose, fructose was degraded after a lag phase, in which fructose-1-phosphate kinase activity increased . Suspensions of glucose-grown cells consumed initially only glucose rather than fructose, those of fructose-grown cells degraded fructose rather than glucose . Upon longer incubation times, glucose- and fructose-grown cells also metabolized the alternate hexoses . The data indicate that, in the archaeon H . saccharolyticus, the isomeric hexoses glucose and fructose are degraded via inducible, functionally separated glycolytic pathways: glucose via a modified ED pathway, and fructose via a modified EM pathway.

FEMS Microbiol Lett, 2001 Mar 15, 196(2), 113 - 8
Purification and characterisation of a possible assimilatory nitrite reductase from the halophile archaeon Haloferax mediterranei; Martinez-Espinosa RM et al.; The nitrite reductase from the extreme halophilic archaeon, Haloferax mediterranei, has been purified and characterised . H . mediterranei is capable of growing in a minimal medium (inorganic salts and glucose as a carbon source) with nitrate as the only nitrogen source . The overall purification was 46-fold with about 4% recovery of activity . The enzyme is a monomeric protein of approximately 66 kDa . A pH of 7.5 and high temperatures up to 60 degrees C are necessary for optimum activity . Reduced methyl viologen has been found to be an electron donor as effective as ferredoxin . NADPH and NADH, which are electron donors in nitrite reductases from different non-photosynthetic bacteria, were not effective with nitrite reductase from H . mediterranei.

J Lipid Res, 2001 Mar, 42(3), 352 - 8
Ergosterol biosynthesis in novel melanized fungi from hypersaline environments; Mejanelle L et al.; Halotolerant and halophilic melanized fungi were recently described in hypersaline waters . A close study of the sterol composition of such fungi, namely Hortaea werneckii, Alternaria alternata, Cladosporium sphaerospermum, Cladosporium sp., and Aureobasidium pullulans revealed the dominance of ergosterol and the presence of 29 intermediates of its biosynthesis pathway . The presence or absence of intermediates from distinct synthesis routes gave insight into the operative synthetic pathways from 4,4,14-trimethylcholesta-8,24-dien-3 beta-ol (lanosterol) to ergosterol in melanized fungi and in Saccharomyces cerevisiae, a reference yeast cultured in parallel . In all studied melanized fungi, initial methylation at C-24 took place before C-14 and C-4 demethylation, involving a different reaction sequence from that observed in S . cerevisiae . Further transformation was observed to occur through various routes . In A . alternata, isomerization at C-7 takes place prior to desaturation at C-5 and C-22, and methylene reduction at C-24 . In addition to these pathways in Cladosporium spp., H . werneckii, and A . pullulans, ergosterol may also be synthesized through reduction of the C-24 methylene group before desaturation at C-5 and C-22 or vice versa . Moreover, in all studied melanized fungi except A . alternata, ergosterol biosynthesis may also proceed through C-24 methylene reduction prior to C-4 demethylation . -- Mejanelle, L., J . F . Lopez, N . Gunde-Cimerman, and J . O . Grimalt . Ergosterol biosynthesis in novel melanized fungi from hypersaline environments . J . Lipid Res . 2001 . 42: 352--358.

Microb Ecol, 2001 Jan, 41(1), 1 - 11
Sulfate Reduction Dynamics and Enumeration of Sulfate-Reducing Bacteria in Hypersaline Sediments of the Great Salt Lake (Utah, USA); Brandt KK et al.; Bacterial sulfate reduction activity (SRA) was measured in surface sediments and slurries from three sites in the Great Salt Lake (Utah, USA) using radiolabeled 35S-sulfate . High rates of sulfate reduction (363 +/- 103 and 6,131 +/- 835 nmol cm-3 d-1) were measured at two sites in the moderately hypersaline southern arm of the lake, whereas significantly lower rates (32 +/- 9 nmol cm-3 d-1) were measured in the extremely hypersaline northern arm . Bacterial sulfate reduction was strongly affected by salinity and showed an optimum around 5-6% NaCl in the southern arm and an optimum of around 12% NaCl in the more hypersaline northern arm of the lake . High densities of sulfate-reducing bacteria (SRB) ranging from 2.2 x 107 to 6.7 x 108 cells cm-3 were determined by a newly developed tracer MPN-technique (T-MPN) employing sediment media and 35S-sulfate . Calculation of specific sulfate reduction rates yielded values comparable to those obtained in pure cultures of SRB . However, when using a conventional MPN technique with synthetic media containing high amounts of Fe(II), the numbers of SRB were underestimated by 1-4 orders of magnitude as compared to the T-MPN method . Our results suggest that high densities of slightly to moderately halophilic and extremely halotolerant SRB are responsible for the high rates of sulfate reduction measured in Great Salt Lake sediments.

