Halophilic bacterium

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 at high salt concentration; van de Vossenberg JL et al.; The influence of pH and the salt concentration on the proton and sodium ion permeability of liposomes formed from lipids of the halophile Halobacterium salinarum and the haloalkaliphile Halorubrum vacuolatum were studied . In contrast with liposomes formed from Escherichia coli lipids, liposomes formed from halophilic lipids remained stable up to 4M of NaCl and KCl . The proton permeability of the liposomes from lipids of halophiles was independent of the salt concentration and was essentially constant between pH 7 and pH 9 . The sodium ion permeability increased with the salt concentration but was 10- to 100 fold lower than the proton permeability . It is concluded that the membranes of halophiles are stable over a wide range of salt concentrations and at elevated pH values and are well adapted to the halophilic conditions.

Changgeng Yi Xue Za Zhi, 1999 Sep, 22(3), 508 - 14
Vibrio parahemolyticus bacteremia: case report; Ng TC et al.; Vibrio parahemolyticus (V . parahemolyticus) is a halophilic gram-negative bacillus that lives in the ocean . It is the leading cause of infectious diarrhea in Taiwan and sometimes produces soft tissue infections, but it is rarely a cause of bacteremia . There have been only 11 cases reported in the literature . Most of the cases involved a history of ingestion of seafood or exposure to seawater . In addition, those patients were all immunosuppressed, especially with leukemia and cirrhosis . We report a 60-year-old male patient with chronic hepatitis C and adrenal insufficiency . He developed V . parahemolyticus bacteremia following ingestion of seafood one week prior to admission . His condition was complicated with neck and right lower leg soft tissue infection, as well as multiple organ failure . The patient survived after intravenous ceftazidime, oral doxycycline, and surgical debridement . To our knowledge, this is the 12th reported cases on Medline, and the second bacteremic case in Taiwan . After reviewing the literature, we suggest that all patients with immunosuppressed conditions or adrenal insufficiency should eat foods that are well cooked and avoid raw seafood . Moreover, when patients who are at risk to develop fever, diarrhea, and soft tissue infection after ingestion of seafood, V . parahemolyticus infection should be suspected . All culture specimens should be inoculated on Vibrios selective media.

J Bacteriol, 1999 Nov, 181(22), 7140 - 2
An improved transposon for the halophilic archaeon Haloarcula hispanica; Woods WG et al.; An improved transposon (ThD73) for Haloarcula hispanica is described . Based on the halobacterial insertion sequence ISH28, it showed little target sequence specificity but was biased toward a lower G+C content . Twenty randomly selected ThD73 mutants were analyzed, and the DNA flanking their insertions revealed several recognizable sequences, including two (unrelated) ISH elements.

FEMS Microbiol Lett, 1999 Nov 15, 180(2), 325 - 30
Salt stress affects sterol biosynthesis in the halophilic black yeast Hortaea werneckii; Petrovic U et al.; Hortaea werneckii is a black yeast recently isolated from salterns in Slovenia . Some of the adaptations of halophilic microorganisms to increased salinity and osmolarity of the environment are alterations in membrane properties . By modulating the fluidity, sterols play an important role as a component of eukaryotic biological membranes . We studied the regulation of sterol biosynthesis in H . werneckii through the activity and amount of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG R), a key regulatory enzyme in the biosynthesis of sterols . We found some differences in the characteristics of HMG R and in its regulation by different environmental salinities in H . werneckii when compared to the mesophilic baker's yeast, Saccharomyces cerevisiae . Our results suggest that halophilic black yeast regulates sterol biosynthesis through HMG R in a different way than mesophiles, which might be a consequence of the different ecophysiology of halophilic black yeasts . From this perspective, H . werneckii is an interesting novel model organism for studies on salt stress-responsive proteins as well as on sterol biosynthesis in eukaryotes.

J Mol Biol, 1999 Nov 19, 294(1), 93 - 101
Resistance mutations in 23 S rRNA identify the site of action of the protein synthesis inhibitor linezolid in the ribosomal peptidyl transferase center; Kloss P et al.; Oxazolidinones represent a novel class of antibiotics that inhibit protein synthesis in sensitive bacteria . The mechanism of action and location of the binding site of these drugs is not clear . A new representative of oxazolidinone antibiotics, linezolid, was found to be active against bacteria and against the halophilic archaeon Halobacterium halobium . The use of H . halobium, which possess only one chromosomal copy of rRNA operon, allowed isolation of a number of linezolid-resistance mutations in rRNA . Four types of linezolid-resistant mutants were isolated by direct plating of H . halobium cells on agar medium containing antibiotic . In addition, three more linezolid-resistant mutants were identified among the previously isolated mutants of H . halobium containing mutations in either 16 S or 23 S rRNA genes . All the isolated mutants were found to contain single-point mutations in 23 S rRNA . Seven mutations affecting six different positions in the central loop of domain V of 23 S rRNA were found to confer resistance to linezolid . Domain V of 23 S rRNA is known to be a component of the ribosomal peptidyl transferase center . Clustering of linezolid-resistance mutations within this region strongly suggests that the binding site of the drug is located in the immediate vicinity of the peptidyl transferase center . However, the antibiotic failed to inhibit peptidyl transferase activity of the H . halobium ribosome, supporting the previous conclusion that linezolid inhibits translation at a step different from the catalysis of the peptide bond formation .

Arch Microbiol, 1999 Nov, 172(5), 321 - 9
Osmoadaptation in halophilic and alkaliphilic methanotrophs
Khmelenina VN, Kalyuzhnaya MG, Sakharovsky VG, Suzina NE, Trotsenko YA, Gottschalk G.
By using (1)H- and (13)C-NMR spectroscopy, an accumulation of sucrose and two cyclic amino acids {ectoine (1,4,5, 6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) and 5-oxoproline (pyrrolidone carboxylic acid)} was detected in the halotolerant methanotrophs Methylobacter alcaliphilus 20Z and Methylobacter modestohalophilus 10S . The organic solute pool was found to increase upon raising the NaCl concentration . In M . alcaliphilus 20Z, the intracellular level of the total solutes was shown to be sufficient to balance the osmotic pressure of the medium, whereas in M . modestohalophilus 10S their content was several times lower . Additionally, phosphatidylglycerol and phosphatidylcholine were predominant cell phospholipids in salt-adapted M . alcaliphilus 20Z . However, no phosphatidylcholine was found in M . modestohalophilus 10S, and the portion of phosphatidylglycerol increased while phosphatidylethanolamine decreased upon elevated external NaCl concentrations . Regularly arranged glycoprotein surface layers (S-layers) of hexagonal and linear (p2) symmetry were observed on the outer cell walls of M . alcaliphilus 20Z and M . modestohalophilus 10S . The S-layer in M alcaliphilus 20Z consisting of tightly packed, cup-shaped subunits was lost during growth at pH 7.2 (the lowest possible pH) in the absence of NaCl . Hence, osmoadaptation in the methanotrophs studied involves structure/function alterations of cell envelopes and changes in the chemical composition of membranes as well as de novo synthesis of compatible solutes.

