Appl Microbiol Biotechnol, 2000 Sep, 54(3), 319 - 25 Polymer production by two newly isolated extremely halophilic archaea: application of a novel corrosion-resistant bioreactor; Hezayen FF et al.; A novel corrosion-resistant bioreactor composed of polyetherether ketone (PEEK), tech glass and silicium nitrite ceramics was constructed and applied for the cultivation of two newly isolated, extremely halophilic archaea producing poly(gamma-glutamic acid) (PGA), or poly(beta-hydroxy butyric acid) (PHB), respectively . These bacteria were isolated from hypersaline soil close to Aswan (Egypt) . The isolate strain 40, which is related to the genus Natrialba, produced large amounts of PGA when cultivated on solid medium . Culture conditions were optimised applying the corrosion-resistant bioreactor . PGA production was dependent on NaCl concentration and occurred about at 20% (w/v) NaCl in the medium . A maximum cell density of about 1.6 g cell dry matter/l was obtained when the bioreactor was stirred and aerated in a batch fermentation process using proteose-peptone medium . The supernatant was monitored with respect to PGA formation, and after 90 h a maximum of 470 mg/l culture volume was detected by HPLC analysis . Culture conditions were optimized for the isolate 56, which accumulated PHB as intracellular granules . Batch fermentations in the stirred and aerated bioreactor applying acetate and n-butyric acid as carbon sources led to cell density of 2.28 g cell dry matter/l and a maximum PHB accumulation contributing to about 53% of cellular dry weight . About 4.6 g PHB were isolated from 10.6 g dried cells of strain 56, which exhibited a weight average molar mass of 2.3 x 10(5) g mol(-1) and a polydispersity of about 1.4. Biophys Chem, 2000 Aug 30, 86(2-3), 155 - 64 Halophilic enzymes: proteins with a grain of salt; Mevarech M et al.; Halophilic enzymes, while performing identical enzymatic functions as their non-halophilic counterparts, have been shown to exhibit substantially different properties, among them the requirement for high salt concentrations, in the 1-4 M range, for activity and stability, and a high excess of acidic over basic amino residues . The following communication reviews the functional and structural properties of two proteins isolated from the extremely halophilic archaeon Haloarcula marismortui: the enzyme malate-dehydrogenase (hMDH) and the 2Fe-2S protein ferredoxin . It is argued that the high negative surface charge of halophilic proteins makes them more soluble and renders them more flexible at high salt concentrations, conditions under which non-halophilic proteins tend to aggregate and become rigid . This high surface charge is neutralized mainly by tightly bound water dipoles . The requirement of high salt concentration for the stabilization of halophilic enzymes, on the other hand, is due to a low affinity binding of the salt to specific sites on the surface of the folded polypeptide, thus stabilizing the active conformation of the protein. Biophys J, 2000 Oct, 79(4), 2132 - 7 Early intermediates in the photocycle of the Glu46Gln mutant of photoactive yellow protein: femtosecond spectroscopy; Devanathan S et al.; Transient absorption spectroscopy in the time range from -1 ps to 4 ns, and over the wavelength range from 420 to 550 nm, was applied to the Glu46Gln mutant of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila . This has allowed us to elucidate the kinetic constants of excited state formation and decay and photochemical product formation, and the spectral characteristics of stimulated emission and the early photocycle intermediates . Both the quantum efficiency ( approximately 0.5) and the rate constants for excited state decay and the formation of the initial photochemical intermediate (I(0)) were found to be quite similar to those obtained for wild-type PYP . In contrast, the rate constants for the formation of the subsequent photocycle intermediates (I(0)(double dagger) and I(1)), as well as for I(2) and for ground state regeneration as determined in earlier studies, were found to be from 3- to 30-fold larger . The structural implications of these results are discussed. Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 12176 - 81 Genome sequence of Halobacterium species NRC-1; Ng WV et al.; We report the complete sequence of an extreme halophile, Halobacterium sp . NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes . The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported . Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodium-proton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms . Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria . The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea. J Membr Biol, 2000 Oct 1, 177(3), 199 - 208 Vacuolar chloride transport in Mesembryanthemum crystallinum L . measured using the fluorescent dye lucigenin; Wissing F et al.; To study vacuolar chloride (Cl(-)) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl(-) uptake into isolated tonoplast vesicles was measured using the Cl(-)-sensitive fluorescent dye lucigenin (N,N'-dimethyl-9,9'-bisacridinium dinitrate) . Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl(-), with a Stern-Volmer constant of 173 m(-1) in standard assay buffer . While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions . However, the fluorescence intensity and Cl(-)-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion . Chloride transport into tonoplast vesicles of M . crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K(m) of 17.2 mm and a V(max) of 4.8 mm min(-1) . Vacuolar Cl(-) transport was not affected by sulfate, malate, or nitrate . In the presence of 250 microm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl(-) transport was actually significantly increased by 24% . To determine absolute fluxes of Cl(-) using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 x 10(7) m(-1) . After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl(-) transport of 31 nmol m(-2) sec(-1) was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques. Mikrobiologiia, 2000 Jul-Aug, 69(4), 465 - 70 {Synthesis of osmoprotectors by halophilic and alkalophilic methanotrophs}; Khmelenina VN et al.; The 1H-NMR analysis of methanol extracts of halophilic and halotolerant alkaliphilic methanotrophs isolated from the soda lakes of Southern Transbaikal and Tuva showed that bacterial cells grown at an optimum salinity accumulated mainly sucrose and 5-oxo-1-proline, whereas cells adapted to 0.5-1.0 M NaCl additionally synthesized ectoine . A more detailed study showed that nitrogen deficiency in the growth medium of Methylobacter alcaliphilus 20Z decreased the synthesis of nitrogen-containing osmoprotectants, ectoine and 5-oxo-1-proline . M . alcaliphilus 20Z cells exhibited activities of UDP-glucose pyrophosphorylase and sucrose-phosphate synthase involved in sucrose synthesis . Glutamine synthetase in vitro did not require NH4+ ions, which implies that this enzyme is involved in 5-oxo-1-proline synthesis . Cells grown at high salinity exhibited elevated levels of aspartate kinase, aspartate-semialdehyde dehydrogenase, and ectoine synthase . This suggests that ectoine is synthesized via aspartate and aspartate-semialdehyde, i.e., via the route earlier established for extremely halophilic bacteria. Science, 2000 Sep 15, 289(5486), 1902 - 6 Bacterial rhodopsin: evidence for a new type of phototrophy in the sea; Beja O et al.; Extremely halophilic archaea contain retinal-binding integral membrane proteins called bacteriorhodopsins that function as light-driven proton pumps . So far, bacteriorhodopsins capable of generating a chemiosmotic membrane potential in response to light have been demonstrated only in halophilic archaea . We describe here a type of rhodopsin derived from bacteria that was discovered through genomic analyses of naturally occuring marine bacterioplankton . The bacterial rhodopsin was encoded in the genome of an uncultivated gamma-proteobacterium and shared highest amino acid sequence similarity with archaeal rhodopsins . The protein was functionally expressed in Escherichia coli and bound retinal to form an active, light-driven proton pump . The new rhodopsin exhibited a photochemical reaction cycle with intermediates and kinetics characteristic of archaeal proton-pumping rhodopsins . Our results demonstrate that archaeal-like rhodopsins are broadly distributed among different taxa, including members of the domain Bacteria . Our data also indicate that a previously unsuspected mode of bacterially mediated light-driven energy generation may commonly occur in oceanic surface waters worldwide. Biochim Biophys Acta, 2000 Aug 30, 1460(1), 119 - 32 Lipidic cubic phase crystallization of bacteriorhodopsin and cryotrapping of intermediates: towards resolving a revolving photocycle; Pebay-Peyroula E et al.; Bacteriorhodopsin is a small retinal protein found in the membrane of the halophilic bacterium Halobacterium salinarum, whose function is to pump protons across the cell membrane against an electrostatic potential, thus converting light into a proton-motive potential needed for the synthesis of ATP . Because of its relative simplicity, exceptional stability and the fundamental importance of vectorial proton pumping, bacteriorhodopsin has become one of the most important model systems in the field of bioenergetics . Recently, a novel methodology to obtain well-diffracting crystals of membrane proteins, utilizing membrane-like bicontinuous lipidic cubic phases, has been introduced, providing X-ray structures of bacteriorhodopsin and its photocycle intermediates at ever higher resolution . We describe this methodology, the new insights provided by the higher resolution ground state structures, and review the mechanistic implications of the structural intermediates reported to date . A detailed understanding of the mechanism of vectorial proton transport across the membrane is thus emerging, helping to elucidate a number of fundamental issues in bioenergetics. Environ Manage, 2000 Nov, 26(5), 539 - 552 Riparian Vegetation and Water Chemistry in a Basin Under Semiarid Mediterranean Climate, Andarax River, Spain; SALINAS MJ et al.; A study has been made of the relationships between the characteristics of the riparian vegetation (floristic composition, structure and diversity) and the spatial-temporal variation of the quality of the stream waters in a basin under a semiarid Mediterranean climate in the southeastern Iberian Peninsula . The plant communities of the high reaches present greater specific richness and diversity (S(mean) = 7.0 +/- 3.4 and H'(mean) = 2.0 +/- 0.7) than do those of the middle and low reaches (S(mean) = 4.5 +/- 1.6 and H'(mean) = 1.8 +/- 0.6) . One zone reached the highest specific richness (S = 12, H' = 3.2), which, apart from being situated in the intermediate stretch of the basin, represents a transitional state (ecotone) between the Salix and Tamarix communities . The characteristics of the waters analyzed indicate very high rates of erosion and runoff due to the nature of the soils (easily eroded marls) and to agricultural expansion and mining since the 16th century . The present-day riparian vegetation is not adequate to absorb the nitrates added to the basin by crop fertilization, reaching extremely high values, particularly during the dry period (between 1.2 and 42.5 mg/liter) . Sewage dumping at three sampling stations did not appear to affect the specific composition of the woody vegetation . In the zones with watercourses, water salinity was low during the period of greater water flow, but considerably higher in the dry season (the upper limit was some 1.2 mS/m), resulting in a predominance of salt cedars over willows . Three types of saltcedar areas were distinguished: subhalophilous, which barely changes its chemical composition over the season; halophilous, which develops over strongly mineralized waters and markedly alters in chemical composition during the dry season; and hyperhalophilous, where salinity is extraordinarily high and quite constant throughout the year . A direct relationship was found between the dominance of Tamarix africana and abundance of NaCl. J, Exp . Mar . Biol . Ecol. . 