Syst Appl Microbiol, 2000 Dec, 23(4), 563 - 72
A nested array of rRNA targeted probes for the detection and identification of enterococci by reverse hybridization; Behr T et al.; Complete 23S and almost complete 16S rRNA gene sequences were determined for the type strains of the validly described Enterococcus species, Melissococcus pluton and Tetragenococcus halophilus . A comprehensive set of rRNA targeted specific oligonucleotide hybridization probes was designed according to the multiple probe concept . In silico probe design and evaluation was performed using the respective tools of the ARB program package in combination with the ARB databases comprising the currently available 16S as well as 23S rRNA primary structures . The probes were optimized with respect to their application for reverse hybridization in microplate format . The target comprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chain reaction) using general primers targeting a wide spectrum of bacteria . Alternatively, amplification of two adjacent rDNA fragments of enterococci was performed by using specific primers . In vitro evaluation of the probe set was done including all Enterococcus type strains, and a selection of other representatives of the gram-positive bacteria with a low genomic DNA G+C content . The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants.

Biochim Biophys Acta, 2001 May 1, 1505(1), 169 - 78
Role of sodium bioenergetics in Vibrio cholerae; Hase CC et al.; The ability of the bacterium to use sodium in bioenergetic processes appears to play a key role in both the environmental and pathogenic phases of Vibrio cholerae . Aquatic environments, including fresh, brackish, and coastal waters, are an important factor in the transmission of cholera and an autochthonous source . The organism is considered to be halophilic and has a strict requirement for Na(+) for growth . Furthermore, expression of motility and virulence factors of V . cholerae is intimately linked to sodium bioenergetics and to each other . Several lines of evidence indicated that the activity of the flagellum of V . cholerae might have an impact on virulence gene regulation . As the V . cholerae flagellum is sodium-driven and the Na(+)-NQR enzyme is known to create a sodium motive force across the bacterial membrane, it was recently suggested that the increased toxT expression observed in a nqr-negative strain is mediated by affecting flagella activity . It was suggested that the V . cholerae flagellum might respond to changes in membrane potential and the resulting changes in flagellar rotation might serve as a signal for virulence gene expression . However, we recently demonstrated that although the flagellum of V . cholerae is not required for the effects of ionophores on virulence gene expression, changes in the sodium chemical potential are sensed and thus alternative mechanisms, perhaps involving the TcpP/H proteins, for the detection of these conditions must exist . Analyzing the underlying mechanisms by which bacteria respond to changes in the environment, such as their ability to monitor the level of membrane potential, will probably reveal complex interplays between basic physiological processes and virulence factor expression in a variety of pathogenic species.

Biochim Biophys Acta, 2001 May 1, 1505(1), 37 - 44
Recent progress in the Na(+)-translocating NADH-quinone reductase from the marine Vibrio alginolyticus; Hayashi M et al.; The respiratory chain of Gram-negative marine and halophilic bacteria has a Na(+)-dependent NADH-quinone reductase that functions as a primary Na(+) pump . The Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of six structural genes (nqrA to nqrF) . The NqrF subunit has non-covalently bound FAD . There are conflicting results on the existence of other flavin cofactors . Recent studies revealed that the NqrB and NqrC subunits have a covalently bound flavin, possibly FMN, which is attached to a specified threonine residue . A novel antibiotic, korormicin, was found to specifically inhibit the NQR complex . From the homology search of the nqr operon, it was found that the Na(+)-pumping NQR complex is widely distributed among Gram-negative pathogenic bacteria.