J Mol Biol, 1999 Nov 12, 293(5), 1029 - 38
Unique recognition style of tRNA(Leu) by Haloferax volcanii leucyl-tRNA synthetase; Soma A et al.; The recognition manner of tRNA(Leu), a class II tRNA characterized by a long variable arm, by leucyl-tRNA synthetase from an extreme halophilic archaea, Haloferax volcanii, was studied using the in vitro transcription system . It was found that the discriminator base (A73) and the long variable arm, especially the specific loop sequence A47CG47D and U47H at the base of this helix, are significant for recognition by LeuRS . An appropriate stem length of the variable arm was also required . Base substitutions in the anticodon arm did not affect the leucylation activity . Transplantation of both the discriminator base and the variable arm of tRNA(Leu) was not sufficient to introduce leucylation activity to tRNA(Ser) . Insertion of an additional nucleotide into the D-loop, which is not involved in the direct interaction with LeuRS, converted tRNA(Ser) to an efficient leucine acceptor . This suggests that differences in the tertiary structure play a key role in eliminating tRNA(Ser) . The sequence-specific recognition of the long variable arm of tRNA(Leu) has not been observed in any of other organisms reported, such as Escherichia coli, yeast or human . On the other hand, the mode of discrimination from non-cognate tRNAs is similar to that in E . coli in that differences in the tertiary structure play a key role . Similarity extends to the substrate stringency, exemplified by a cross-species aminoacylation study showing that no class II tRNAs from E . coli or yeast can be leucylated by H . volcanii LeuRS . Our results have implications for the understanding of the evolution of the recognition system of class II tRNA .

Biochem Biophys Res Commun, 1999 Oct 14, 264(1), 24 - 8
Cloning, sequencing, and characterization of ribosomal protein and RNA polymerase genes from the region analogous to the alpha-operon of escherichia coli in halophilic archaea, halobacterium halobium; Sano K et al.; A determination was made of the nucleotide sequence of the 3215-bp region of a ribosomal protein gene cluster (HS13, HS4, HS11, and HeL18), RNA polymerase (RNA poly D), and tRNA genes (tRNAser and tRNAarg) of halophilic Archaea Halobacterium halobium, which is analogous to the alpha-operon of Escherichia coli (tRNAser-HS13-HS4-HS11-RNA poly D-tRNAarg-HeL18) . The seven-gene string was preceded by a pseudoknot-like structure similar to the proposed S4 ribosomal protein binding site of the alpha-operon mRNA leader in E . coli . Using an inducible expression system H . halobium HS4 was produced in large amounts in E . coli, and immunoblot analysis showed the S4 to constitute a 21-kDa polypeptide component of the ribosome . Analysis of the deduced amino acids sequence revealed that the HS13, HS4, and HS11 sequences including the RNA polymerase subunit are more similar to their eukaryotic than to their bacterial counterparts . HeL18, located downstream of the gene cluster analogous to the E . coli alpha-operon (S13-S11-S4-RNA poly D-L17), was similar to both the eukaryotic (eL18) and eubacterial ribosomal protein L15 located in the spc-operon, but not to L17 positioned as the terminal gene of the bacterial alpha-operon .

Arch Microbiol, 1999 Oct, 172(4), 264 - 7
Chloride dependence of endospore germination in halobacillus halophilus
Dohrmann AB, Muller V V.
The ion requirement for germination and outgrowth of endospores from the moderately halophilic salt marsh bacterium Halobacillus halophilus was studied . Germination and outgrowth of endospores plated onto nutrient broth was dependent on the salt concentration in the artificial seawater used as the source of ions . Maximal germination and outgrowth were observed when double-concentrated artificial seawater was used . Replacement of chloride salts in the artificial seawater by other salts resulted in a complete loss of germination and outgrowth that was restored upon addition of chloride . To analyze the role of chloride more directly and quantitatively, a defined growth medium was used in which the artificial seawater was substituted by a solution of magnesium sulfate and sodium chloride . Spore germination and outgrowth were strictly dependent on the chloride concentration; maximal germination and outgrowth were observed at approximately 1.3 M Cl(-) . Chloride could be substituted by bromide, but not by sulfate or nitrate . Microscopic examinations of single spores clearly showed that germination is the chloride-dependent step . This first report on chloride dependence of spore germination in any endospore-forming bacterium adds another function to chloride in H . halophilus apart from its being essential for the physiology of the vegetative cell . de/link/service/journals/00203/bibs/9172004/91720264.htm</HEA

Biochemistry, 1999 Oct 12, 38(41), 13766 - 72
Dual photoactive species in Glu46Asp and Glu46Ala mutants of photoactive yellow protein: a pH-driven color transition; Devanathan S et al.; Photoactive yellow protein (PYP) is a blue light sensor present in the purple photosynthetic bacterium Ectothiorhodospira halophila, which undergoes a cyclic series of absorbance changes upon illumination at its lambda(max) of 446 nm . The anionic p-hydroxycinnamoyl chromophore of PYP is covalently bound as a thiol ester to Cys69, buried in a hydrophobic pocket, and hydrogen-bonded via its phenolate oxygen to Glu46 and Tyr42 . The chromophore becomes protonated in the photobleached state (I(2)) after it undergoes trans-cis isomerization, which results in breaking of the H-bond between Glu46 and the chromophore and partial exposure of the phenolic ring to the solvent . In previous mutagenesis studies of a Glu46Gln mutant, we have shown that a key factor in controlling the color and photocycle kinetics of PYP is this H-bonding system . To further investigate this, we have now characterized Glu46Asp and Glu46Ala mutants . The ground-state absorption spectrum of the Glu46Asp mutant shows a pH-dependent equilibrium (pK = 8.6) between two species: a protonated (acidic) form (lambda(max) = 345 nm), and a slightly blue-shifted deprotonated (basic) form (lambda(max) = 444 nm) . Both of these species are photoactive . A similar transition was also observed for the Glu46Ala mutant (pK = 7.9), resulting in two photoactive red-shifted forms: a basic species (lambda(max) = 465 nm) and a protonated species (lambda(max) = 365 nm) . We attribute these spectral transitions to protonation/deprotonation of the phenolate oxygen of the chromophore . This is demonstrated by FT Raman spectra . Dark recovery kinetics (return to the unphotolyzed state) were found to vary appreciably between these various photoactive species . These spectral and kinetic properties indicate that the hydrogen bond between Glu46 and the chromophore hydroxyl group is a dominant factor in controlling the pK values of the chromophore and the glutamate carboxyl.