2000 Jul 30, 250(1-2), 133 - 167 Review of nitrogen and phosphorus metabolism in seagrasses; Touchette BW et al.; Within the past few decades, major losses of seagrass habitats in coastal waters impacted by cultural eutrophication have been documented worldwide . In confronting a pressing need to improve the management and protection of seagrass meadows, surprisingly little is known about the basic nutritional physiology of these critical habitat species, or the physiological mechanisms that control their responses to N and P gradients . The limited available evidence to date already has revealed, for some seagrass species such as the north temperate dominant Zostera marina, unusual responses to nutrient enrichment in comparison to other vascular plants . Seagrasses derive N and P from sediment pore water (especially ammonium) and the water column (most nitrate) . The importance of leaves versus roots in nutrient acquisition depends, in part, on the enrichment conditions . For example, a shift from reliance on sediment pore water to increased reliance on the overlying water for N and P supplies has been observed under progressive water-column nutrient enrichment . Seagrasses may be N-limited in nutrient-poor waters with sandy or (less so) organic sediments, and P-limited in carbonate sediments . On the basis of data from few species, seagrasses appear to have active uptake systems for NO(3)(-) and PO(4)(-3), but NH(4)(+) uptake may involve both low- and high-affinity systems . P(i) uptake affinities reported thus far are much lower than values for active ammonium uptake, but comparable to values for nitrate uptake by leaf tissues . Beyond such basic information, seagrass species have shown considerable variation in nutritional response . Dominance of acropetal versus basipetal nutrient translocation appears to vary among species as an innate trait . While some species follow classic Michaelis-Menten kinetics for N(i) uptake, others have exhibited sustained linear uptake with limited or negligible product feedback inhibition, perhaps in adaptation to oligotrophic environments . Zostera marina also is able to maintain nitrate reductase (NR) activity during dark periods if adequate carbohydrate reserves and substrate are available . Thus, this species can respond to nitrate pulses throughout a diel cycle, rather than being limited as most plants to nitrate uptake during the light period . Further adaptations may have occurred for seagrasses in extremely nitrate-depauperate conditions . For example, Halophila decipiens and H . stipulacea lack inducible NR and apparently have lost the ability to reduce nitrate; and a biphasic rather than hyperbolic P(i) uptake curve, with 'surge' uptake, has been described for Zostera noltii . Many seagrasses respond favorably to low or moderate N and/or P enrichment . However, excessive N(i) loading to the water column can inhibit seagrass growth and survival, not only as an indirect effect by stimulating algal overgrowth and associated light reduction, but-for some species-as a direct physiological effect . The latter direct impact has been most pronounced for plants growing in sandy (nutrient-poor) sediments, and is exacerbated by elevated temperatures and/or light reduction . Ammonia toxicity, known for many vascular plants, has been reported in seagrasses Ruppia drepanensis and Z . marina (125 microM water-column NH(4)(+), 5 weeks) . Z . marina has shown to be inhibited, as well, by pulsed water-column nitrate enrichment (as low as 3.5-7 microM NO(3)(-), 3-5 weeks), which is actively taken up without apparent product feedback inhibition . Inhibition by elevated nitrate has also been reported, with description of the underlying physiological mechanisms, in certain macroalgae and microalgae . In Z . marina, this effect has been related to the high, sustained energy demands of nitrate uptake, and to inducement of internal carbon limitation by the concomitant 'carbon drain' into amino acid assimilation . In contrast, nitrate enrichment can stimulate growth of Z . marina when the sediment, rather than the water column, is the source . Because seagrass species have shown considerable variation in nutritional response, inferences about one well-studied species, from one geographic location, should not be applied a priori to that species in other regions or to seagrasses in general . Most of the available information has been obtained from study of a few species, and the basic nutritional physiology of many seagrasses remains to be examined and compared across geographic regions . Nonetheless, the relatively recent gains in general understanding about the physiological responses of some seagrass species to nutrient gradients already have proven valuable in both basic and applied research . For example, physiological variables such as tissue C:N:P content have begun to be developed as integrative indicators of nutrient conditions and anthropogenic nutrient enrichment . To strengthen insights for management strategies to optimize seagrass survival in coastal waters adjacent to exponential human population growth and associated nutrient inputs, additional emphasis is critically needed to assess the role of variable interactions-among inorganic as well as organic N, P and C, environmental factors such as temperature, light, and other community components-in controlling the physiology, growth and survival of these ecologically important marine angiosperms. Rapid Commun Mass Spectrom, 2000, 14(17), 1586 - 91 Characterisation of membrane phospholipids and glycolipids from a halophilic archaebacterium by high-performance liquid chromatography/electrospray mass spectrometry; Qiu D et al.; Combined high-performance liquid chromatography and electrospray mass spectrometry (LC/ES-MS) has been used for direct characterisation of the polar membrane lipids in total lipid extracts from Halobacterium salinarium, a species of halophilic archaebacterium . The principle phospholipids found were the diphytanyl archaeol phosphatidylglycerol and diphytanyl archaeol phosphatidylglycerolphosphate methyl ester . The application of LC/ES-MS revealed the additional presence of diphytanyl archaeol phosphatidylglycerol sulphate The extracts also contained an archaeol glycolipid, initially detected in preliminary offline ES-MS studies, which was further characterised by LC/ES-MS and by product ion tandem mass spectrometry (MS/MS) as a sulphate ester of diglycosyl-2,3-di-O-phytanyl-sn-glycerol . Whilst archaeol phospho- and glycolipids containing a (C(20)-C(20))-isopranyl glycerol ether core predominated, LC/ES-MS of the extracts from Halobacterium salinarium indicated the presence of an analogue containing one double bond in its isoprenyl ether core as a minor component of the phosphatidylglycerolphosphate methyl ester fraction, providing a further example of the previously recognised existence of isoprenologues of diphytanyl archaeols which occur as minor components of archaebacterial membrane lipids . The value of these techniques in compositional analysis of archaebacterial lipid extracts is discussed . Nature, 2000 Aug 10, 406(6796), 653 - 7 Molecular mechanism of vectorial proton translocation by bacteriorhodopsin; Subramaniam S et al.; Bacteriorhodopsin, a membrane protein with a relative molecular mass of 27,000, is a light driven pump which transports protons across the cell membrane of the halophilic organism Halobacterium salinarum . The chromophore retinal is covalently attached to the protein via a protonated Schiff base . Upon illumination, retinal is isomerized . The Schiff base then releases a proton to the extracellular medium, and is subsequently reprotonated from the cytoplasm . An atomic model for bacteriorhodopsin was first determined by Henderson et al, and has been confirmed and extended by work in a number of laboratories in the last few years . Here we present an atomic model for structural changes involved in the vectorial, light-driven transport of protons by bacteriorhodopsin . A 'switch' mechanism ensures the vectorial nature of pumping . First, retinal unbends, triggered by loss of the Schiff base proton, and second, a protein conformational change occurs . This conformational change, which we have determined by electron crystallography at atomic (3.2 A in-plane and 3.6 A vertical) resolution, is largely localized to helices F and G, and provides an 'opening' of the protein to protons on the cytoplasmic side of the membrane. J Bacteriol, 2000 Sep, 182(17), 5020 - 4 Glycine betaine transport in the obligate halophilic archaeon Methanohalophilus portucalensis; Lai MC et al.; Transport of the osmoprotectant glycine betaine was investigated using the glycine betaine-synthesizing microbe Methanohalophilus portucalensis (strain FDF1), since solute uptake for this class of obligate halophilic methanogenic Archaea has not been examined . Betaine uptake followed a Michaelis-Menten relationship, with an observed K(t) of 23 microM and a V(max) of 8 nmol per min per mg of protein . The transport system was highly specific for betaine: choline, proline, and dimethylglycine did not significantly compete for {(14)C}betaine uptake . The proton-conducting uncoupler 2, 4-dinitrophenol and the ATPase inhibitor N, N-dicyclohexylcarbodiimide both inhibited glycine betaine uptake . Growth of cells in the presence of 500 microM betaine resulted in faster cell growth due to the suppression of the de novo synthesis of the other compatible solutes, alpha-glutamate, beta-glutamine, and N(epsilon)-acetyl-beta-lysine . These investigations demonstrate that this model halophilic methanogen, M . portucalensis strain FDF1, possesses a high-affinity and highly specific betaine transport system that allows it to accumulate this osmoprotectant from the environment in lieu of synthesizing this or other osmoprotectants under high-salt growth conditions. Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1621 - 7 Haloanaerobium fermentans sp . nov., a strictly anaerobic, fermentative halophile isolated from fermented puffer fish ovaries; Kobayashi T et al.; A strain of strictly anaerobic and moderately halophilic bacteria isolated from salted puffer fish ovaries was studied phenotypically, genotypically and phylogenetically . On the basis of its physiological and morphological characteristics, the new isolate is considered to be a member of the genus Haloanaerobium . It is a motile, rod-shaped, non-spore-forming, gram-negative, obligate anaerobe that grows in the presence of 25% (w/v) NaCl . The optimum salt concentration for growth is 10% (w/v) . It grows well at 15 and 45 degrees C, but not at 10 or 50 degrees C . The optimum temperature for growth is 35 degrees C . It grows at pH 6.0-9.0 and the optimum pH for growth is 7.5 . It ferments N-acetylglucosamine, cellobiose, fructose, galactose, D-glucose, lactose, maltose, D-mannose, raffinose, D-ribose, sucrose and D-xylose . It ferments D-glucose with the production of hydrogen, carbon dioxide, ethanol and organic acids such as acetate, formate and lactate . 16S rRNA gene sequence information confirmed the phylogenetic position of the new isolate, strain R-9T, as a member of the genus Haloanaerobium . DNA-DNA hybridization data revealed that isolate R-9T exhibited low levels of reassociation (less than 30%) with previously described Haloanaerobium species . Based on these results, the new isolate appears to represent a new Haloanaerobium species, for which the name Haloanaerobium fermentans sp . nov . is proposed . The type strain is R-9T (= JCM 10494T). Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1563 - 89 Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence; Anzai Y et al.; The broad and vague phenotypic definition allowed the genus Pseudomonas to become a dumping ground for incompletely characterized polarly flagellated, gram-negative, rod-shaped, aerobic bacteria, and a large number of species have been accommodated in the genus Pseudomonas . The 16S rRNA sequences of 128 valid and invalid Pseudomonas species, which included almost valid species of the genus Pseudomonas listed in the Approved Lists of Bacterial Names, were obtained: sequences of 59 species were determined and those of 69 species were obtained from the GenBank/EMBL/DDBJ databases . These sequences were compared with the sequences of other species of the Proteobacteria . Fifty-seven valid or invalid species including Pseudomonas aeruginosa (type species of the genus Pseudomonas Migula 1894) belonged to the genus Pseudomonas (sensu stricto) . Seven subclusters were formed in the cluster of the genus Pseudomonas (sensu stricto), and the resulting clusters conformed well to the rRNA-DNA hybridization study by Palleroni (1984) . The other species did not belong to the genus Pseudomonas (sensu stricto) and were related to other genera, which were placed in four subclasses of the Proteobacteria (alpha, beta, gamma and gamma-beta subclasses) . Twenty-six examined species, which were not included in the cluster of the Pseudomonas (sensu stricto) and have not been transferred to other genera as yet, are listed alphabetically: 'Pseudomonas abikonensis', Pseudomonas antimicrobica, Pseudomonas beijerinckii, Pseudomonas beteli, Pseudomonas boreopolis, 'Pseudomonas butanovora', Pseudomonas carboxydohydrogena, Pseudomonas cissicola, Pseudomonas doudoroffii, Pseudomonas echinoides, Pseudomonas elongata, Pseudomonas flectens, Pseudomonas geniculata, Pseudomonas halophila, Pseudomonas hibiscicola, Pseudomonas huttiensis, Pseudomonas iners, Pseudomonas lanceolata, Pseudomonas lemoignei, Pseudomonas mephitica, Pseudomonas pictorum, Pseudomonas saccharophila, Pseudomonas spinosa, Pseudomonas stanier, Pseudomonas syzygii and Pseudomonas woodsii . The phylogenetic affiliations of these 26 pseudomonads species are shown. Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1501 - 3 Reclassification of Bacillus marismortui as Salibacillus marismortui comb . nov; Arahal DR et al.; Recently, the features of a group of strains isolated from Dead Sea enrichments obtained in 1936 by one of us (B . E . Volcani) were described . They were gram-positive, moderately halophilic, spore-forming rods, and were placed in a new species, Bacillus marismortui . At the same time, the new genus Salibacillus was proposed for the halophilic species Bacillus salexigens . B . marismortui and Salibacillus salexigens have similar phenotypic characteristics and the same peptidoglycan type . Phylogenetic analysis based on 16S rRNA sequence comparisons showed that they are sufficiently closely related (96.6% similarity) as to warrant placement in the same genus . However, DNA-DNA hybridization experiments showed that they constitute two separate species (41% DNA similarity) . Therefore the reclassification of Bacillus marismortui as Salibacillus marismortui comb . nov . is proposed. FEMS Microbiol Lett, 2000 Aug 15, 189(2), 211 - 4 Purification and characterization of a membrane-associated ATPase from Natronococcus occultus, a haloalkaliphilic archaeon; Eddy ML et al.; Isolated membranes of the extreme haloalkaliphilic archaeon Natronococcus occultus were able to hydrolyze ATP via an ATPase, which required the presence of Mg(2+), high concentrations of NaCl, and a pH value of 9 . The native molecular mass of the purified ATPase was 130 kDa and was composed of 74- and 61-kDa subunits . Enzyme activity was specific for the hydrolysis of ATP with slight activity towards GTP, CTP, and ITP . The enzyme required NaCl for maximal activity but Na(2)SO(4) and (NH(4))(2)SO(4) could substitute . The enzyme showed no activity if Na(2)SO(3) or sodium citrate was substituted for NaCl . The ATPase from N . occultus was inhibited by NBD-Cl, NaN(3), and ouabain, and was sensitive to nitrate, vanadate, DCCD, and bafilomycin A(1) . It was not inhibited by NEM in contrast to other previously characterized halophile ATPases . The ATPase had a K(M) of 0.5 mM and appeared to be non-competitively inhibited by NaN(3) with a K(I) of 