Nucleic Acids Res . 2001 Mar 15;29(6):E30.
OliI, a unique restriction endonuclease that recognizes the discontinuous sequence 5'-CACNN NGTG-3'; Padegimiene E et al.; A new type II restriction endonuclease designated OLI:I has been partially purified from the halophilic bacterium Oceanospirillum linum 4-5D . OLI:I recognizes the interrupted hexanucleotide palindrome 5'-CACNN NNGTG-3' and cleaves it in the center generating blunt-ended DNA fragments.

Proc Natl Acad Sci U S A, 2001 Feb 27, 98(5), 2521 - 5 Epub 2001 Feb 20.
Genomic and genetic dissection of an archaeal regulon; Baliga NS et al.; The extremely halophilic archaeon Halobacterium sp . NRC-1 can grow phototrophically by means of light-driven proton pumping by bacteriorhodopsin in the purple membrane . Here, we show by genetic analysis of the wild type, and insertion and double-frame shift mutants of Bat that this transcriptional regulator coordinates synthesis of a structural protein and a chromophore for purple membrane biogenesis in response to both light and oxygen . Analysis of the complete Halobacterium sp . NRC-1 genome sequence showed that the regulatory site, upstream activator sequence (UAS), the putative binding site for Bat upstream of the bacterio-opsin gene (bop), is also present upstream to the other Bat-regulated genes . The transcription regulator Bat contains a photoresponsive cGMP-binding (GAF) domain, and a bacterial AraC type helix-turn-helix DNA binding motif . We also provide evidence for involvement of the PAS/PAC domain of Bat in redox-sensing activity by genetic analysis of a purple membrane overproducer . Five additional Bat-like putative regulatory genes were found, which together are likely to be responsible for orchestrating the complex response of this archaeon to light and oxygen . Similarities of the bop-like UAS and transcription factors in diverse organisms, including a plant and a gamma-proteobacterium, suggest an ancient origin for this regulon capable of coordinating light and oxygen responses in the three major branches of the evolutionary tree of life . Finally, sensitivity of four of five regulon genes to DNA supercoiling is demonstrated and correlated to presence of alternating purine-pyrimidine sequences (RY boxes) near the regulated promoters.

Trends Plant Sci, 2001 Feb, 6(2), 66 - 71
Plant salt tolerance; Zhu JK; Soil salinity is a major abiotic stress in plant agriculture worldwide . This has led to research into salt tolerance with the aim of improving crop plants . However, salt tolerance might have much wider implications because transgenic salt-tolerant plants often also tolerate other stresses including chilling, freezing, heat and drought . Unfortunately, suitable genetic model systems have been hard to find . A recently discovered halophytic plant species, Thellungiella halophila, now promises to help in the detection of new tolerance determinants and operating pathways in a model system that is not limited to Arabidopsis traits or ecotype variations.

FEBS Lett, 2001 Feb 2, 489(2-3), 125 - 8
Chloride dependence of glycine betaine transport in Halobacillus halophilus; Roessler M et al.; Growth of Halobacillus halophilus is strictly chloride-dependent but the physiological basis for the chloride dependence remains to be elucidated . To address the function of Cl(-) in H . halophilus, a physiological study was performed . It was found that uptake of the compatible solute glycine betaine under isoosmotic conditions was stimulated by increasing salt concentrations . Uptake of glycine betaine required both, Na(+) and Cl(-) . Cl(-) could be substituted by nitrate and bromide, but not by sulfate . Glycine betaine transport was optimal at around 0.7 M Cl(-) . Cells responded to an osmotic upshock by accumulating glycine betaine, but only in the presence of chloride . These studies revealed the first chloride-dependent glycine betaine transporter in a prokaryote.