Curr Opin Microbiol, 1999 Oct, 2(5), 542 - 7
Archaeal genomics; Gaasterland T; Four euryarchaeal genomes have been completely sequenced and are publicly available: Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Pyrococcus horikoshii and Archaeoglobus fulgidus . Four more genome sequences, two crenarchaeal and two pyrococci, will soon be released . In addition, seven more archaeal genome sequencing projects are under way, including two halophiles, two Thermoplasma, and a methanogen . These projects cover all branches of the archaeal domain and will lead to new insights into archaeal metabolism, DNA processing, and evolutionary relationships with the Bacteria and Eukarya.

J Mol Evol, 1999 Oct, 49(4), 439 - 52
DNA supercoiling and temperature adaptation: A clue to early diversification of life?
Lopez-Garcia P.
Cellular systems to control an appropriate DNA geometry for function probably evolved simultaneously with DNA genomes . Such systems are basically DNA topoisomerases and DNA-binding proteins . Therefore, their distribution in extant organisms may be a source of information on early evolution and the nature of the last common ancestor (cenancestor) . Most living beings need the strand-opening potential of negative DNA supercoiling to allow transcription and other DNA-dependent processes . Mesophiles have global negatively supercoiled DNA, essentially due to gyrase (introducing negative supercoils) in bacteria and to DNA wrapping around histone cores in eukaryotes . Mesophilic archaea, halophilic methanogens, and halophiles might use a gyrase, whereas some methanogens might use histone wrapping . The existence of these two distinct mechanisms suggests that mesophily appeared at least twice in evolution . On the other hand, only one system which is based on reverse gyrase (introducing positive supercoils) appears to be required for hyperthermophilic life . Archaeal hyperthermophiles lacking gyrase have relaxed to positively supercoiled DNA, but hyperthermophilic bacteria of the genus Thermotoga, which have both gyrase and reverse gyrase, have negative supercoiling . This suggests that reverse gyrase is necessary at least locally, but whereas these hyperthermophilic bacteria favor general melting potential and stability at critical active regions, hyperthermophilic archaea favor general linking excess and local melting . In this context, the existence of a thermophilic (60-80 degrees C) ancestor endowed with only relaxing topoisomerases is hypothesized . Such temperatures allow a compromise between melting potential and stability, i.e., an appropriate DNA geometry for function . Subsequent duplication and functional specialization of existing DNA topoisomerases would then have facilitated adaptation to hyperthermophily and mesophily in archaea and bacteria, respectively . If reverse gyrase is an ancient character in hyperthermophilic bacteria, the cenancestor would have already been a hyperthermophile . Histone sequence homology and similarities of nucleosome structural dynamics suggest that eukaryotes inherited this system for DNA structural homeostasis from methanogenic euryarchaea . Some mesophilic archaea would have improved their adaptability to mesophily by importing gyrase from bacteria.

FEMS Microbiol Lett, 1999 Sep 1, 178(1), 183 - 90
Sequence and transcriptional studies of five clustered flagellin genes from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1; Nagahisa K et al.; Five clustered genes (flaB1, flaB2, flaB3, flaB4 and flaB5) for multiple subunits of flagellar filaments from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 were cloned and sequenced . Deduced amino acid sequences were aligned and it was revealed that five flagellin genes are homologous especially in the N-terminal hydrophobic region which might be important for interaction among flagellin subunits . Phylogenetic analysis was performed among archaeal flagellins from methanogens, an extreme halophile and our hyperthermophile, indicating that KOD1 flagellins were grouped together with methanogenic counterparts and were distinguishable from halophilic flagellins . Northern analysis of transcripts from flagellin genes from P . kodakaraensis KOD1 revealed that four major transcripts (0.98, 3.7, 5.4 and 9.2 kb) initiating from immediately upstream of flaB1 encode different combinations of five flagellins.

J Bacteriol, 1999 Sep, 181(18), 5814 - 24
Halophilic 20S proteasomes of the archaeon Haloferax volcanii: purification, characterization, and gene sequence analysis; Wilson HL et al.; A 20S proteasome, composed of alpha(1) and beta subunits arranged in a barrel-shaped structure of four stacked rings, was purified from a halophilic archaeon Haloferax volcanii . The predominant peptide-hydrolyzing activity of the 600-kDa alpha(1)beta-proteasome on synthetic substrates was cleavage carboxyl to hydrophobic residues (chymotrypsin-like {CL} activity) and was optimal at 2 M NaCl, pH 7.7 to 9.5, and 75 degrees C . The alpha(1)beta-proteasome also hydrolyzed insulin B-chain protein . Removal of NaCl inactivated the CL activity of the alpha(1)beta-proteasome and dissociated the complex into monomers . Rapid equilibration of the monomers into buffer containing 2 M NaCl facilitated their reassociation into fully active alpha(1)beta-proteasomes of 600 kDa . However, long-term incubation of the halophilic proteasome in the absence of salt resulted in hydrolysis and irreversible inactivation of the enzyme . Thus, the isolated proteasome has unusual salt requirements which distinguish it from any proteasome which has been described . Comparison of the beta-subunit protein sequence with the sequence deduced from the gene revealed that a 49-residue propeptide is removed to expose a highly conserved N-terminal threonine which is proposed to serve as the catalytic nucleophile and primary proton acceptor during peptide bond hydrolysis . Consistent with this mechanism, the known proteasome inhibitors carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132) and N-acetyl-leucinyl-leucinyl-norleucinal (calpain inhibitor I) were found to inhibit the CL activity of the H . volcanii proteasome (K(i) = 0.2 and 8 microM, respectively) . In addition to the genes encoding the alpha(1) and beta subunits, a gene encoding a second alpha-type proteasome protein (alpha(2)) was identified . All three genes coding for the proteasome subunits were mapped in the chromosome and found to be unlinked . Modification of the methods used to purify the alpha(1)beta-proteasome resulted in the copurification of the alpha(2) protein with the alpha(1) and beta subunits in nonstoichometric ratios as cylindrical particles of four stacked rings of 600 kDa with CL activity rates similar to the alpha(1)beta-proteasome, suggesting that at least two separate 20S proteasomes are synthesized . This study is the first description of a prokaryote which produces two separate 20S proteasomes and suggests that there may be distinct physiological roles for the two different alpha subunits in this halophilic archaeon.