3.1 mM. Appl Environ Microbiol, 2000 Aug, 66(8), 3269 - 76 Distribution and diversity of archaea corresponding to the limnological cycle of a hypersaline stratified lake (Solar lake, Sinai, Egypt); Cytryn E et al.; The vertical and seasonal distribution and diversity of archaeal sequences was investigated in a hypersaline, stratified, monomictic lake, Solar Lake, Sinai, Egypt, during the limnological development of stratification and mixing . Archaeal sequences were studied via phylogenetic analysis of 16S rDNA sequences as well as denaturing gradient gel electrophoresis analysis . The 165 clones studied were grouped into four phylogenetically different clusters . Most of the clones isolated from both the aerobic epilimnion and the sulfide-rich hypolimnion were defined as cluster I, belonging to the Halobacteriaceae family . The three additional clusters were all isolated from the anaerobic hypolimnion . Cluster II is phylogenetically located between the genera Methanobacterium and Methanococcus . Clusters III and IV relate to two previously documented groups of uncultured euryarchaeota, remotely related to the genus Thermoplasma . No crenarchaeota were found in the water column of the Solar Lake . The archaeal community in the Solar Lake under both stratified and mixed conditions was dominated by halobacteria in salinities higher than 10% . During stratification, additional clusters, some of which may possibly relate to uncultured halophilic methanogens, were found in the sulfide- and methane-rich hypolimnion. J Clin Microbiol, 2000 Aug, 38(8), 3123 - 4 Human infection with Halomonas venusta following fish bite; von Graevenitz A et al.; Halomonas venusta, a moderately halophilic, nonfermentative gram-negative rod, is reported for the first time as a human pathogen in a wound that originated from a fish bite. J Biol Chem, 2000 Jul 21, 275(29), 22196 - 201 Extreme halophiles synthesize betaine from glycine by methylation; Nyyssola A et al.; Glycine betaine is a compatible solute, which is able to restore and maintain osmotic balance of living cells . It is synthesized and accumulated in response to abiotic stress . Betaine acts also as a methyl group donor and has a number of important applications including its use as a feed additive . The known biosynthetic pathways of betaine are universal and very well characterized . A number of enzymes catalyzing the two-step oxidation of choline to betaine have been isolated . In this work we have studied a novel betaine biosynthetic pathway in two phylogenically distant extreme halophiles, Actinopolyspora halophila and Ectothiorhodospira halochloris . We have identified a three-step series of methylation reactions from glycine to betaine, which is catalyzed by two methyltransferases, glycine sarcosine methyltransferase and sarcosine dimethylglycine methyltransferase, with partially overlapping substrate specificity . The methyltransferases from the two organisms show high sequence homology . E . halochloris methyltransferase genes were successfully expressed in Escherichia coli, and betaine accumulation and improved salt tolerance were demonstrated. Arch Microbiol, 2000 May-Jun, 173(5-6), 445 - 8 Novel glycoproteins of the halophilic archaeon Haloferax volcanii; Eichler J; Archaea possess many eukaryote-like properties, including the ability to glycosylate proteins . Using oligosaccharide staining and lectin binding, this study revealed the existence of several glycosylated Haloferax volcanii membrane proteins, besides the previously reported surface layer (S-layer) glycoprotein . While the presence of glycoproteins in archaeal S-layers and flagella is well-documented, few archaeal glycoproteins that are not part of these structures have been reported . The glycosylated 150, 98, 58 and 54 kDa protein species detected were neither precursors nor breakdown products of the 190 kDa S-layer glycoprotein . Furthermore, these novel glycoproteins were outwardly oriented and intimately associated with the membrane. J Bacteriol, 2000 Aug, 182(15), 4328 - 36 Eight of fourteen gvp genes are sufficient for formation of gas vesicles in halophilic archaea; Offner S et al.; The minimal number of genes required for the formation of gas vesicles in halophilic archaea has been determined . Single genes of the 14 gvp genes present in the p-vac region on plasmid pHH1 of Halobacterium salinarum (p-gvpACNO and p-gvpDEFGHIJKLM) were deleted, and the remaining genes were tested for the formation of gas vesicles in Haloferax volcanii transformants . The deletion of six gvp genes (p-gvpCN, p-gvpDE, and p-gvpHI) still enabled the production of gas vesicles in H . volcanii . The gas vesicles formed in some of these gvp gene deletion transformants were altered in shape (Delta I, Delta C) or strength (Delta H) but still functioned as flotation devices . A minimal p-vac region (minvac) containing the eight remaining genes (gvpFGJKLM-gvpAO) was constructed and tested for gas vesicle formation in H . volcanii . The minvac transformants did not form gas vesicles; however, minvac/gvpJKLM double transformants contained gas vesicles seen as light refractile bodies by phase-contrast microscopy . Transcript analyses demonstrated that minvac transformants synthesized regular amounts of gvpA mRNA, but the transcripts derived from gvpFGJKLM were mainly short and encompassed only gvpFG(J), suggesting that the gvpJKLM genes were not sufficiently expressed . Since gvpAO and gvpFGJKLM are the only gvp genes present in minvac/JKLM transformants containing gas vesicles, these gvp genes represent the minimal set required for gas vesicle formation in halophilic archaea . Homologs of six of these gvp genes are found in Anabaena flos-aquae, and homologs of all eight minimal halobacterial gvp genes are present in Bacillus megaterium and in the genome of Streptomyces coelicolor. Syst Appl Microbiol, 2000 Apr, 23(1), 124 - 31 Nucleotide sequence of the 235 rRNA from Haloferax mediterranei and phylogenetic analysis of halophilic archaea based on LSU rRNA; Briones C et al.; 23S rRNA gene from the halophilic archaeon Haloferax mediterranei (strain ATCC 33500) was cloned and sequenced . Proceeding from the 2,912 nucleotides long sequence, the secondary structure of Haloferax genus large subunit rRNA was proposed . Haloferax mediterranei intergenic spacers 16S/23S and 23S/5S were also sequenced, and found to be 382 and 116 nucleotides long respectively . The 16S/23S spacer showed an Ala-tRNA intervening sequence, which is a common feature in Euryarchaeota . Sequence analysis of 23S rRNA and 16S rRNA was performed for the six organisms from the family Halobacteriaceae with both available gene sequences . Phylogenetic trees with completely different topology were obtained using both molecules. Appl Environ Microbiol, 2000 Jul, 66(7), 3052 - 7 Extremely halophilic bacteria in crystallizer ponds from solar salterns; Anton J et al.; It is generally assumed that hypersaline environments with sodium chloride concentrations close to saturation are dominated by halophilic members of the domain Archaea, while Bacteria are not considered to be relevant in this kind of environment . Here, we report the high abundance and growth of a new group of hitherto-uncultured Bacteria in crystallizer ponds (salinity, from 30 to 37%) from multipond solar salterns . In the present study, these Bacteria constituted from 5 to 25% of the total prokaryotic community and were affiliated with the Cytophaga-Flavobacterium-Bacteroides phylum . Growth was demonstrated in saturated NaCl . A provisional classification of this new bacterial group as "Candidatus Salinibacter gen . nov." is proposed . The perception that Archaea are the only ecologically relevant prokaryotes in hypersaline aquatic environments should be revised. J Biotechnol, 2000 May 26, 79(3), 193 - 203 Stability and stabilization of globular proteins in solution; Jaenicke R; Proteins are multifunctional: their amino acid sequences simultaneously determine folding, function and turnover . Correspondingly, evolution selected for compromises between rigidity (stability) and flexibility (folding/function/degradation), to the result that generally the free energy of stabilization of globular proteins in solution is the equivalent to only a few weak intermolecular interactions . Additional increments may come from extrinsic factors such as ligands or specific compatible solutes . Apart from the enthalpic effects, entropy may play a role by reducing the flexibility (cystine bridges, increased proline content), or by water release from residues buried upon folding and association . Additional quaternary interactions and closer packing are typical characteristics of proteins from thermophiles . In halophiles, protein stability and function are maintained by increased ion binding and glutamic acid content, both allowing the protein inventory to compete for water at high salt . Acidophiles and alkalophiles show neutral intracellular pH; proteins facing the outside extremes of pH possess anomalously high contents in ionizable amino acids . Global comparisons of the amino acid compositions and sequences of proteins from mesophiles and extremophiles did not result in general rules of protein stabilization, even after including complete genome sequences into the search . Obviously, proteins are individuals that optimize internal packing and external solvent interactions by very different mechanisms, each protein in its own way . Strategies deduced from specific ultrastable proteins allow stabilizing point mutations to be predicted. FEMS Microbiol Ecol, 2000 Jun 1, 32(3), 235 - 240 Hypersaline waters in salterns - natural ecological niches for halophilic black yeasts; Gunde-Cimermana N et al.; Hypersaline waters in salterns have so far been considered to be populated only with halophilic algae and bacteria and completely lacking halophilic fungi . In this paper we present population dynamics of polymorphic black yeasts, isolated from hypersaline waters (3-30% NaCl) of a saltern, in relation to different physicochemical parameters . Hortaea werneckii, Phaeotheca triangularis, Trimmatostroma salinum, Aureobasidium pullulans and Cladosporium spp . were detected with the highest frequency just before the peak of halite (NaCl) concentration . Since H . werneckii, P . triangularis and T . salinum are not known outside saline environments, these results suggest that hypersaline water is their natural ecological niche. Int J Food Microbiol, 2000 Jun 1, 56(2-3), 211 - 8 Differentiation of Tetragenococcus populations occurring in products and manufacturing processes of puffer fish ovaries fermented with rice-bran; Kobayashi T et al.; Tetragenococcus strains isolated from the manufacturing process of Japanese puffer fish ovaries fermented with rice-bran were characterized and differentiated phenotypically and genotypically . A total of 413 Tetragenococcus isolates were evaluated . On the basis of five representative substrates, the isolates were grouped into seven groups . An RFLP (restriction fragment length polymorphisms) analysis of the 16S rRNA gene of representative strains of major groups revealed that they could be grouped into two groups: one was identified as the most prominent halophilic lactic acid coccus, Tetragenococcus halophilus, and the other as T . muriaticus, which has recently been added to the genus Tetragenococcus as a new species . Physiologically, the major differences between the two groups were found in the ability to grow in medium not supplemented with NaCl and the fermentation of L-arabinose, sucrose and D-mannitol, and several other carbohydrates. Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1297 - 303 Natrinema versiforme sp . nov., an extremely halophilic archaeon from Aibi salt lake, Xinjiang, China; Xin H et al.; A novel extremely halophilic archaeon, strain XF10T, was isolated from a salt lake in China . This organism was neutrophilic, non-motile and pleomorphic, and was rod, coccus or irregularly shaped . It required at least 1.5 M NaCl for growth and grew in a wide range of MgCl2 concentrations (0.005-0.5 M) . Lipid extract of whole cells contained two glycolipids with the same chromatographic properties as two unidentified glycolipids found in the two described Natrinema species, Natrinema pellirubrum and Natrinema pallidum . Phylogenetic analysis based on 16S rDNA sequence comparison revealed that strain XF10T clustered with the two described Natrinema species and several other strains (strains T5.7, GSL-11 and Haloterrigena turkmenica JCM 9743) with more than 98.1% sequence similarities, suggesting that strain XF1OT belongs to the genus Natrinema . Comparative analysis of phenotypic properties and DNA-DNA hybridization between strain XF10T and the Natrinema species supported the conclusion that strain XF10T is a novel species within the genus Natrinema . The name Natrinema versiforme sp . nov . is proposed for this strain . The type strain is XF10T (=JCM 10478T=AS 1.2365T=ANMR 0149T). Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1229 - 37 Halothiobacillus kellyi sp . nov., a mesophilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium isolated from a shallow-water hydrothermal vent in the Aegean Sea, and emended description of the genus Halothiobacillus; Sievert SM et al.; A new mesophilic, chemolithoautotrophic, sulfur-oxidizing bacterium, strain Milos-BII1T, was isolated from a sediment sample taken from a shallow-water hydrothermal vent in the Aegean Sea with thiosulfate as electron donor and CO2 as carbon source . Based on the almost complete sequence of the 16S rRNA gene, strain Milos-BII1T forms a phylogenetic cluster with Thiobacillus hydrothermalis, Thiobacillus neapolitanus, Thiobacillus halophilus and Thiobacillus sp . W5, all of which are obligately chemolithoautotrophic bacteria . Because of their phylogenetic relatedness and their physiological similarities it is proposed to transfer these organisms to a newly established genus within the gamma-subclass of the Proteobacteria, Halothiobacillus gen . nov . (Kelly and Wood 2000) . Strain Milos-BII1T represents a new species of this genus, named Halothiobacillus kellyi . Cells were Gram-negative rods and highly motile . The organism was obligately autotrophic and strictly aerobic . Nitrate was not used as electron acceptor . Chemolithoautotrophic growth was observed with thiosulfate, tetrathionate, sulfur and sulfide . Growth was observed between pH values of 3.5 and 8.5, with an optimum at pH 6.5 . The temperature limits for growth were 3.5 and 49 degrees C, with an optimum between 37 and 42 degrees C . Growth occurred between 0 and 2 M NaCl, with an optimum NaCl concentration between 400 and 500 mM . The mean maximum specific growth rate on thiosulfate was 0.45 h(-1). Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1065 - 71 Haloterrigena thermotolerans sp . nov., a halophilic archaeon from Puerto Rico; Montalvo-Rodriguez R et al.; An extremely halophilic Archaeon belonging to the order Halobacteriales was isolated from the solar salterns of Cabo Rojo, Puerto Rico . The organism, designated strain PR5T, is rod-shaped, non-motile and requires at least 12% (w/v) NaCl to grow . The strain is highly thermotolerant: its temperature optimum is 50 degrees C and growth is possible up to 60 degrees C . Polar lipid analysis revealed the presence of the bis-sulfated glycolipid S2-DGD-1 as sole glycolipid and the absence of the glycerol diether analogue of phosphatidylglycerosulfate . Both C20,C20 and C20,C25 core lipids are present . The G+C content of the DNA is 63.3 mol% . According to 16S rDNA sequence data, strain PR5T is closely related to the representatives of the genera Haloterrigena and Natrinema, but on the basis of its phenotypic properties, 16S rDNA sequence and DNA-DNA hybridization studies, strain PR5T cannot be assigned to any of the recognized species within these genera . On the basis of its polar lipid composition, the isolate has been assigned to the genus Haloterrigena . The creation of a new species, Haloterrigena thermotolerans, is therefore proposed to accommodate this isolate . The type strain is strain PR5T (= DSM 11552T = ATCC 700275T). J Protein Chem, 1999 Nov, 18(8), 837 - 44 Identification and partial purification of DnaK homologue from extremely halophilic archaebacteria, Halobacterium cutirubrum; Tokunaga H et al.; The levels of synthesis of six proteins were increased at elevated growth temperature of the extremely halophilic archaebacterium Halobacterium cutirubrum . One of these proteins, with an apparent molecular mass of 97 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), bound to an ATP-agarose column in the presence of 4 M NaCl, but not in the absence of salt, indicating that this protein retained its ATP-binding activity only at high salt concentration . The NH2-terminal sequence of this protein and the internal sequences of the tryptic peptides covering 1/3 of the total number of residues coincided with that deduced from the nucleotide sequence of the dnaK gene isolated from H . cutirubrum . The results strongly suggest that this apparent 97-kDa protein is the gene product of dnaK, although the molecular mass calculated from the nucleotide sequence is only 68,495, much smaller than the value of this protein determined by SDS-PAGE . Ferguson plot analysis indicated that this protein showed anomalous mobility on SDS-PAGE . We have purified DnaK homologue to greater than 90% homogeneity with stepwise elution from an ATP-agarose column. Microbiology, 2000 May, 146 ( Pt 5), 1061 - 9 2-Oxoacid dehydrogenase multienzyme complexes in the halophilic Archaea? Gene sequences and protein structural predictions; Jolley KA et al.; All Archaea catalyse the conversion of pyruvate to acetyl-CoA via a simple pyruvate oxidoreductase . This is in contrast to the Eukarya and most aerobic bacteria, which use the pyruvate dehydrogenase multienzyme complex {PDHC}, consisting of multiple copies of three component enzymes: E1 (pyruvate decarboxylase), E2 (lipoate acetyl-transferase) and E3 (dihydrolipoamide dehydrogenase, DHLipDH) . Until now no PDHC activity has been found in the Archaea, although DHLipDH has been discovered in the extremely halophilic Archaea and its gene sequence has been determined . In this paper, the discovery and sequencing of an operon containing the DHLipDH gene in the halophilic archaeon Haloferax volcanii are reported . Upstream of the DHLipDH gene are 3 ORFs which show highest sequence identities with the E1alpha, E1beta and E2 genes of the PDHC from gram-positive organisms . Structural predictions of the proposed protein product of the E2 gene show a domain structure characteristic of the E2 component in PDHCs, and catalytically important residues, including the lysine to which the lipoic acid cofactor is covalently bound, are conserved . Northern analyses indicate the transcription of the whole operon, but no PDHC enzymic activity could be detected in cell extracts . The presence in the E2 gene of an insertion (equivalent to approximately 100 aa) not found in bacterial or eukaryal E2 proteins, might be predicted to prevent multienzyme complex assembly . This is the first detailed report of the genes for a putative 2-oxoacid dehydrogenase complex in the Archaea, and the evolutionary and metabolic consequences of these findings are discussed. Appl Environ Microbiol, 2000 Jun, 66(6), 2438 - 44 Characterization of a salt-tolerant family 42 beta-galactosidase from a psychrophilic antarctic Planococcus isolate; Sheridan PP et al.; We isolated a gram-positive, halotolerant psychrophile from a hypersaline pond located on the McMurdo Ice Shelf in Antarctica . A phylogenetic analysis of the 16S rRNA gene sequence of this organism showed that it is a member of the genus Planococcus . This assignment is consistent with the morphology and physiological characteristics of the organism . A gene encoding a beta-galactosidase in this isolate was cloned in an Escherichia coli host . Sequence analysis of this gene placed it in glycosidase family 42 most closely related to an enzyme from Bacillus circulans . Even though an increasing number of family 42 glycosidase sequences are appearing in databases, little information about the biochemical features of these enzymes is available . Therefore, we purified and characterized this enzyme . The purified enzyme did not appear to have any metal requirement, had an optimum pH of 6.5 and an optimum temperature of activity at 42 degrees C, and was irreversibly inactivated within 10 min when it was incubated at 55 degrees C . The enzyme had an apparent K(m) of 4.9 micromol of o-nitrophenyl-beta-D-galactopyranoside, and the V(max) was 467 micromol of o-nitrophenol produced/min/mg of protein at 39 degrees C . Of special interest was the finding that the enzyme remained active at high salt concentrations, which makes it a possible reporter enzyme for halotolerant and halophilic organisms. Biometals, 2000 Mar, 13(1), 23 - 8 Purification and characterization of a ferredoxin from Haloarcula japonica strain TR-1; Sugimori D et al.; A ferredoxin (Fd) was purified from the extremely halophilic archaeon, Haloarcula japonica strain TR-1, to electrophoretic homogeneity . The apparent molecular weight (Mr) of the Fd was estimated to be 24,000 on SDS-polyacrylamide gel electrophoresis . The amino acid composition analysis revealed that the Fd composed of a number of acidic amino acids (uncorrected for amides) . The N-terminal amino acid sequence (30 residues) was determined to be: PTVEYLNYEVVDDNGWDMYDDDVFAEASDM . The iron content was 3.42+/-0.04 mol/mol-Fd on the basis of the apparent Mr value . The absorption and ESR spectra of the Fd showed similarity to those of Fds from plant and Halobacterium halobium . These results led us to conclude that the H . japonica Fd contained a {2Fe-2S} cluster. Biosci Biotechnol Biochem, 2000 Apr, 64(4), 751 - 6 Characterization of a phage resistance plasmid, pLKS, of silage-making Lactobacillus plantarum NGRI0101; Eguchi T et al.; Phage contamination has resulted in abnormal fermentation in silage . We isolated a phage-resistant strain, Lactobacillus plantarum NGRI0101 from silage . The strain carried two plasmids, pLKL (6.8 kb) and pLKS (2.0 kb) . By curing and retransformation of the plasmids, we clarified that pLKS has phage resistant activity, characterized as no adsorption inhibition . pLKS has 2,025 bp and three orfs, orfl23, orf132, and orf918 . The predicted amino acid sequence of the orf918 product showed high similarity to those of Rep proteins of Pediococcus halophilus plasmid pUCL287 and Lactobacillus acidophilus plasmid pLA103 . The replication origin (ori) was upstream from orf918 . There was no gene similar to typical phage resistant genes encoded by known plasmids . The phage resistance of L . plantarum NGRI0101 may possibly be due to a plasmid-encoded abortive infection. Curr Microbiol, 2000 Jun, 40(6), 418 - 22 Recovery of Bacillus thuringiensis from marine sediments of Japan; Maeda M et al.; Marine sediments from a Japanese bay were examined for the occurrence of Bacillus thuringiensis . Of 1313 colonies belonging to the Bacillus cereus/B . thuringiensis group, 22 (1.7%) were allocated to B . thuringiensis . Marine isolates of B . thuringiensis consisted of heterogeneous multiple H serogroups; 10 isolates were assigned to the eight serovars (kurstaki, sumiyoshiensis, sotto, aizawai, darmstadiensis, thompsoni, neoleonensis, and higo); two motile isolates failed to react with the reference antisera; and the others were serologically untestable . Insecticidal activities were associated with two kurstaki isolates (toxic to both Lepidoptera and Diptera) and a higo isolate (Diptera-specific) . None of the parasporal inclusion proteins of the 22 isolates exhibited in vitro cytotoxic activity against two vertebrate cells, sheep erythrocytes and HeLa cells . All B . thuringiensis isolates had no halophilism, although seawater-based medium supported their growth, sporulation, and formation of parasporal inclusions. Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 183 - 90 Halorhabdus utahensis gen . nov., sp . nov., an aerobic, extremely halophilic member of the Archaea from Great Salt Lake, Utah; Waino M et al.; Strain AX-2T (T = type strain) was isolated from sediment of Great Salt Lake, Utah, USA . Optimal salinity for growth was 27% (w/v) NaCl and only a few carbohydrates supported growth of the strain . Strain AX-2T did not grow on complex substrates such as yeast extract or peptone . 16S rRNA analysis revealed that strain AX-2T 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 . The polar lipid composition comprising phosphatidyl glycerol, the methylated derivative of diphosphatidyl glycerol, triglycosyl diethers and sulfated triglycosyl diethers, but not phosphatidyl glycerosulfate, was not identical to that of any other aerobic, halophilic species . On the basis of the data presented, it is proposed that strain AX-2T should be placed in a new taxon, for which the name Halorhabdus utahensis is appropriate . The type strain is strain AX-2T (= DSM 12940T). Comp Biochem Physiol B Biochem Mol Biol, 2000 Feb, 125(2), 205 - 10 Characterization of acetohydroxy acid synthase activity in the archaeon Haloferax volcanii; Vyazmensky M et al.; Whereas the biochemistry of acetohydroxy acid synthase has been extensively studied in bacteria and eukaryotes, relatively little is known about the enzyme in archaea, the third kingdom of life . The present study biochemically characterizes acetohydroxy acid synthase activity in the halophilic archaea Haloferax volcanii . In addressing ion requirements, enzyme inhibition and antibody labeling, the results reveal that, except for its elevated salt requirements, the haloarchaeal enzyme is remarkably similar to its bacterial counterpart. J Biol Chem, 2000 Jul 28, 275(30), 22839 - 46 Evidence for post-translational membrane insertion of the integral membrane protein bacterioopsin expressed in the heterologous halophilic archaeon Haloferax volcanii; Ortenberg R et al.; The gene coding for the integral membrane protein bacterioopsin (Bop), that is composed of seven transmembrane helices, was expressed in the halophilic archaeon Haloferax volcanii as a fusion protein with the halobacterial enzyme dihydrofolate reductase and with the cellulose binding domain of Clostridium thermocellum cellulosome . In each case, bacterioopsin was present both in the membrane and in the cytoplasmic fractions . Pulse-chase labeling experiments showed that the fusion protein in the cytoplasmic fraction is the precursor of the membrane-bound species . Bacterioopsin mutants that lack the seventh helix (BopDelta7) were found to accumulate only in the cytoplasmic fraction, whereas bacterioopsin mutants that lack either helices four and five (BopDelta4-5), or helices one and two (BopDelta1-2), were found in the cytoplasmic as well as in the membrane fractions . The seventh helix, when expressed alone, could target in trans the insertion of a separately expressed bacterioopsin mutant protein that has only the first six helices . These results support a model in which bacterioopsin is produced in H . volcanii as a soluble protein and in which its insertion into the membrane occurs post-translationally . According to this model, membrane insertion is directed by the seventh helix. J Struct Biol, 2000 May, 130(1), 10 - 26 The cell surface glycoprotein layer of the extreme halophile Halobacterium salinarum and its relation to Haloferax volcanii: cryo-electron tomography of freeze-substituted cells and projection studies of negatively stained envelopes; Trachtenberg S et al.; We have studied the surface layer (S-layer) of Halobacterium salinarum (formerly Halobacterium halobium), an extreme halophile requiring high concentrations of sodium, by electron microscopy of (a) isolated, negatively stained, flattened envelopes and (b) cryo-fixation of intact cells in their high-salt growth medium followed by freeze substitution and tomography of thin sections . From the negatively stained isolated envelopes we have calculated a two-dimensional, projection map that is strikingly similar to that of Haloferax volcanii, an extreme halophile requiring high