Biochimie, 2000 Dec, 82(12), 1143 - 50
2-Hydroxyacid dehydrogenase from Haloferax mediterranei, a D-isomer-specific member of the 2-hydroxyacid dehydrogenase family; Bonete MJ et al.; An NAD-dependent D-2-hydroxyacid dehydrogenase (EC 1.1.1.) was isolated and characterized from the halophilic Archaeon Haloferax mediterranei . The enzyme is a dimer with a molecular mass of 101.4 +/- 3.3 kDa . It is strictly NAD-dependent and exhibits its highest activity in 4 M NaCl . The enzyme is characterized by a broad substrate specificity 2-ketoisocaproate and 2-ketobutyrate being the substrates with the higher Vmax/Km . When pyruvate and 2-ketobutyrate were the substrates the optimal pH was acidic (pH 5) meanwhile for 2-ketoisocaproate maximum activity was achieved at basic pH between 7.5 and 8.5 . The optimum temperature was 52 degrees C and at 65 degrees C there was a pronounced activity decrease . This new enzyme can be used for the production of D-2-hydroxycarboxylic acid.

J Bacteriol, 2001 Jan, 183(1), 287 - 91
Secreted euryarchaeal microhalocins kill hyperthermophilic crenarchaea; Haseltine C et al.; Few antibiotics targeting members of the archaeal domain are currently available for genetic studies . Since bacterial antibiotics are frequently directed against competing and related organisms, archaea by analogy might produce effective antiarchaeal antibiotics . Peptide antibiotic (halocin) preparations from euryarchaeal halophilic strains S8a, GN101, and TuA4 were found to be toxic for members of the hyperthermophilic crenarchaeal genus Sulfolobus . No toxicity was evident against representative bacteria or eukarya . Halocin S8 (strain S8a) and halocin R1 (strain GN101) preparations were cytostatic, while halocin A4 (strain TuA4) preparations were cytocidal . Subsequent studies focused on the use of halocin A4 preparations and Sulfolobus solfataricus . Strain TuA4 cell lysates were not toxic for S . solfataricus, and protease (but not nuclease) treatment of the halocin A4 preparation inactivated toxicity, indicating that the A4 toxic factor must be a secreted protein . Potassium chloride supplementation of the Sulfolobus assay medium potentiated toxicity, implicating use of a salt-dependent mechanism . The utility of halocin A4 preparations for genetic manipulation of S . solfataricus was assessed through the isolation of UV-induced resistant mutants . The mutants exhibited stable phenotypes and were placed into distinct classes based on their levels of resistance.

Biochim Biophys Acta, 2000 Dec 15, 1524(2-3), 149 - 54
Expression and fast-flow purification of a polyhistidine-tagged myoglobin-like aerotaxis transducer; Piatibratov M et al.; A Co(2+)-affinity, fast-flow perfusion chromatography method to purify a polyhistidine-tagged myoglobin-like aerotaxis transducer HemAT-Hs has been developed . The method relies upon a six-histidine affinity tag fused to the C-terminus and N-terminus of HemAT-Hs for expression in the native host, an extremely halophilic Archaeon Halobacterium salinarum, and in the heterologous host Escherichia coli, respectively . The His-tagged HemAT-Hs can be purified rapidly using either low or high ionic strength buffers . Purified His-tagged HemAT-Hs in high or low salt buffers demonstrated no difference in spectral characteristics and retained reversible oxygen binding capacity . This fast-flow Co(2+)-affinity perfusion chromatography provides a simple method for preparation of halophilic heme containing soluble proteins for biophysical and structural studies.