FEBS Lett, 1999 Sep 17, 458(2), 252 - 6
Kinetics of and intermediates in a photocycle branching reaction of the photoactive yellow protein from Ectothiorhodospira halophila; Hendriks J et al.; We have studied the kinetics of the blue light-induced branching reaction in the photocycle of photoactive yellow protein (PYP) from Ectothiorhodospira halophila, by nanosecond time-resolved UV/Vis spectroscopy . As compared to the parallel dark recovery reaction of the presumed blue-shifted signaling state pB, the light-induced branching reaction showed a 1000-fold higher rate . In addition, a new intermediate was detected in this branching pathway, which, compared to pB, showed a larger extinction coefficient and a blue-shifted absorption maximum . This substantiates the conclusion that isomerization of the chromophore is the rate-controlling step in the thermal photocycle reactions of PYP and implies that absorption of a blue photon leads to cis-->trans isomerization of the 4-hydroxy-cinnamyl chromophore of PYP in its pB state.

J Microbiol Methods, 1999 Sep, 37(3), 231 - 43
Application of a tetrazolium dye as an indicator of viability in anaerobic bacteria; Bhupathiraju VK et al.; The use of the redox dye 5-cyano-2,3,-ditolyl tetrazolium chloride (CTC) for evaluating the metabolic activity of aerobic bacteria has gained wide application in recent years . In this study, we examined the utility of CTC in capturing the metabolic activity of anaerobic bacteria . In addition, the factors contributing to abiotic reduction of CTC were also examined . CTC was used in conjunction with the fluorochrome 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF), that targets bacterial cell wall proteins, to quantitate the active fraction of total bacterial numbers . Facultative anaerobic bacteria, including Escherichia coli grown fermentatively, and Pseudomonas chlorophis, P . fluorescens, P . stutzeri, and P . pseudoalcalegenes subsp . pseudoalcalegenes grown under nitrate-reducing conditions, actively reduced CTC during all phases of growth . Greater than 95% of these cells accumulated intracellular CTC-formazan crystals during the exponential phase . Obligate anaerobic bacteria, including Syntrophus aciditrophicus grown fermentatively, Geobacter sulfurreducens grown with fumarate as the electron acceptor, Desulfovibrio desulfuricans subsp . desulfuricans and D . halophilus grown under sulfate-reducing conditions, Methanobacterium formicicum grown on formate, H2 and CO2, and Methanobacterium thermoautotrophicum grown autotrophically on H2 and CO2 all reduced CTC to intracellular CTC-formazan crystals . The optimal CTC concentration for all organisms examined was 5 mM . Anaerobic CTC incubations were not required for quantification of anaerobically grown cells . CTC-formazan production by all cultures examined was proportional to biomass production, and CTC reduction was observed even in the absence of added nutrients . CTC was reduced by culture fluids containing ferric citrate as electron acceptor following growth of either G . metallireducens or G . sulfurreducens . Abiotic reduction of CTC was observed in the presence of ascorbic acid, cysteine hydrochloride, dithiothreitol, NADH, NADPH, Fe(II)Cl2, sodium thioglycolic acid and sodium sulfide . These results suggest that while CTC can be used to capture the metabolic activity of anaerobic bacteria, care must be taken to avoid abiotic reduction of CTC.

Appl Environ Microbiol, 1999 Sep, 65(9), 3774 - 9
Role of Ngamma-acetyldiaminobutyrate as an enzyme stabilizer and an intermediate in the biosynthesis of hydroxyectoine; Canovas D et al.; Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain Halomonas elongata DSM 3043 that is affected in the ectoine synthase gene (ectC) . This strain accumulates large amounts of Ngamma-acetyldiaminobutyrate (NADA), the precursor of ectoine (D . Canovas, C . Vargas, F . Iglesias-Guerra, L . N . Csonka, D . Rhodes, A . Ventosa, and J . J . Nieto, J . Biol . Chem . 272:25794-25801, 1997) . Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant . Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities . The effect of the growth stage on the accumulation of solutes was also investigated . NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation . The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate . A (1)H NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (ectA::Tn1732) and CHR63 (ectC::Tn1732) indicated that H . elongata can synthesize hydroxyectoine by two different pathways-directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine.

Microbios, 1999, 98(391), 141 - 7
Effect of Halococcus morrhuae and Halobacterium saccharovorum on the activation of human peripheral blood lymphocytes; Montes MJ et al.; The immunomodulator properties of two species of halophilic Archaebacteria, Halobacterium saccharovorum and Halococcus rnorrhuae, were analysed by the study of lymphocyte activation . Two methods were used to detect activation in lymphocytes, namely incorporation of the radioactive nucleotide {3H}-thymidine, and CD25 expression . H . morrhuae had a stimulatory effect on human lymphocytes, but this action was observed only with the {3H}-thymidine uptake method, whereas H . saccharovorum produced no immunomodulator effect.