concentrations of magnesium; both projection maps show the hexagonal arrangement of the morphological units with an identical center-to-center spacing of 150 A; each of the morphological units of the two species has six subunits with a similar density distribution and apparent domain organization . In contrast to the two-dimensional map, the tomographic reconstruction of Halob . salinarum does not agree in a straightforward way with the three-dimensional, electron crystallographic map of negatively stained Halof . volcanii envelopes, although the main features of the lattice and the morphological units are evident . The tomographic reconstruction of sections from epoxy-embedded material suffers from directional compression due to sectioning stress and continuous dimensional changes and mass loss due to electron irradiation . This communication consists, therefore, of three parts: (a) a comparison of the projection maps of negatively stained envelopes of Halof . volcanii and Halob . salinarum; (b) a comparison of the three-dimensional maps obtained by electron crystallography (Halof . volcanii) and low-dose cryo-tomography (Halob . salinarum); and (c) a methodological study of mass loss and dimensional changes of plastic-embedded material under low-dose conditions at room and liquid nitrogen temperatures . Extremophiles, 2000 Apr, 4(2), 91 - 8 Halophilic adaptation of enzymes; Madern D et al.; It is now clear that the understanding of halophilic adaptation at a molecular level requires a strategy of complementary experiments, combining molecular biology, biochemistry, and cellular approaches with physical chemistry and thermodynamics . In this review, after a discussion of the definition and composition of halophilic enzymes, the effects of salt on their activity, solubility, and stability are reviewed . We then describe how thermodynamic observations, such as parameters pertaining to solvent-protein interactions or enzyme-unfolding kinetics, depend strongly on solvent composition and reveal the important role played by water and ion binding to halophilic proteins . The three high-resolution crystal structures now available for halophilic proteins are analyzed in terms of haloadaptation, and finally cellular response to salt stress is discussed briefly. FEMS Microbiol Lett, 2000 May 15, 186(2), 171 - 5 Organophosphonate metabolism by a moderately halophilic bacterial isolate; Hayes VE et al.; A Gram-negative halophile isolated from soil beneath a road gritting salt pile grew optimally at 10% (w/v) NaCl and was shown most likely to be Chromohalobacter marismortui or Pseudomonas beijerinckii on the basis of 16S rRNA analysis . The strain utilised phosphonoacetate, 2-aminoethyl-, 3-aminopropyl-, 4-aminobutyl-, methyl- and ethyl-phosphonates as phosphorus sources for growth . Differences were observed in the growth rate on different phosphonates and the range of phosphonates utilised at elevated NaCl concentrations, possibly as a result of differentially-induced transport mechanisms . An assay of cell-free extracts of 2-aminoethylphosphonate (2AEP) grown cells showed no detectable 2AEP:pyruvate aminotransferase or phosphonoacetaldehyde hydrolase activity. Syst Appl Microbiol, 1999 Dec, 22(4), 520 - 9 Genetic organization of the mobilization region of the plasmid pHE1 from Halomonas elongata; Vargas C et al.; The mobilization (mob) region of the non-self transmissible 4.2-kb plasmid pHE1 from the moderately halophilic bacterium Halomonas elongata ATCC 33174 has been identified and characterized . Analysis of the sequence revealed the presence of four open reading frames (mobCABD) which show a complex organization with two of them (mobB and mobD) entirely overlapped by a third (mobA) . The deduced proteins appeared to have a high degree of homology to Mob proteins of CoIE1 and closely related plasmids . To assess the functionality of the mob region, the hybrid vector pHS134 was constructed, consisting of the complete plasmid pHEI, the E . coli vector pKS(-) and a streptomycin-resistance gene for positive selection in Halomonas . Vector pHS134 was found to be mobilizable from E . coli to H . elongata assisted by pRK600 . Upstream of the mob genes, an oriT region with a putative nick sequence highly homologous to that of CoIE1 plasmids was identified . To our knowledge, this is the first mobilizable plasmid found in moderate halophiles . This property, together with its small size, the availability of its complete sequence, and its broad host range in moderately halophilic strains, makes pHE1 a good candidate for the construction of cloning and expression vectors for these extremophiles. Microb Ecol, 2000 Jan, 39(1), 22 - 31 Phylogenetic Analysis of Bacterial Communities Associated with Leaves of the Seagrass Halophila stipulacea by a Culture-Independent Small-Subunit rRNA Gene Approach; Weidner S et al.; The phylogenetic diversity of the bacterial community associated with leaves of the marine plant Halophila stipulacea in the northern Gulf of Elat was examined by 16S rRNA gene (rDNA) sequence analyses of a clone library . For 59 clones corresponding to 51 ARDRA (amplified rDNA restriction analysis) groups, the sequence of approximately 1 kb was determined, and the fraction of the corresponding ARDRA groups of the leaf library was calculated . The class Proteobacteria was represented by 62.6% of the clone sequences . Most sequences originated from members of the gamma-subclass (27.3%), affiliated with members of the genera Pseudomonas, Vibrio, Marinomonas, Oceanospirillum, and other marine groups . Affiliation to the alpha-subclass was determined for 24.2% of the sequences . They were related to the genera Hyphomonas, Roseobacter, Ruegeria, and Rhizobiaceae . Several alpha-proteobacterial sequences were distantly related to known sequences . Only 4% of the clone sequences were related to beta-Proteobacteria . Additionally, 7.1% of the sequences possibly belonged to the class Proteobacteria, but branched deeply from known subclasses . Several sequences were affiliated to members of the orders Verrucomicrobiales and Planctomycetales, the Holophaga/Acidobacterium phylum, and chloroplasts of marine diatoms . </hea Microbiology, 2000 Apr, 146 ( Pt 4), 861 - 8 The alpha-amylase gene amyH of the moderate halophile Halomonas meridiana: cloning and molecular characterization; Coronado MJ et al.; Two types of Tn1732-induced mutants defective in extracellular amylase activity were isolated from the moderate halophile Halomonas meridiana DSM 5425 . Type I mutants displayed amylase activity in the periplasm, and were unable to use any of the carbon sources tested, including starch and its hydrolysis product maltose . The type II mutant was affected in the gene responsible for the synthesis of the extracellular alpha-amylase . This gene (amyH) was isolated by functional complementation of mutant II and sequenced . The deduced protein (AmyH) showed a high degree of homology to a proposed family of alpha-amylases consisting of enzymes from Alteromonas (Pseudoalteromonas) haloplanktis, Thermomonospora curvata, streptomycetes, insects and mammals . AmyH contained the four highly conserved regions in amylases, as well as a high content of acidic amino acids . The amyH gene was functional in the moderate halophile Halomonas elongata and, when cloned in a multicopy vector, in Escherichia coli . AmyH is believed to be the first extracellular-amylase-encoding gene isolated from a moderate halophile, a group of extremophiles of great biotechnological potential . In addition, H . meridiana and H . elongata were able to secrete the thermostable alpha-amylase from Bacillus licheniformis, indicating that members of the genus Halomonas are good candidates for use as cell factories to produce heterologous extracellular enzymes. Nucleic Acids Symp Ser, 1999, (42), 75 - 6 Molecular cloning of A1-ATPase gene from extremely halophilic archaeon Haloarcula japonica strain TR-1; Yatsunami R et al.; The genes encoding A1-ATPase A- and B-subunits were cloned from Haloarcula japonica strain TR-1 . Nucleotide sequencing analysis of the A1-ATPase gene revealed that the A- and B-subunits consisted of 586 and 473 amino acids, respectively . The deduced amino acid sequences of the A- and B-subunits of Ha . japonica showed high identities with those of Halobacterium salinarum and Haloferax volcanii . The consensus ATP-binding motif was found in the A-subunit. Nucleic Acids Symp Ser, 1999, (42), 73 - 4 Transcriptional regulation of cruxrhodopsin gene from extremely halophilic archaeon Haloarcula japonica strain TR-1; Yatsunami R et al.; Transcription of the cruxrhodopsin (cR) gene in extremely halophilic archaeon Haloarcula japonica strain TR-1 was investigated using Northern analysis to quantify message level . In the cell cultures growing in the dark, cR transcript level remained very low . In contrast, exposure of the cell cultures to light stimulated transcription of the cR gene . In addition, cR gene transcription was also induced when Ha . japonica was grown under high oxygen tension and then shifted to low oxygen tension in the dark . These results suggested that transcription of the cR gene is regulated by high light intensity and low oxygen tension. Acta Crystallogr D Biol Crystallogr, 2000 May, 56 ( Pt 5), 645 - 7 Expression, purification, crystallization and preliminary X-ray diffraction studies of bacterial and archaeal L4 ribosomal proteins; Worbs M et al.; Ribosomal protein L4 is implicated in the peptidyltransferase activity of the ribosome and in certain bacteria it regulates the transcription and translation of the 11-gene S10 operon . The genes for the L4 ribosomal proteins from the hyperthermophilic bacterium Thermotoga maritima and the halophilic archaeon Haloarcula marismortui have been PCR amplified from genomic DNA and cloned under the control of a T7 promoter to generate overexpressing Escherichia coli strains . For both proteins, efficient purification procedures were developed to yield material suitable for crystallization trials . Crystals of T . maritima L4 were obtained in the orthorhombic space group P2(1)2(1)2(1), with one molecule per asymmetric unit, diffracting to 1.7 A resolution with synchrotron radiation . Crystals of H . marismortui L4 belonged to the trigonal space group P3(1)21 or P3(2)21 and diffracted to 3.2 A resolution with a rotating-anode source, presumably containing three molecules per asymmetric unit . The results demonstrate that for certain halophilic proteins the same purification and crystallization procedures can be employed as for conventional proteins. Dis Aquat Organ, 2000 Feb 9, 39(3), 193 - 9 Epidermal lesions and mortality caused by vibriosis in deep-sea Bahamian echinoids: a laboratory study; Bauer JC et al.; When significant mortality of the bathyal spatangoid echinoid Paleopneustes cristatus occurred under laboratory conditions, we investigated the cause and course of the disease by culturing and identifying internal pathogens, then experimentally infecting healthy urchins with isolates of the suspected disease organism . The pathogen was determined to be the gram-negative halophilic bacterium Vibrio alginolyticus . This species was also recovered from frozen post-challenge specimens of P . cristatus and from moribund individuals of Archaeopneustes hystrix, another spatangoid reared under similar in vitro conditions . This is the first experimental study of bacterial disease in any deep-sea invertebrate. Mol Microbiol, 2000 Apr, 36(1), 105 - 13 The gene for a halophilic beta-galactosidase (bgaH) of Haloferax alicantei as a reporter gene for promoter analyses in Halobacterium salinarum; Patenge N et al.; Investigations of transcriptional regulation and the characterization of promoters in homologous expression systems are most easily performed using suitable reporter genes . Presumably because of the high internal salt concentration in halophilic Archaea, the successful application of the commonly used reporter genes has not been reported so far . Recently, the gene for an extremely halophilic beta-galactosidase (bgaH) from Haloferax alicantei has become available . After transformation of Halobacterium salinarum with a vector-carrying bgaH, the enzyme activity in cell lysates could be readily determined by a simple colorimetric assay and colonies could be screened for activity on plates containing Xgal substrate . Expression of bgaH under the control of various halobacterial promoters of known strength led to different specific beta-galactosidase activities in the lysates . Using Northern blot hybridization and semiquantitative RT-PCR, it was shown that the bgaH transcript level corresponded to the specific enzyme activity . Therefore, the bgaH gene of Haloferax alicantei appears to be a useful tool for in vivo studies of gene expression in Halobacterium salinarum and possibly other halophilic Archaea. Mol Microbiol, 2000 Mar, 35(6), 1493 - 505 The extremely halophilic archaeon Haloferax volcanii has two very different dihydrofolate reductases; Ortenberg R et al.; The gene encoding dihydrofolate reductase, hdrA, from the extremely halophilic archaeon Haloferax volcanii was previously isolated from a spontaneous trimethoprim-resistant mutant in a DNA sequence that had undergone amplification . Here, we show that deletion of hdrA did not affect growth in minimal medium and that the strain carrying the deletion remained sensitive to trimethoprim . A spontaneous trimethoprim-resistant colony was isolated in the hdrA deletion strain and found to possess a new DNA amplification . Sequencing of the amplification revealed a second, substantially different, dihydrofolate reductase gene, hdrB, which was found to be located immediately downstream of the thymidylate synthase gene, hts . The physiological role of hDHFR-1 and hDHFR-2 was determined by generating Haloferax volcanii strains in which each gene, hdrA or hdrB, or both genes were deleted . It was found that hdrB alone can support growth of Haloferax volcanii in minimal medium, whereas hdrA alone can support growth of Haloferax volcanii in minimal medium only when the medium is supplemented with thymidine . It was also shown that, in contrast to Escherichia coli, the DeltahdrA, DeltahdrB double deletion mutant is viable in the presence of a functional thymidylate synthase gene . The hdrB gene was overexpressed in Escherichia coli and the enzyme purified to homogeneity . The biochemical properties of the new enzyme (hDHFR-2) are markedly different from those of hDHFR-1 . The use of the dihydrofolate reductase and thymidylate synthase genes as stable selectable markers is described. Plant Physiol, 2000 Apr, 122(4), 1239 - 47 Ectoine, the compatible solute of Halomonas elongata, confers hyperosmotic tolerance in cultured tobacco cells; Nakayama H et al.; 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) functions as a compatible osmolyte in the moderate halophile Halomonas elongata OUT30018 . Ectoine is biosynthesized by three successive enzyme reactions from aspartic beta-semialdehyde . The genes encoding the enzymes involved in the biosynthesis, ectA, ectB, and ectC, encoding L-2,4-diaminobutyric acid acetyltransferase, L-2, 4-diaminobutyric acid transaminase, and L-ectoine synthase, respectively, have been previously cloned . To investigate the function of ectoine as a compatible solute in plant cells, the three genes were individually placed under the control of the cauliflower mosaic virus 35S promoter and introduced together into cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells . The transgenic BY2 cells accumulated a small quantity of ectoine (14-79 nmol g(-1) fresh weight) and showed increased tolerance to hyperosmotic shock (900 mOsm) . Furthermore, the transgenic BY2 cells exhibited a normal growth pattern even under hyperosmotic conditions (up to 530 mOsm), in which the growth of the untransformed BY2 (wild type) cells was obviously delayed . These results suggest that genetically engineered synthesis of ectoine results in the increased hyperosmotic tolerance of cultured tobacco BY2 cells despite the low level of accumulation of the solute. Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 559 - 64 Thermohalobacter berrensis gen . nov., sp . nov., a thermophilic, strictly halophilic bacterium from a solar saltern; Cayol JL et al.; A new thermophilic, strictly halophilic, anaerobic, non-sporulating rod-shaped bacterium, measuring 0.5 x 3.0-8.0 microns and designated strain CTT3T, was isolated from a solar saltern . Strain CTT3T stained Gram-negative, was motile by means of laterally inserted flagella, had a genome G + C content of 33 mol% and grew optimally at 65 degrees C and pH 7.0 with 5% NaCl . The strain also grew readily at 70 degrees C in the presence of 15% NaCl . Strain CTT3T fermented cellobiose, fructose, glucose, maltose, mannitol, mannose, sucrose, glycerol, N-acetylglucosamine, starch, pyruvate and bio-Trypticase . It produced acetate, ethanol, H2 and presumably CO2 from glucose . 16S rRNA gene sequence analysis indicated that it is a member of cluster XII of the Clostridiales and related genera of the subphylum of the Gram-positive bacteria containing genomes of low G + C content . Its phenotypic and phylogenetic characteristics clearly differentiated it from all other members of this cluster . Based on the findings it is proposed that strain CTT3T be designated as a new species of a new genus, Thermohalobacter berrensis gen . nov., sp . nov . The type strain is CTT3T (= CNCM 105955T). J Appl Microbiol, 2000 Mar, 88(3), 495 - 503 Cloning and expression of alpha-amylase from the hyperthermophilic archaeon Pyrococcus woesei in the moderately halophilic bacterium Halomonas elongata; Frillingos S et al.; An extracellular alpha-amylase gene from the hyperthermophilic archaeon Pyrococcus woesei has been cloned and sequenced . The 1.4-kb protein-coding sequence is identical to that of the corresponding alpha-amylase gene of the closely related species P . furiosus . By using a shuttle cloning vector for halophilic bacteria, the P . woesei alpha-amylase was expressed in the moderate halophile Halomonas elongata, under the control of a native H . elongata promoter . The hyperthermophilic amylase activity expressed in the halophilic host was recovered completely in the crude membrane fraction of cell homogenates, suggesting the formation of inclusion bodies or that the secretion machinery of H . elongata may fail to recognize and release the pyrococcal alpha-amylase to the extracellular medium . However, thermal stability, metal ion interactions, optimal temperature and pH values for the crude and purified recombinant alpha-amylase were comparable with those of the native pyrococcal enzyme . The P . woesei amylase activity expressed in H . elongata was consistently detected in the cells upon growth on a wide range of NaCl concentrations (0.7-2.5 mol l-1) . To our knowledge, this is the first report on the expression of an archaeal gene (P . woesei alpha-amylase) in a moderate halophilic host which serves as a cell factory able to grow under extreme salt conditions and with very simple nutritional requirements. Int J Food Microbiol, 2000 Mar 10, 54(1-2), 81 - 9 Strictly anaerobic halophiles isolated from canned Swedish fermented herrings (Surströmming); Kobayashi T et al.; Strictly anaerobic halophiles were isolated from canned Swedish fermented herrings (Surstromming) . All isolates were phenotypically uniform with some exceptions and were identified as the genus Haloanaerobium and assigned to either Haloanaerobium praevalens or Haloanaerobiuim alcaliphilum . A comparative analysis of 16S rDNA sequences revealed that the representative strain S-8 of the isolates was identical to that of Haloanaerobium praevalens DSM 2228T . Furthermore, this strain exhibited high levels (> 80%) of DNA-DNA homology with Haloanaerobium praevalens DSM 2228T . This is a novel report of halophilic anaerobes isolated from a food product . Such anaerobes may contribute to the intense flavor and the swollen can characteristics of Swedish fermented herring. Int J Food Microbiol, 2000 Mar 10, 54(1-2), 9 - 18 Occurrence and expression of virulence-related properties of Vibrio species isolated from widely consumed seafood products; Baffone W et al.; In this study, widely consumed fresh seafood products were examined for the presence of Vibrio spp . Thirteen percent of the samples examined were found to be contaminated with halophilic vibrios belonging to the species V . alginolyticus (81.48%), V . parahaemolyticus (14.8%) and V . cholerae non 0:1 (3.7%) . A greater isolation frequency (18.9%) was found for mussels . Significant adhesiveness and strong cytotoxicity factors were revealed in a significant number of the Vibrio spp . isolated . These results confirm that the presence of Vibrio spp . in seafood products is common, and suggest that routine examination of such products for these pathogenic agents would be advisable. Appl Environ Microbiol, 2000 Apr, 66(4), 1572 - 9 Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions; Barth S et al.; The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures . The major disadvantage of this technique is the low yield of active protein . Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli . rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes . Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations . Adding 10 mM glycine betaine for the cultivation of E . coli under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs . Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations . The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments . This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems. Extremophiles, 2000 Feb, 4(1), 35 - 41 Halocins: are they involved in the competition between halobacteria in saltern ponds? Kis-Papo T, Oren A. Many representatives of the family Halobacteriaceae ("halobacteria") excrete halophilic bacteriocins (halocins) that inhibit the growth of other halobacteria . In spite of the fact that halocin production is widespread among the Halobacteriaceae, no information is available on their ecological significance . To test whether halocins may play a role in the interspecies competition between different types of halobacteria in saltern crystallizer ponds inhabited by dense communities of these red halophiles, we assayed for halocins active against a variety of halobacteria in salterns from different locations worldwide . Detection of halocin activity was based on the inhibition of growth of indicator organisms on agar plates, the decreased incorporation of radiolabeled substrates, and microscopic examinations . No halocin activity was detected in any of the brines examined, in spite of the fact that halocin production was demonstrated in cultures of most microorganisms isolated from these brines . Thus, the contribution of halocins in the competition between different halobacteria in hypersaline aquatic environments is probably negligible. Immunopharmacol Immunotoxicol, 2000 Feb, 22(1), 131 - 41 Effect of exopolysaccharide V2-7, isolated from Halomonas eurihalina, on the proliferation in vitro of human peripheral blood lymphocytes; Perez-Fernandez ME et al.; The immunomodulatory activity of the exopolysaccharide V2-7, a sulfated polymer excreted by the moderately halophilic bacteria Halomonas eurihalina, was studied in vitro . {3H}thymidine incorporation and flow-cytometry measurements showed that this exopolysaccharide enhanced the unspecific proliferation of human lymphocytes in response to the presence of anti-CD3 monoclonal antibody . It was effective at concentrations of less than 1 microg/ml, maximum activity being achieved at 0.2 microg/ml. Biochem J, 2000 Mar 1, 346 Pt 2, 251 - 4 The ShBle resistance determinant from Streptoalloteichus hindustanus is expressed in Haloferax volcanii and confers resistance to bleomycin; Nuttall SD et al.; We have designed a gene cassette for expression of the bleomycin-resistance protein from Streptoalloteichus hindustanus (ShBle) in the extremely halophilic archaeon Haloferax volcanii, and shown that transformed haloarchaea are resistant to bleomycin . Recombinant ShBle was purified by a one-step affinity-chromatography procedure as a correctly folded, dimeric protein . ShBle thus provides a useful haloarchaeal selectable marker and represents the first non-halophilic and soluble heterologous protein to be expressed in the Haloarchaea. Res Microbiol, 2000 Jan-Feb, 151(1), 13 - 8 Transposon mutagenesis in Halomonas eurihalina; Llamas I et al.; We have established a transposon mutagenesis procedure for the moderate halophile Halomonas eurihalina, a bacteria that produces an exopolysaccharide (EPS) of considerable biotechnological interest . We used suicide plasmids pUT and pSUP102 to introduce the transposons mini-Tn5 and Tn1732 into H . eurihalina via Escherichia coli mediated conjugation . Southern hybridization analysis demonstrated that insertions of the transposon mini-Tn5 into H . eurihalina occurred randomly at single sites in the chromosome, whereas Tn1732 insertion also took place at random, but simultaneously, at several sites . Phenotypic analysis revealed that different mutants were generated by using mini-Tn5 . The isolation of exopolysaccharide-defective strains is the first stage towards carrying out genetic studies on EPS production by this microorganism. Gene, 2000 Jan 25, 242(1-2), 357 - 67 The ftsZ gene of Haloferax mediterranei: sequence, conserved gene order, and visualization of the FtsZ ring; Poplawski A et al.; We sequenced the ftsZ gene region of the halophilic archaeon Haloferax mediterranei and mapped the transcription start sites for the ftsZ gene . The gene encoded a 363-amino-acid long FtsZ protein with a predicted molecular mass of 38 kDa and an isoelectric point of 4.2 . A high level of similarity to the FtsZ protein of Haloferax volcanii was apparent, with 97 and 90% identity at the amino acid and nucleotide levels, respectively . Structural conservation at the protein level was shown by visualization of the FtsZ ring structure in H . mediterranei cells using an antiserum raised against FtsZ of H . volcanii . FtsZ rings were observed in cells in different stages of division, including cells with pleomorphic shapes and cells that appeared to be undergoing asymmetric division . Cells were also observed that displayed constriction-like invaginations in the absence of an FtsZ ring, indicating that morphological data are not sufficient to determine whether pleomorphic Haloferax cells are undergoing cell division . Both the upstream and downstream gene order in the ftsZ region was found to be conserved within the genus Haloferax . Furthermore, the downstream gene order, which includes the secE and nusG genes, is conserved in almost all euryarchaea sequenced to date . The secE and nusG genes are likely to be transcriptionally and translationally coupled in Haloferax, and this co-expression may have been a selective force that has contributed to keeping the gene cluster intact. Can J Microbiol, 2000 Feb, 46(2), 180 - 7 The effects of ultraviolet radiation on the moderate halophile Halomonas elongata and the extreme halophile Halobacterium salinarum; Martin EL et al.; Both the moderately halophilic bacterium, Halomonas elongata, and the extremely halophilic archaea, Halobacterium salinarum, can be found in hypersaline environments (e.g., salterns) . On complex media, H . elongata grows over a salt range of 0.05-5.2 M, whereas, H . salinarum multiplies over a salt range of 2.5-5.2 M . The purpose of this study was to illustrate the effect that solar (UV-A and UV-B) and germicidal radiation (UV-C) had on the growth patterns of these bacteria at varied salt concentrations . Halomonas elongata grown on a complex medium at 0.05, 1.37, and 4.3 M NaCl was found to be more sensitive to UV-A and UV-B radiation, as the salt concentration of the medium increased . Halobacterium salinarum grown on a complex medium at 3.0 and 4.3 M NaCl did not show a significant drop in viability after 39.3 kJ.m-2 of UV-A and UV-B exposure . When exposed to UV-C, H . elongata exhibited substantially more sensitivity than H . salinarum . In H . elongata, differential