Int Microbiol, 1999 Dec, 2(4), 259 - 66
Aerobic and facultative anaerobic heterotrophic bacteria associated to Mediterranean oysters and seawater; Pujalte MJ et al.; A comparative study on the composition and seasonal fluctuations of the main heterotrophic bacterial groups and species isolated from Mediterranean oysters and their growing-seawater was carried out . For the study we used 574 strains isolated from Marine Agar (MA) and submitted to numerical analysis of phenotypic traits in previous studies, plus 323 isolates recovered on Thiosulphate Citrate Bile Sucrose (TCBS) agar from the same samples and identified in this study . Oyster samples were dominated by halophilic fermentative bacteria during most of the year with predominance of two Vibrio species, V . splendidus (at temperatures lower than 20 degrees C), and V . harveyi (at higher temperatures) . On the contrary, Vibrio spp . was not the predominant microbiota of seawater, where most isolates had remained unidentified but corresponded to alpha-Proteobacteria, as shown by rDNA hybridization with phylogenetic probes in this study . Among the strict aerobes that could be identified, none of them showed a clear dominance, and many different groups were represented in very low percentages, in contrast with the major species from oyster samples . Shannon-Weaver diversity index revealed significant differences between both types of samples . No apparent seasonality was found in the distribution of seawater species, in sharp contrast with oyster-associated bacteria.

Syst Appl Microbiol, 2000 Oct, 23(3), 376 - 85
Taxonomic analysis of extremely halophilic archaea isolated from 56-years-old dead sea brine samples; Arahal DR et al.; A taxonomic study comprising both phenotypic and genotypic characterization, has been carried out on a total of 158 extremely halophilic aerobic archaeal strains . These strains were isolated from enrichments prepared from Dead Sea water samples dating from 1936 that were collected by B . E . Volcani for the demonstration of microbial life in the Dead Sea . The isolates were examined for 126 morphological, physiological, biochemical and nutritional tests . Numerical analysis of the data, by using the S(J) coefficient and UPGMA clustering method, showed that the isolates clustered into six phenons . Twenty-two out of the 158 strains used in this study were characterized previously (ARAHAL et al., 1996) and were placed into five phenotypic groups . The genotypic study included both the determination of the guanineplus-cytosine content of the DNA and DNA-DNA hybridization studies . For this purpose, representative strains from the six phenons were chosen . These groups were found to represent some members of three different genera - Haloarcula (phenons A, B, and C), Haloferax (phenons D and E) and Halobacterium (phenon F) - of the family Halobacteriaceae, some of them never reported to occur in the Dead Sea, such as Haloarcula hispanica, while Haloferax volcanii (phenons D and E) was described in the Dead Sea by studies carried out several decades later than Volcani's work.

FEMS Microbiol Ecol, 2000 Dec 1, 34(2), 113 - 119
5S rRNA fingerprints of marine bacteria, halophilic archaea and natural prokaryotic assemblages along a salinity gradient; Casamayor EO et al.; Natural prokaryotic assemblages from two multi-pond solar salterns and pure cultures of both marine bacteria and halophilic archaea were analyzed and compared by electrophoretic analysis of 5S rRNAs . A salinity gradient from seawater (3.7%) to NaCl precipitation (37%) was studied . The culture-independent, PCR-free, fingerprinting analysis covered two objectives: (i) to compare natural assemblages among them and with results previously obtained through a PCR-dependent approach and (ii) to estimate the in situ relevance of those prokaryotic groups obtained with classical culture methodologies . Natural assemblages were analyzed through cluster analysis of quantitative 5S rRNA band patterns . The resulting groups were in accordance with environmental parameters (i.e., NaCl concentration) and with the clustering obtained after a PCR-dependent approach, showing the formation of three salinity-based groups of samples (<10%, 10-25% and >25% salinity) . Similarities between the laboratory strains tested and dominant community members were studied by comparing 5S rRNA band patterns . The lack of match obtained after cluster analysis indicated that the prokaryotic populations relevant in the ponds below 25% salinity were neither Flavobacteria nor haloarchaeal strains belonging to the genera Halococcus, Haloarcula and Halobacterium . Members of Proteobacteria and Gram-positive bacteria were found to match bands in these samples . The 5S rRNA fingerprint from the dominant community members in the ponds above 30% salinity did not fit any of the cultured halophilic archaea studied, in agreement with earlier PCR results . This is consistent with a greater bias introduced by culture-dependent methods than by those based on PCR, especially for archaeal populations.