Arch Biochem Biophys, 1999 Sep 1, 369(1), 143 - 8
Amino acid sequences of two high-potential iron-sulfur proteins (HiPIPs) from the moderately halophilic purple phototrophic bacterium, Rhodospirillum salinarum; Ambler RP et al.; The amino acid sequences of two very different high-potential iron-sulfur protein (HiPIP) isozymes have been determined from the moderately halophilic purple phototrophic bacterium, Rhodospirillum salinarum . Iso-1 HiPIP, which is monomeric and contains 57 amino acid residues, is most similar to the Thiobacillus ferrooxidans iron-oxidizing enzyme (45% identity and a 6-residue deletion) . On the other hand, iso-2 HiPIP, which is isolated as an oligomer, contains a peptide chain with 54 amino acid residues . It is the smallest reported to date and is only 31% identical to iso-1 HiPIP . A massive deletion of 17 residues is found at the N-terminus, such that only 2 residues remain prior to the first cysteine . Iso-2 HiPIP also has a 12-residue insertion and a 5-residue deletion . Prior to this study, there were only 2 absolutely conserved residues (Tyr 19 and Gly 75, Chromatium numbering) in addition to the 4 iron-sulfur cluster binding cysteine residues among the 13 HiPIPs sequenced to date . We found that Tyr 19 is absent in iso-2 HiPIP along with the entire N-terminal loop . Moreover, Gly 75 is substituted in both R . salinarum HiPIPs . These characteristics make the R . salinarum HiPIPs, and especially iso-2, the most divergent yet characterized .

Biochim Biophys Acta, 1999 Aug 5, 1428(2-3), 446 - 54
In situ analysis of sulfur in the sulfur globules of phototrophic sulfur bacteria by X-ray absorption near edge spectroscopy; Prange A et al.; During the oxidation of sulfide and thiosulfate purple and green sulfur bacteria accumulate globules of 'elemental' sulfur . Although essential for a thorough understanding of sulfur metabolism in these organisms, the exact chemical nature of the stored sulfur is still unclear . We applied sulfur K-edge X-ray absorption near edge spectroscopy (XANES) to probe the forms of sulfur in intact cells . Comparing XANES spectra of Allochromatium vinosum, Thiocapsa roseopersicina, Marichromatium purpuratum, Halorhodospira halophila and Chlorobium vibrioforme grown photolithoautotrophically on sulfide with reference probes (fingerprint method), we found sulfur chains with the structure R-S(n)-R . Evidence for the presence of sulfur rings, polythionates and anionic polysulfides in the sulfur globules of these bacteria was not obtained.

Genetics, 1999 Aug, 152(4), 1363 - 72
Control of ribosomal protein L1 synthesis in mesophilic and thermophilic archaea; Kraft A et al.; The mechanisms for the control of ribosomal protein synthesis have been characterized in detail in Eukarya and in Bacteria . In Archaea, only the regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10, and MvaL12) of the mesophilic Methanococcus vannielii has been extensively investigated . As in Bacteria, regulation takes place at the level of translation . The regulator protein MvaL1 binds preferentially to its binding site on the 23S rRNA, and, when in excess, binds to the regulatory target site on its mRNA and thus inhibits translation of all three cistrons of the operon . The regulatory binding site on the mRNA, a structural mimic of the respective binding site on the 23S rRNA, is located within the structural gene about 30 nucleotides downstream of the ATG start codon . MvaL1 blocks a step before or at the formation of the first peptide bond of MvaL1 . Here we demonstrate that a similar regulatory mechanism exists in the thermophilic M . thermolithotrophicus and M . jannaschii . The L1 gene is cotranscribed together with the L10 and L11 gene, in all genera of the Euryarchaeota branch of the Archaea studied so far . A potential regulatory L1 binding site located within the structural gene, as in Methanococcus, was found in Methanobacterium thermoautotrophicum and in Pyrococcus horikoshii . In contrast, in Archaeoglobus fulgidus a typical L1 binding site is located in the untranslated leader of the L1 gene as described for the halophilic Archaea . In Sulfolobus, a member of the Crenarchaeota, the L1 gene is part of a long transcript (encoding SecE, NusG, L11, L1, L10, L12) . A previously suggested regulatory L1 target site located within the L11 structural gene could not be confirmed as an L1 binding site.

Genetics, 1999 Aug, 152(4), 1285 - 97
Genetic diversity of archaea in deep-sea hydrothermal vent environments; Takai K et al.; Molecular phylogenetic analysis of naturally occurring archaeal communities in deep-sea hydrothermal vent environments was carried out by PCR-mediated small subunit rRNA gene (SSU rDNA) sequencing . As determined through partial sequencing of rDNA clones amplified with archaea-specific primers, the archaeal populations in deep-sea hydrothermal vent environments showed a great genetic diversity, and most members of these populations appeared to be uncultivated and unidentified organisms . In the phylogenetic analysis, a number of rDNA sequences obtained from deep-sea hydrothermal vents were placed in deep lineages of the crenarchaeotic phylum prior to the divergence of cultivated thermophilic members of the crenarchaeota or between thermophilic members of the euryarchaeota and members of the methanogen-halophile clade . Whole cell in situ hybridization analysis suggested that some microorganisms of novel phylotypes predicted by molecular phylogenetic analysis were likely present in deep-sea hydrothermal vent environments . These findings expand our view of the genetic diversity of archaea in deep-sea hydrothermal vent environments and of the phylogenetic organization of archaea.

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1231 - 40
Description of Cellulophaga baltica gen . nov., sp . nov . and Cellulophaga fucicola gen . nov., sp . nov . and reclassification of {Cytophaga} lytica to Cellulophaga lytica gen . nov., comb . nov; Johansen JE et al.; Phenotypic data indicate that gliding, yellow/orange-pigmented, agar-digesting bacterial strains were members of the Cytophaga-Flavobacterium-Bacteroides (CFB) group . The strains were isolated from the surface of the marine benthic macroalga Fucus serratus L . and the surrounding seawater at three localities in Danish waters . The bacteria were Gram-negative, flexirubin-negative, aerobic, catalase-positive and oxidase-negative and were psychrophilic and halophilic . All strains utilized D-fructose, L-fucose and alpha-ketobutyric acid and degraded alginic acid, carrageenan, starch and autoclaved yeast cells . Amplification with primers specific for repetitive extragenic palindromic elements by PCR divided the strains of this study into two groups . Both groups showed unique PCR amplification patterns compared to reference strains of the CFB group . Phylogenetic analysis of 16S rDNA sequences showed association of these organisms and {Cytophaga} lytica at the genus level . Hybridization of total chromosomal DNA revealed that the new strains and {Cytophaga} lytica ATCC 23178T were clearly distinct from each other and other previously described species of the CFB group . A new genus is described, Cellulophaga gen . nov . comprising two new species, Cellulophaga baltica gen . nov., sp . nov . (NN015840T = LMG 18535T) and Cellulophaga fucicola gen . nov., sp . nov . (NN015860T = LMG 18536T), as well as the emendation of {Cytophaga} lytica to Cellulophaga lytica gen . nov., comb . nov.