sensitivity to UV-C was observed . At 0.05 M NaCl, H . elongata was less sensitive to UV-C than at 1.37 and 4.3 M NaCl . Both bacteria showed some photoreactivation when incubated under visible light following both UV-A, UV-B, and UV-C exposure . Mutagenesis following UV-C exposure was demonstrated by both organisms. Mol Microbiol, 2000 Mar, 35(5), 1168 - 79 The archaeal halophilic virus-encoded Dam-like methyltransferase M . phiCh1-I methylates adenine residues and complements dam mutants in the low salt environment of Escherichia coli; Baranyi U et al.; The genome of the archaeal virus phiCh1, infecting Natrialba magadii (formerly Natronobacterium magadii), is composed of 58.5 kbp linear ds DNA . Virus particles contain several RNA species in sizes of 100-800 nucleotides . A fraction of phiCh1 genomes is modified within 5'-GATC-3' and related sequences, as determined by various restriction enzyme digestion analyses . High performance liquid chromatography revealed a fifth base, in addition to the four nucleosides, which was identified as N6-methyladenosine . Genetic analyses and subsequent sequencing led to the identification of a DNA (N6-adenine) methyltransferase (mtase) gene . The protein product was designated M.phiCh1-I . By the localization of the most conserved motifs (a DPPY motif occurring before FxGxG), the enzyme was placed within the beta-subgroup of the (N6-adenine) methyltransferase class . The mtase gene of phiCh1 was classified as a 'late' gene, as determined by measuring the kinetics of mRNA and protein expression in N . magadii during the lytic cycle of phiCh1 . After infection of cells, M.phiCh1-I mRNA and protein could be detected in lower amounts than in the situation of virus induction from lysogenic cells . Consequently, only about 5% of the phiCh1 progeny genomes after infection of N . magadii carry the M.phiCh1-I methylation in contrast to 50% of virus genomes generated by induction of phiCh1-lysogenic N . magadii cells . Heterologous expression of the mtase from a halophile with 3 M cytoplasmic salt concentration showed an unexpected feature: the protein was active in the low environment of Escherichia coli and was able to methylate DNA in vivo . Interestingly, it seemed to exhibit a higher sequence specificity in E . coli that resulted in adenine methylation exclusively in the sequence 5'-GATC-3' . Additionally, expression of M.phiCh1-I in dam- E . coli cells led to a complete substitution of the function of M.Dam in DNA mismatch repair. Microbiology, 2000 Feb, 146 ( Pt 2), 455 - 63 Genes for the synthesis of the osmoprotectant glycine betaine from choline in the moderately halophilic bacterium Halomonas elongata DSM 3043, USA; Canovas D et al.; The genes involved in the oxidative pathway of choline to glycine betaine in the moderate halophile Halomonas elongata DSM 3043 were isolated by functional complementation of an Escherichia coli strain defective in glycine betaine synthesis . The cloned region was able to mediate the oxidation of choline to glycine betaine in E . coli, but not the transport of choline, indicating that the gene(s) involved in choline transport are not clustered with the glycine betaine synthesis genes . Nucleotide sequence analysis of a 4.6 kb segment from the cloned DNA revealed the occurrence of three ORFs (betIBA) apparently arranged in an operon . The deduced betI gene product exhibited features typical for DNA-binding regulatory proteins . The deduced BetB and BetA proteins showed significant similarity to soluble glycine betaine aldehyde dehydrogenases and membrane-bound choline dehydrogenases, respectively, from a variety of organisms . Evidence is presented that BetA is able to oxidize both choline and glycine betaine aldehyde and therefore can mediate both steps in the synthesis of glycine betaine. RNA, 2000 Feb, 6(2), 296 - 306 Mapping posttranscriptional modifications in 5S ribosomal RNA by MALDI mass spectrometry; Kirpekar F et al.; We present a method to screen RNA for posttranscriptional modifications based on Matrix Assisted Laser Desorption/Ionization mass spectrometry (MALDI-MS) . After the RNA is digested to completion with a nucleotide-specific RNase, the fragments are analyzed by mass spectrometry . A comparison of the observed mass data with the data predicted from the gene sequence identifies fragments harboring modified nucleotides . Fragments larger than dinucleotides were valuable for the identification of posttranscriptional modifications . A more refined mapping of RNA modifications can be obtained by using two RNases in parallel combined with further fragmentation by Post Source Decay (PSD) . This approach allows fast and sensitive screening of a purified RNA for posttranscriptional modification, and has been applied on 5S rRNA from two thermophilic microorganisms, the bacterium Bacillus stearothermophilus and the archaeon Sulfolobus acidocaldarius, as well as the halophile archaea Halobacterium halobium and Haloarcula marismortui . One S . acidocaldarius posttranscriptional modification was identified and was further characterized by PSD as a methylation of cytidine32 . The modified C is located in a region that is clearly conserved with respect to both sequence and position in B . stearothermophilus and H . halobium and to some degree also in H . marismortui . However, no analogous modification was identified in the latter three organisms . We further find that the 5' end of H . halobium 5S rRNA is dephosphorylated, in contrast to the other 5S rRNA species investigated . The method additionally gives an immediate indication of whether the expected RNA sequence is in agreement with the observed fragment masses . Discrepancies with two of the published 5S rRNA sequences were identified and are reported here. Mol Microbiol, 2000 Feb, 35(3), 647 - 56 BasT, a membrane-bound transducer protein for amino acid detection in Halobacterium salinarum; Kokoeva MV et al.; Halophilic archaea, such as eubacteria, use methyl-accepting chemotaxis proteins (MCPs) to sense their environment . We show here that BasT is a halobacterial transducer protein (Htp) responsible for chemotaxis towards five attractant amino acids . The C-terminus of the protein exhibits the highly conserved regions that are diagnostic for MCPs: the signalling domain for communication with the histidine kinase and the methylation sites that interact with the methylation/demethylation enzymes for adaptation . Hydropathy analysis predicts an enterobacterial-type transducer protein topology for BasT, with an extracellular putative ligand-binding domain flanked by two transmembrane helices and a cytoplasmic domain . BasT-inactivated mutant cells are missing a membrane protein radiolabelled with L-{methyl-3H}-methionine in wild-type cells, confirming that BasT is methylatable and membrane bound . Behavioural analysis of the basT mutant cells by capillary and chemical-in-plug assays demonstrates complete loss of chemotactic responses towards five (leucine, isoleucine, valine, methionine and cysteine) of the six attractant amino acids for Halobacterium salinarum, whereas they still respond to arginine . The volatile methyl group production assays also corroborate these findings and confirm that BasT signalling induces methyl group turnover . Our data identify BasT as the chemotaxis transducer protein for the branched chain amino acids leucine, isoleucine and valine as well as for methionine and cysteine . Thus, BasT and the arginine sensor Car cover the entire spectrum of chemotactic responses towards attractant amino acids in H . salinarum. Yakugaku Zasshi, 2000 Jan, 120(1), 16 - 27 {Bioenergetics of marine bacteria--respiration-coupled Na+ pump}; Unemoto T; Marine bacteria are unique in the requirement for Na+ for optimal growth . Using a marine bacterium Vibrio alginolyticus, it was confirmed that Na+ is essential for the active uptake of all amino acids . Furthermore, the respiratory chain of V . alginolyticus was found to require Na+ for the maximum activity . The site of Na(+)-dependent activation is localized in the NADH-quinone reductase segment, where Na+ is extruded from the cells as a direct result of redox reaction . Thus, marine bacteria are able to directly generate sodium-motive force by respiratory chain activity . The sodium-motive force is directly coupled to the active uptake of nutrients and to the rotation of polar flagella . In addition to the energy coupling by proton circulation, marine bacteria are unique in utilizing Na+ circulation for the energy coupling . The latter mode of energy coupling is superior to proton circulation especially at alkaline and Na(+)-rich conditions . The respiration-coupled Na+ pump is widely distributed among Gram-negative marine and moderately halophilic bacteria . Recently, it was found that the same type of Na+ pump is distributed in the Gram-negative pathogenic bacteria . Since the presence of Na+ pump widens the adaptability of bacteria to grow at harsh environments, Na+ pump is likely to be helpful for the growth of pathogenic bacteria in the host cells to manifest their pathogenicity. Appl Environ Microbiol, 2000 Feb, 66(2), 509 - 17 Glycine betaine, carnitine, and choline enhance salinity tolerance and prevent the accumulation of sodium to a level inhibiting growth of Tetragenococcus halophila; Robert H et al.; Natural-abundance (13)C-nuclear magnetic resonance was used to probe the intracellular organic solute content of the moderately halophilic bacterium Tetragenococcus halophila . When grown in complex growth media supplemented or not with NaCl, T . halophila accumulates glycine betaine and carnitine . Unlike other moderate halophiles, T . halophila was not able to produce potent osmoprotectants (such as ectoines and glycine betaine) through de novo synthesis when cultured in defined medium under hyperosmotic constraint . Addition of 2 mM carnitine, glycine betaine, or choline to defined medium improved growth parameters, not only at high salinity (up to 2.5 M NaCl) but also in media lacking NaCl . These compounds were taken up when available in the surrounding medium . The transport activity occurred at low and high salinities and seems to be constitutive . Glycine betaine and carnitine were accumulated by T . halophila in an unmodified form, while exogenously provided choline led to an intracellular accumulation of glycine betaine . This is the first evidence of the existence of a choline-glycine betaine pathway in a lactic acid bacterium . An assay showed that the compatible solutes strikingly repressed the accumulation of glutamate and slightly increased the intracellular potassium level only at high salinity . Interestingly, osmoprotectant-treated cells were able to maintain the intracellular sodium concentration at a relatively constant level (200 to 300 nmol/mg {dry weight}), independent of the NaCl concentration of the medium . In contrast, in the absence of osmoprotectant, the intracellular sodium content increased sharply from 200 to 2,060 nmol/mg (dry weight) when the salinity of the medium was raised from 1 to 2 M . Indeed, the imported compatible solutes play an actual role in regulating the intracellular Na(+) content and confer a much higher salt tolerance to T . halophila. Biochemistry, 2000 Feb 8, 39(5), 1001 - 10 Insights into the molecular relationships between malate and lactate dehydrogenases: structural and biochemical properties of monomeric and dimeric intermediates of a mutant of tetrameric L-{LDH-like} malate dehydrogenase from the halophilic archaeon Haloarcula marismortui; Madern D et al.; L-Malate (MalDH) and L-lactate (LDH) dehydrogenases belong to the same family of NAD-dependent enzymes . LDHs are tetramers, whereas MalDHs can be either dimeric or tetrameric . To gain insight into molecular relationships between LDHs and MalDHs, we studied folding intermediates of a mutant of the LDH-like MalDH (a protein with LDH-like structure and MalDH enzymatic activity) from the halophilic archaeon Haloarcula marismortui (Hm MalDH) . Crystallographic analysis of Hm MalDH had shown a tetramer made up of two dimers interacting mainly via complex salt bridge clusters . In the R207S/R292S Hm MalDH mutant, these salt bridges are disrupted . Its structural parameters, determined by neutron scattering and analytical centrifugation under different conditions, showed the protein to be a tetramer in 4 M NaCl . At lower salt concentrations, stable oligomeric intermediates could be trapped at a given pH, temperature, or NaCl solvent concentration . The spectroscopic properties and enzymatic behavior of monomeric, dimeric, and tetrameric species were thus characterized . The properties of the dimeric intermediate were compared to those of dimeric intermediates of LDH and dimeric MalDHs . A detailed analysis of the putative dimer-dimer contact regions in these enzymes provided an explanation of why some can form tetramers and others cannot . The study presented here makes Hm MalDH the best characterized example so far of an LDH-like MalDH. Biochemistry, 2000 Feb 8, 39(5), 992 - 1000 Halophilic adaptation: novel solvent protein interactions observed in the 2.9 and 2.6 A resolution structures of the wild type and a mutant of malate dehydrogenase from Haloarcula marismortui; Richard SB et al.; Previous biophysical studies of tetrameric malate dehydrogenase from the halophilic archaeon Haloarcula marismortui (Hm MalDH) have revealed the importance of protein-solvent interactions for its adaptation to molar salt conditions that strongly affect protein solubility, stability, and activity, in general . The structures of the E267R stability mutant of apo (-NADH) Hm MalDH determined to 2.6 A resolution and of apo (-NADH) wild type Hm MalDH determined to 2.9 A resolution, presented here, highlight a variety of novel protein-solvent features involved in halophilic adaptation . The tetramer appears to be stabilized by ordered water molecule networks and intersubunit complex salt bridges "locked" in by bound solvent chloride and sodium ions . The E267R mutation points into a central ordered water cavity, disrupting protein-solvent interactions . The analysis of the crystal structures showed that halophilic adaptation is not aimed uniquely at "protecting" the enzyme from the extreme salt conditions, as may have been expected, but, on the contrary, consists of mechanisms that harness the high ionic concentration in the environment. FEMS Microbiol Lett, 2000 Feb 1, 183(1), 81 - 8 The microbial composition of three limnologically disparate hypersaline Antarctic lakes; Bowman JP et al.; 16S rRNA clone library analysis was used to examine the biodiversity and community structure within the sediments of three hypersaline Antarctic lakes . Compared to sediment of low to moderate salinity Antarctic lakes the species richness of the hypersaline lake sediments was 2-20 times lower . The community of Deep Lake (32% salinity, average sediment temperature -15 degrees C) was made up almost entirely of halophilic Archaea . The sediment communities of two meromictic hypersaline lakes, Organic Lake (20% salinity, -7 degrees C) and Ekho Lake (15% salinity, 15 degrees C) were more complex, containing phylotypes clustering within the Proteobacteria and Cytophagales divisions and with algal chloroplasts . Many phylotypes of these lakes were related to taxa more adapted to marine-like salinity and perhaps derive from bacteria exported into the sediment from the lower salinity surface waters . The Ekho Lake clone library contained several major phylotypes related to the Haloanaerobiales, the growth of which appears to be promoted by the comparatively high in situ temperature of this lake. FEMS Microbiol Lett, 2000 Feb 1, 183(1), 67 - 71 Production and biochemical characterization of an alpha-amylase from the moderate halophile Halomonas meridiana; Coronado M et al.; Extracellular amylase production by the moderate halophile Halomonas meridiana was optimized and the enzyme was characterized biochemically . The highest amylase production was achieved by growing H . meridiana cultures in media with 5% salts and starch, in the absence of glucose until the end of the exponential phase . The amylase exhibited maximal activity at pH 7.0, being relatively stable in alkaline conditions . Optimal temperature and salinity for activity were 37 degrees C and 10% NaCl, respectively . Moreover, activity at salinity as high as 30% salts was detected . Maltose and maltotriose were the main end products of starch hydrolysis, indicating an alpha-amylase activity. Structure Fold Des, 1999 Dec 15, 7(12), 1575 - 83 The Escherichia coli large ribosomal subunit at 7.5 A resolution; Matadeen R et al.; BACKGROUND: In recent years, the three-dimensional structure of the ribosome has been visualised in different functional states by single-particle cryo-electron microscopy (cryo-EM) at 13-25 A resolution . Even more recently, X-ray crystallography has achieved resolution levels better than 10 A for the ribosomal structures of thermophilic and halophilic organisms . We present here the 7.5 A solution structure of the 50S large subunit of the Escherichia coli ribosome, as determined by cryo-EM and angular reconstitution . RESULTS: The reconstruction reveals a host of new details including the long alpha helix connecting the N- and C-terminal domains of the L9 protein, which is found wrapped like a collar around the base of the L1 stalk . A second L7/L12 dimer is now visible below the classical L7/L12 'stalk', thus revealing the position of the entire L8 complex . Extensive conformational changes occur in the 50S subunit upon 30S binding; for example, the L9 protein moves by some 50 A . Various rRNA stem-loops are found to be involved in subunit binding: helix h38, located in the A-site finger; h69, on the rim of the peptidyl transferase centre cleft; and h34, in the principal interface protrusion . CONCLUSIONS: Single-particle cryo-EM is rapidly evolving towards the resolution levels required for the direct atomic interpretation of the structure of the ribosome . Structural details such as the minor and major grooves in rRNA double helices and alpha helices of the ribosomal proteins can already be visualised directly in cryo-EM reconstructions of ribosomes frozen in different functional states. Cornea, 2000 Jan, 19(1), 26 - 9 Vibrio ocular infections on the U.S . Gulf Coast; Penland RL et al.; PURPOSE: To describe the epidemiology of Vibrio eye infections . METHOD: We reviewed the records of a patient from our institution with V . vulnificus keratitis and conducted a literature search for other cases of ocular infections with Vibrio species . RESULTS: A 39-year-old fisherman was struck in his left eye with an oyster shell fragment, developed suppurative V . vulnificus keratitis, and was successfully treated with combined cefazolin and gentamicin . Including our patient, 17 cases of eye infections with Vibrio spp . have been reported, and 11 (65%) involved exposure to seawater or shellfish . Of the seven cases due to V . vulnificus (six keratitis and one endophthalmitis), six had known exposure to shellfish or seawater along the U.S . coast of the Gulf of Mexico . Of five cases of V . alginolyticus conjunctivitis, three had been exposed to fish or shellfish . Three infections with V . parahaemolyticus (one keratitis and two endophthalmitis) were reported; two of these occurred in people exposed to brackish water on or near the Gulf Coast . Two cases of postsurgical endophthalmitis, one with V . albensis and one with V . fluvialis, also were reported . CONCLUSIONS: In addition to septicemia, gastroenteritis, and wound infections, halophilic noncholera Vibrio species can cause sight-threatening ocular infections . Ocular trauma by shellfish from contaminated water is the most common risk factor for Vibrio conjunctivitis and keratitis . Nearly one half of reported Vibrio infections of the eye occurred along the U.S . coast of the Gulf of Mexico. J Biol Inorg Chem, 1999 Dec, 4(6), 727 - 41 Simultaneous interpretation of Mössbauer, EPR and 57Fe ENDOR spectra of the {Fe4S4} cluster in the high-potential iron protein I from Ectothiorhodospira halophila; Dilg AW et al.; Mossbauer spectra of the oxidized {Fe4S4}3+ and the reduced {Fe4S4}2+ clusters in the high-potential iron protein I from Ectothiorhodospira halophila were measured in a temperature range from 5 K to 240 K . EPR measurements and 57Fe electron-nuclear double resonance (ENDOR) experiments were carried out with the oxidized protein . In the oxidized state the cluster has a net spin S = 1/2 and is paramagnetic . As common in {Fe4S4}3+ clusters, the Mossbauer spectrum was simulated with two species contributing equally to the absorption area: two Fe3+ atoms couple to the "ferric-ferric" pair, and one Fe2+ and one Fe3+ atom give the "ferric-ferrous pair" . For the simulation of the Mossbauer spectrum, g-values were taken from EPR measurements . A-tensor components were determined by 57Fe ENDOR experiments that turned out to be a necessary source of estimating parameters independently . In order to obtain a detailed agreement of Mossbauer and ENDOR data, electronic relaxation has to be taken into account . Relaxing the symmetry condition in a way that the electric field gradient tensor does not coincide with g- and A-tensors yielded an even better agreement of experimental and theoretical Mossbauer spectra . Spin-spin and spinlattice relaxation times were estimated by pulsed EPR; the former turned out to be the dominating mechanism at T = 5 K . Relaxation times measured by pulsed EPR and obtained from the Mossbauer fit were compared and yield nearly identical values . The reduced cluster has one additional electron and has a diamagnetic (S = 0) ground state . All the four irons are indistinguishable in the Mossbauer spectrum, indicating a mixed-valence state of Fe2.5+ for each. J Bacteriol, 2000 Jan, 182(2), 532 - 5 Motility and flagellum synthesis in Halobacillus halophilus are chloride dependent; Roessler M et al.; The motility of Halobacillus halophilus as observed on swarm agar plates was strictly dependent on the chloride concentration . Cl(-) was apparently not used as the coupling ion for flagellar rotation . Cells grown in the absence of chloride were devoid of flagella, but flagellation was restored upon the addition of chloride . These experiments indicate that chloride is involved in synthesis of flagella in H . halophilus. Microbiology, 1999 Dec, 145 ( Pt 12), 3565 - 74 Very similar strains of Halococcus salifodinae are found in geographically separated permo-triassic salt deposits; Stan-Lotter H et al.; The authors have previously isolated a novel extremely halophilic archaeon, Halococcus salifodinae Blp, from Austrian rock salt deposited about 250 million years ago . In this study they compared strain Blp with two other halococci isolated independently from geographically distant salt deposits of similar age, and with two recent isolates (N1 and H2) from the same site as strain Blp . Strain BG2/2 was from a salt mine in Germany and strain Br3 from a halite deposit in England; both resembled Hc . salifodinae Blp in cellular and colonial morphology . Strains Blp, BG2/2 and Br3 had identical 16S rRNA sequences, very similar whole-cell protein patterns, which were different from those of other halococci, similar G+C contents and identical sequences in a 108-base insertion in their 5S rRNA gene . Other similarities included composition and relative abundances of polar lipids, antibiotic susceptibility, enzymic activities and Fourier-transform infrared spectra . Strains N1 and H2 showed similar morphology, whole-cell protein patterns and biochemical characteristics as strains Blp, Br3 and BG2/2 . Their partial 16S rRNA sequences (682 and 641 bases, respectively) were indistinguishable from those of strains Blp, Br3 and BG2/2 . Therefore strains N1 and H2 can be considered as reisolates of Hc . salifodinae which were obtained 8 years after the first samples were taken from that mine . The results presented suggest that viable halophilic archaea, which belong to the same species, occur in widely separated evaporite locations of similar geological age, and support the notion that these halophilic isolates from subterranean salt deposits may be the remnants of populations which inhabited ancient hypersaline seas. Syst Appl Microbiol, 1999 Sep, 22(3), 360 - 5 Polyphasic taxonomy of a novel Halobacillus, Halobacillus thailandensis sp . nov . isolated from fish sauce; Chaiyanan S et al.; In order to speed up fish sauce production, a more complete understanding of the microorganisms associated with the fermentation was needed . This study was undertaken to meet that need . A bacterium was isolated from a fish sauce production line containing 25% NaCl . It is a Gram-positive, rod-shaped bacillus with pointed ends, occurring as single cells, pairs, or short chains . Endospores are produced on a low nutrient medium and, in old cultures, the cells round up, even when undergoing division . The cell wall is relatively amorphous and similar to that of Gram-positive bacteria in structure and composition . Cells grown in a medium containing 10-20% salt possess thicker cell walls than those grown in a medium with 3% salt . Based on 16S rRNA sequence and DNA/DNA hybridization data, we conclude that the bacterium is a species of Halobacillus . This bacterium shares 99.2% and 97.2% 16S rRNA similarity with Halobacillus litoralis and Halobacillus halophilus respectively and DNA/DNA homology was lower than 70%, considered indicative of species similarity . Three highly expressed extra-cellular proteolytic enzymes with M(r) of approximately 100 kDa, 42 kDa and 17 kDa, respectively, were detected by gelatin-polyacrylamide gel electrophoresis . Activity of the 100 kDa and 17 kDa proteases was inhibited by phenylmethanesulphonyl fluoride (PMSF), without being affected by L-trans epoxysuccinyl-leucylamide 4-guanidino-butane (E-64), pepstatin, EDTA, or 1, 10-phenanthroline, leading to the conclusion that these enzymes are serine proteases . The 42-kDa protease was inhibited by EDTA and 1,10-phenanthroline, but not by PMSF, thus, being classified a metalloprotease . The strain has been successfully employed to improve fermentation in industrial production of fish sauce in Thailand. Arch Microbiol, 1999 Dec, 172(6), 401 - 6 Phosphofructokinase activities within the order spirochaetales and the characterisation of the pyrophosphate-dependent phosphofructokinase from spirochaeta thermophila Ronimus RS, Morgan HW, Ding YR. The subtype of phosphofructokinase activity, either ATP-, ADP- or pyrophosphate-dependent, present in members of three genera from the Spirochaetales was investigated . The individual species/strains examined included Spirochaeta alkalica, S . asiatica, S . halophila, S . isovalerica, S . litoralis, S . zuelzerae, S . thermophila, two thermophilic spirochetes, Treponema bryantii, T . denticola, paragraph signT . pectinovorum, Leptospira biflexa and L . interrogans . All of the Spirochaeta strains, regardless of their phenotype, possessed primarily a pyrophosphate-dependent phosphofructokinase . In contrast, T . bryantii, T . denticola and L . biflexa had predominantly an ATP-dependent activity, whereas no activity was detected in T . pectinovorum or paragraph signL . interrogans . The results suggest that pyrophosphate-dependent phosphofructokinase activity may be a reliable phenotypic marker for the genus Spirochaeta and that there are potentially interesting differences in how the catabolism of saccharides is controlled among members of genera within the Spirochaetales . The pyrophosphate-dependent phosphofructokinase from S . thermophila strain RI 19.B1 was purified (303-fold) to homogeneity and biochemically characterised . The S . thermophila enzyme displayed hyperbolic kinetics with respect to both the forward and reverse cosubstrates and was not significantly affected by traditional activators or inhibitors of phosphofructokinase . The biochemical characterisation represents the first spirochete phosphofructokinase to be described. Extremophiles, 1999 Nov, 3(4), 253 - 7 Lipid membranes from halophilic and alkali-halophilic Archaea have a low H+ and Na+ permeability a