Biochemistry, 2000 Nov 28, 39(47), 14392 - 9
Probing the nature of the blue-shifted intermediate of photoactive yellow protein in solution by NMR: hydrogen-deuterium exchange data and pH studies; Craven CJ et al.; The nature of the pB intermediate of photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been probed by NMR . pH-dependent changes in the NMR spectrum of the dark state of PYP are shown to closely mimic exchange broadening effects observed previously in the NMR spectrum of the pB intermediate in solution . Amide H-D exchange data show that while pB retains a solid protected core, two regions become significantly less protected than the dark state . The amide exchange data help to rationalize why the conformational exchange process affects the N-terminal 28-residue segment of the protein, which is not close to the site of chromophore rearrangement . At very low pH (pH 1.7), the dark state NMR spectrum displays approximately 30 very sharp signals, which are characteristic of a portion of the molecule becoming unfolded . Similarities between the dark state spectra at pH approximately 3.2 and the spectra of pB suggest a model for pB in solution where the protein exists in an equilibrium between a well-ordered state and a state in which a region is unfolded . Such a two-state model accounts for the exchange phenomena observed in the NMR spectra of pB, and the hydrophobic exposure and lability inferred from thermodynamic data . It is likely that in the crystalline environment the ordered form of pB is strongly favored.

J Biol Chem, 2001 Feb 2, 276(5), 3660 - 5 Epub 2000 Oct 31.
Importance of the anticodon sequence in the aminoacylation of tRNAs by methionyl-tRNA synthetase and by valyl-tRNA synthetase in an Archaebacterium; Ramesh V et al.; The mode of recognition of tRNAs by aminoacyl-tRNA synthetases and translation factors is largely unknown in archaebacteria . To study this process, we have cloned the wild type initiator tRNA gene from the moderate halophilic archaebacterium Haloferax volcanii and mutants derived from it into a plasmid capable of expressing the tRNA in these cells . Analysis of tRNAs in vivo show that the initiator tRNA is aminoacylated but is not formylated in H . volcanii . This result provides direct support for the notion that protein synthesis in archaebacteria is initiated with methionine and not with formylmethionine . We have analyzed the effect of two different mutations (CAU-->CUA and CAU-->GAC) in the anticodon sequence of the initiator tRNA on its recognition by the aminoacyl-tRNA synthetases in vivo . The CAU-->CUA mutant was not aminoacylated to any significant extent in vivo, suggesting the importance of the anticodon in aminoacylation of tRNA by methionyl-tRNA synthetase . This mutant initiator tRNA can, however, be aminoacylated in vitro by the Escherichia coli glutaminyl-tRNA synthetase, suggesting that the lack of aminoacylation is due to the absence in H . volcanii of a synthetase, which recognizes the mutant tRNA . Archaebacteria lack glutaminyl-tRNA synthetase and utilize a two-step pathway involving glutamyl-tRNA synthetase and glutamine amidotransferase to generate glutaminyl-tRNA . The lack of aminoacylation of the mutant tRNA indicates that this mutant tRNA is not a substrate for the H . volcanii glutamyl-tRNA synthetase . The CAU-->GAC anticodon mutant is most likely aminoacylated with valine in vivo . Thus, the anticodon plays an important role in the recognition of tRNA by at least two of the halobacterial aminoacyl-tRNA synthetases.

Gene, 2000 Oct 3, 256(1-2), 319 - 26
FK506-binding protein-type peptidyl-prolyl cis-trans isomerase from a halophilic archaeum, Halobacterium cutirubrum; Iida T et al.; The halophilic archaeum, Halobacterium cutirubrum, has been shown to have a cyclophilin-type peptidyl-prolyl cis-trans isomerase (PPIase) . Because most archaeal genomes studied only have genes for FK506-binding proteins (FKBPs) as a PPIase, it has been unclear whether H . cutirubrum has an FKBP-type PPIase or not . In the present study, a gene encoding an FKBP-type PPIase was cloned from genomic DNA of H . cutirubrum and then sequenced . This FKBP was deduced to be composed of 303 amino acid residues with a molecular mass of 33.3kDa . Alignment of its amino acid sequence with those of other reported FKBPs showed that it contained two insertion sequences in the regions corresponding to the bulge and flap of human FKBP12, which are common to archaeal FKBPs . Its C-terminal amino acid sequence was approximately 130 amino acids longer than the FKBPs of Methanococcus thermolithotrophicus and Thermococcus sp . KS-1 . Among the 14 conserved amino acid residues that form the FK506 binding pocket, only three were found in this FKBP . This gene was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli, and the N-terminal GST portion was removed by protease digestion . The purified recombinant FKBP showed a weak PPIase activity with a low sensitivity to FK506 . This FKBP suppressed aggregation of the unfolded protein.

Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1909 - 13
Nocardiopsis kunsanensis sp . nov., a moderately halophilic actinomycete isolated from a saltern; Chun J et al.; A moderately halophilic actinomycete, designated HA-9T, was isolated from a saltern in Kunsan, Republic of Korea, and was the subject of polyphasic identification . Analysis of 16S rDNA indicated that the isolate belonged to the genus Nocardiopsis, but differed genetically from other Nocardiopsis species . Strain HA-9T contained meso-diaminopimelic acid, no diagnostic sugars, hexa- or octa-hydrogenated menaquinones with 10 isoprene units, straight-chain saturated or monounsaturated, iso-, anteiso-, 10-methyl branched fatty acids with 13-18 carbons and type III phospholipids . All of these characters consistently assign the isolate to the genus Nocardiopsis . All of the validly described Nocardiopsis species, including moderately halophilic Nocardiopsis halophila, can be differentiated from the saltern isolate using morphological and physiological traits . On the basis of polyphasic evidence, the name Nocardiopsis kunsanensis sp . nov . is proposed for strain HA-9T (= KCTC 9831T), which is designated the type strain.

Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1849 - 59
Methylarcula marina gen . nov., sp . nov . and Methylarcula terricola sp . nov.: novel aerobic, moderately halophilic, facultatively methylotrophic bacteria from coastal saline environments; Doronina NV et al.; A new genus, Methylarcula, with two new species, Methylarcula marina and Methylarcula terricola, are proposed for strains h1T and h37T of moderately halophilic facultatively methylotrophic bacteria isolated from the coastal saline habitats . These methylobacteria are aerobic, Gram-negative, asporogenous, non-motile, colourless rods that multiply by binary fission . Their cellular fatty acids profiles consist primarily of straight-chain unsaturated (C18:1; 70-80%), saturated (C18:0; 14-16%) and cyclopropane (C19:0; 5-6%) acids . The major ubiquinone is Q-10 . The dominant phospholipids are phosphatidylethanolamine and phosphatidylcholine . Both strains could use methylamine, some sugars and organic acids as carbon and energy sources . They grew well under optimal conditions (29-35 degrees C, pH 7.5-8.5, 0.5-1.0 M NaCl) and accumulated intracellularly poly-beta-hydroxybutyrate and the compatible solute ectoine . The ectoine pool was found to increase upon increasing the external NaCl concentration and accounted for 18% of the dry cellular weight . Both strains oxidized methylamine by the N-methylglutamate (N-MG) pathway enzymes (gamma-glutamylmethylamide synthetase/lyase and N-MG synthetase/lyase) to formaldehyde and assimilated it via the icl- serine pathway . The DNA G+C content was 60-4 mol% for Methylarcula marina h1T and 57.1 mol% for Methylarcula terricola h37T . The DNA-DNA hybridization value between strains hl and h37 was 25-30%, although they had a low level of DNA relatedness (5-7%) with the type strains of the serine pathway methylobacteria belonging to the genera Methylobacterium, Aminobacter, Methylorhabdus and Methylopila . A comparative 16S rDNA sequence-based phylogenetic analysis placed the two species of Methylarcula into a separate branch of the alpha-3 subclass of the Proteobacteria . The type strains of the new species are Methylarcula marina h1T (= VKM B-2159T) and Methylarcula terricola h37T (= VKM B-2160T).

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