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1141 - 7
Fusibacter paucivorans gen . nov., sp . nov., an anaerobic, thiosulfate-reducing bacterium from an oil-producing well; Ravot G et al.; A strictly anaerobic, halotolerant, spindle-shaped rod, designated strain SEBR 4211T, was isolated from an African saline oil-producing well . Cells stain Gram-positive, which was confirmed by electron microscopy observations . Strain SEBR 4211T was motile by means of one to four peritrichous flagella, had a G+C content of 43 mol% and grew optimally at 37 degrees C, pH 7.3, with 0 to 3% (w/v) NaCl . It utilized a limited number of carbohydrates (cellobiose, glucose, fructose, mannitol and ribose) and produced acetate, butyrate, CO2 and H2 as end products from glucose fermentation . It reduced thiosulfate to sulfide . In the presence of thiosulfate, a decrease in butyrate and an increase in acetate production was observed . Phylogenetically, strain SEBR 4211T was related to members of the low G+C Clostridiales order with Clostridium halophilum as the closest relative (16S rDNA sequence similarity of 90%) . On the basis of phenotypic, genotypic and phylogenetic characteristics of the isolate, it is proposed to designate it as a new species of a new genus, Fusibacter gen . nov., as Fusibacter paucivorans sp . nov . The type strain is SEBR 4211T (= DSM 12116T).

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1053 - 73
Phylogeny and polyphasic taxonomy of Caulobacter species . Proposal of Maricaulis gen . nov . with Maricaulis maris (Poindexter) comb . nov . as the type species, and emended description of the genera Brevundimonas and Caulobacter; Abraham WR et al.; The genus Caulobacter is composed of prosthecate bacteria often specialized for oligotrophic environments . The taxonomy of Caulobacter has relied primarily upon morphological criteria: a strain that visually appeared to be a member of the Caulobacter has generally been called one without challenge . A polyphasic approach, comprising 16S rDNA sequencing, profiling restriction fragments of 16S-23S rDNA interspacer regions, lipid analysis, immunological profiling and salt tolerance characterizations, was used to clarify the taxonomy of 76 strains of the genera Caulobacter . Brevundimonas, Hyphomonas and Mycoplana . The described species of the genus Caulobacter formed a paraphyletic group with Caulobacter henricii, Caulobacter fusiformis, Caulobacter vibrioides and Mycoplana segnis (Caulobacter segnis comb . nov.) belonging to Caulobacter sensu stricto . Caulobacter bacteroides (Brevundimonas bacteroides comb . nov.), C . henricii subsp . aurantiacus (Brevundimonas aurantiaca comb . nov.), Caulobacter intermedius (Brevundimonas intermedia comb . nov.), Caulobacter subvibrioides (Brevundimonas subvibrioides comb . nov.), C . subvibrioides subsp . albus (Brevundimonas alba comb . nov.), Caulobacter variabilis (Brevundimonas variabilis comb . nov.) and Mycoplana bullata belong to the genus Brevundimonas . The halophilic species Caulobacter maris and Caulobacter halobacteroides are different from these two genera and form the genus Maricaulis gen . nov . with Maricaulis maris as the type species . Caulobacter leidyia was observed to cluster with species of the genus Sphingomonas . Caulobacter crescentus is synonymous with C . vibrioides and C . halobacteroides is synonymous with Maricaulis maris as determined by these analyses and DNA-DNA hybridization . Biomarkers discerning these different genera were determined . The necessary recombinations have been proposed and a description of Maricaulis is presented.

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1039 - 44
Fermentative bacteria from estuarine mud: phylogenetic position of Acidaminobacter hydrogenoformans and description of a new type of gram-negative, propionigenic bacterium as Propionibacter pelophilus gen . nov., sp . nov; Meijer WG et al.; The phylogenetic positions of two strains of fermentative bacteria that had been isolated from the highest positive tubes inoculated with serial dilutions of estuarine mud in agar media with either glutamate or aspartate as substrate were determined by comparative sequence analysis of their 16S rRNA genes . The strain isolated with glutamate (glu 65) utilized several substrates, including a number of amino acids but no sugars . The degradation of certain substrates was enhanced by or dependent upon co-cultivation with a hydrogen-utilizing partner . In earlier work this strain was assigned to the new genus and species Acidaminobacter hydrogenoformans . On the basis of its 16S rRNA gene sequence Acidaminobacter hydrogenoformans has now been identified as a member of cluster XI of the Clostridium subphylum with Clostridium halophilum as its closest relative . The aspartate-fermenting strain asp 66T was a Gram-negative, rather aerotolerant anaerobe which utilized a wide range of substrates in a propionic fermentation and had the ability to fix molecular nitrogen . Strain asp 66T was shown to be a new member of the beta-subclass of the Proteobacteria with Azoarcus sp . strain 6a3 and Rhodocyclus tenuis as its closest relatives . It is described as Propionibacter pelophilus gen . nov., sp . nov., with the type strain asp 66T (= DSM 12018T).

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 953 - 60
Haloanaerobium kushneri sp . nov., an obligately halophilic, anaerobic bacterium from an oil brine; Bhupathiraju VK et al.; Three strains, designated VS-751T, VS-511 and VS-732, of a strictly anaerobic, moderately halophilic, Gram-negative, rod-shaped bacterium were isolated from a highly saline (15-20%) brine from an oil reservoir in central Oklahoma, USA . The optimal concentration of NaCl for growth of these three strains was 2 M (12%), and the strains also grew in the presence of an additional 1 M MgCl2 . The strains were mesophilic and grew at a pH range of 6-8 . Carbohydrates used by all three strains included glucose, fructose, arabinose, galactose, maltose, mannose, cellobiose, sucrose and inulin . Glucose fermentation products included ethanol, acetate, H2 and CO2, with formate produced by two of the three strains . Differences were noted among strains in the optimal temperature and pH for growth, the maximum and minimum NaCl concentration that supported growth, substrate utilization and cellular fatty acid composition . Despite the phenotypic differences among the three strains, analysis of the 16S rRNA gene sequences and DNA-DNA hybridizations showed that these three strains were members of the same genospecies which belonged to the genus Haloanaerobium . The phenotypic and genotypic characteristics of strains VS-751T, VS-511 and VS-732 are different from those of previously described species of Haloanaerobium . It is proposed that strain VS-751T (ATCC 700103T) be established as the type strain of a new species, Haloanaerobium kushneri.

Biophys J, 1999 Aug, 77(2), 1017 - 23
Femtosecond spectroscopic observations of initial intermediates in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila; Devanathan S et al.; Femtosecond time-resolved absorbance measurements were used to probe the subpicosecond primary events of the photoactive yellow protein (PYP), a 14-kD soluble photoreceptor from Ectothiorhodospira halophila . Previous picosecond absorption studies from our laboratory have revealed the presence of two new early photochemical intermediates in the PYP photocycle, I(0), which appears in </=3 ps, and I(0)(double dagger), which is formed in 220 ps, as well as stimulated emission from the PYP excited state . In the present study, kinetic measurements at two excitation wavelengths (395 nm and 460 nm) on either side of the PYP absorption maximum (446 nm) were undertaken using 100-fs pump and probe pulses . Global analysis over a range of probe wavelengths yielded time constants of 1.9 ps for the photochemical formation of the I(0) intermediate via the PYP excited state, and 3.4 ps for the repopulation of the ground state from the excited state . In addition to these pathways, 395 nm excitation also initiated an alternative route for PYP excitation and photochemistry, presumably involving a different excited electronic state of the chromophore . No photochemical intermediates formed before I(0) were observed . Based on these data, a quantum yield of 0.5-0.6 for I(0) formation was determined . The structural and mechanistic aspects of these results are discussed.

Biochemistry, 1999 Jul 13, 38(28), 9039 - 47
Relative role of anions and cations in the stabilization of halophilic malate dehydrogenase; Ebel C et al.; Halophilic malate dehydrogenase unfolds at low salt, and increasing the salt concentration stabilizes, first, the folded form and then, in some cases, destabilizes it . From inactivation and fluorescence measurements performed on the protein after its incubation in the presence of various salts in a large range of concentrations, the apparent effects of anions and cations were found to superimpose . A large range of ions was examined, including conditions that are in general not of physiological relevance, to explore the physical chemistry driving adaptation to extreme environments . The order of efficiency of cations and anions to maintain the folded form is, for the low-salt transition, Ca(2+) approximately Mg(2+) > Li(+) approximately NH(4)(+) approximately Na(+) > K(+) > Rb(+) > Cs(+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-), and for the high-salt transition, NH(4)(+) approximately Na(+) approximately K(+) approximately Cs(+) > Li(+) > Mg(2+) > Ca(2+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-) > Br(-) > I(-) . If a cation or anion is very stabilizing, the effect of the salt ion of opposite charge is limited . Anions of high charge density are always the most efficient to stabilize the folded form, in accordance with the order found in the Hofmeister series, while cations of high charge density are the most efficient only at the lower salt concentrations and tend to denature the protein at higher salt concentrations . The stabilizing efficiency of cations and anions can be related in a minor way to their effect on the surface tension of the solution, but the interaction of ions with sites only present in the folded protein has also to be taken into account . Unfolding at high salt concentrations corresponds to interactions of anions of low charge density and cations of high charge density with the peptide bond, as found for nonhalophilic proteins.

Biotechnol Bioeng, 1999 Jul 5, 64(1), 38 - 45
Expression, reactivation, and purification of enzymes from Haloferax volcanii in Escherichia coli; Connaris H et al.; Enzymes from extreme halophiles have potential as catalysts in biotransformations . We have developed methods for the expression in Escherichia coli and purification of two enzymes from Haloferax volcanii: dihydrolipoamide dehydrogenase and citrate synthase . Both enzymes were expressed in E . coli using the cytoplasmic expression vectors, pET3a and pET3d . Citrate synthase was soluble and inactive, whereas dihydrolipoamide dehydrogenase was expressed as inclusion bodies . Citrate synthase was reactivated following overnight incubation in 2 M KCl, and dihydrolipoamide dehydrogenase was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing 2 M KCl, 10 microM FAD, 1 mM NAD, and 0.3 mM GSSG/3 mM GSH . Maximal activity was obtained after 3 days incubation at 4 degrees C . Purification of the two active enzymes was carried out using high-resolution methods . Dihydrolipoamide dehydrogenase was purified using copper-based metal ion affinity chromatography in the presence of 2 M KCl . Citrate synthase was recovered using dye-affinity chromatography in the presence of salt . A high yield of active enzyme was obtained in both cases . Following purification, characterisation of both recombinant proteins showed that their kinetics and salt-dependence were comparable to those of the native enzymes . Expression of active protein was attempted both by growth of E . coli in the presence of salt and betaine, and also by using periplasmic expression vectors in combination with a high salt growth media . Neither strategy was successful .

Mol Gen Genet, 1999 Jun, 261(4-5), 851 - 61
Analysis of the replication region of the cryptic plasmid pHE1 from the moderate halophile Halomonas elongata; Vargas C et al.; The basic replicon of the narrow-host-range plasmid pHE1 from the moderately halophilic bacterium Halomonas elongata ATCC 33174 has been identified and characterized . The replicon consists of a 1.7-kb DNA fragment, which contains the genetic information required for autonomous replication and stable maintenance . Analysis of its sequence revealed the presence of two ORFs which seem to form one transcription unit . ORF1 encodes a replication initiator protein (RepA), which has a high degree of homology to the theta-replicase (RepA) protein of ColE2 plasmid and to the RepA proteins of a family of replicons from gram-positive and gram-negative bacteria, also related to ColE2 . The product encoded by ORF2 showed a certain similarity to the RepB proteins of the same family of replicons and perhaps represents the pHE1 RepB function . Deletion analysis suggests that the pHE1 origin of replication (ori) is located in an 800-bp region upstream of repA . A third putative gene, incA, was found on the complementary strand to the leader region of the repA mRNA . This, together with the presence in the 5' untranslated region of the repA mRNA of inverted repeats that could form stable stem-loop structures, suggests that the incA gene encodes a small antisense RNA . A possible control mechanism for pHE1 replication is proposed, involving an RNA molecule which sequesters the translational initiation region of the replication protein RepA . The basic replicon characterized here shows very interesting properties that should allow it to be used in the construction of cloning and expression vectors for moderate halophiles.

Curr Microbiol, 1999 Jul, 39(1), 53 - 7
Biomineralization of carbonates by Marinococcus albus and Marinococcus halophilus isolated from the Salar de Atacama (Chile); Rivadeneyra MA et al.; We studied the precipitation of carbonates in 17 strains of moderately halophilic, Gram-positive cocci belonging to two species: Marinococcus halophilus and Marinococcus albus, isolated from the Salar de Atacama (Chile) . They were cultivated in solid and liquid laboratory media for 42 days at salt concentrations (wt/vol) of 3%, 7.5%, 15%, and 20% . The bioliths precipitated were studied by X-ray diffraction and scanning electron microscopy . M . halophilus formed crystals at each of the salt concentrations, with a maximum number of strains capable of precipitating carbonates at 7.5% and 15% salt concentrations . M . albus did not precipitate at 20% and showed a maximum at 7.5% . This behavior is similar to that of other gram-positive bacteria and differs from that found in gram-negative bacteria . The bioliths precipitated were spherical, generally isolated, with a size of 10-100 microm, varying with salinity . They were of magnesium calcite (CO3 Ca1-x Mgx) with Mg content increasing with increasing salinity and Mg/Ca molar ratio of the culture medium . These results demonstrate the active role played by M . halophilus and M . albus in the precipitation of carbonates.

Curr Opin Microbiol, 1999 Jun, 2(3), 265 - 9
Thermophilic and halophilic extremophiles; Madigan MT et al.; The microbiology of extremely hot or saline habitats is a fast moving field with many new successes in the enrichment and isolation of new organisms and in an understanding of molecular factors that impart stability on thermostable and halophilic biomolecules . The results of these studies have shed new light on our understanding of prokaryotic diversity and structural biochemistry.

J Biol Chem, 1999 Jun 18, 274(25), 17655 - 60
Protonation/deprotonation reactions triggered by photoactivation of photoactive yellow protein from Ectothiorhodospira halophila; Hendriks J et al.; Light-dependent pH changes were measured in unbuffered solutions of wild type photoactive yellow protein (PYP) and its H108F and E46Q variants, using two independent techniques: transient absorption changes of added pH indicator dyes and direct readings with a combination pH electrode . Depending on the absolute pH of the sample, a reversible protonation as well as a deprotonation can be observed upon formation of the transient, blue-shifted photocycle intermediate (pB) of this photoreceptor protein . The latter is observed at very alkaline pH, the former at acidic pH values . At neutral pH, however, the formation of the pB state is not paralleled by significant protonation/deprotonation of PYP, as expected for concomitant protonation of the chromophore and deprotonation of Glu-46 during pB formation . We interpret these results as further evidence that a proton is transferred from Glu-46 to the coumaric acid chromophore of PYP, during pB formation . One cannot exclude the possibility, however, that this transfer proceeds through the bulk aqueous phase . Simultaneously, an amino acid side chain(s) (e.g . His-108) changes from a buried to an exposed position . These results, therefore, further support the idea that PYP significantly unfolds in the pB state and resolve the controversy regarding proton transfer during the PYP photocycle.

Biosci Biotechnol Biochem, 1999 Apr, 63(4), 749 - 52
Effect of media constituents on the formation by halophilic yeast of the 2 (or 5)-ethyl-5 (or 2)-methyl-4-hydroxy-3 (2H)-furanone aroma component specific to miso; Sugawara E et al.; The formation of HEMF {2 (or 5)-ethyl-5 (or 2)-methyl-4-hydroxy-3 (2H)-furanone} by yeast was examined in an attempt to investigate its mechanism and involved factors . HEMF formation was promoted by yeast cultivation in a heat-sterilized medium which included glucose, ribose, and a nitrogenous compound such as an extract of shoyu koji, poly-peptone, casamino acid, or an amino acid (glutamic acid, threonine, serine, or alanine).

Extremophiles, 1999 May, 3(2), 163 - 72
Combined effect of the growth temperature and salinity of the medium on the accumulation of compatible solutes by Rhodothermus marinus and Rhodothermus obamensis; Silva Z et al.; In this study we propose revised structures for the two major compatible solutes of Rhodothermus marinus . We have also examined the accumulation of compatible solutes by the type strains of the slightly halophilic and thermophilic species Rhodothermus marinus and Rhodothermus obamensis at several growth temperatures and salinities . The major solutes of R . marinus were identified as alpha-mannosylglycerate (alpha-MG) and alpha-mannosylglyceramide (alpha-MGA), whereas R . obamensis accumulated only alpha-mannosylglycerate . The total osmolyte content was higher during the early exponential phase and decreased abruptly as growth continued into the stationary phase . At low growth temperatures . R . marinus responded to water stress by accumulation of alpha-mannosylglycerate and its amide, in addition to low levels of trehalose, glutamate, and glucose . At the highest growth temperature, alpha-mannosylglycerate was the major compatible solute and alpha-mannosylglyceramide was not detected . When both compounds were present, an increase in the salinity of the growth medium favored the accumulation of alpha-mannosylglyceramide over alpha-mannosylglycerate . The absence of alpha-mannosylglyceramide in R . obamensis at all growth temperatures and salinities constituted the most pronounced difference in the profiles of compatible solute accumulation by the two strains . Trehalose was also a prominent solute in this organism . Both organisms accumulated higher levels of alpha-mannosylglycerate as the temperature was raised . The importance of the two compounds in the mechanisms of thermoadaptation and osmoadaptation is discussed.

Extremophiles, 1999 May, 3(2), 97 - 102
Biodiversity in deep-sea sites located near the south part of Japan; Takami H et al.; We obtained 100 isolates of bacteria from deep-sea mud samples collected at various depths (1050-10897m) . Various types of bacteria such as alkaliphiles, thermophiles, psychrophiles, and halophiles were recovered on agar plates at a frequency of 0.8 x 10(2) to 2.3 x 10(4)/ g of dry sea mud . No acidophiles were recovered . These extremophilic bacteria were widely distributed, being detected at each deep-sea site, and the frequency of isolation of such extremophiles from the deep-sea mud was not directly influenced by the depth of the sampling sites . Phylogenetic analysis of deep-sea isolates based on 16S rDNA sequences revealed that a wide range of taxa were represented in the deep-sea environments . Growth patterns under high hydrostatic pressure were determined for the deep-sea isolates obtained in this study . No extremophilic strains isolated in this study showed growth at 60MPa, although a few of the other isolates grew slightly at this hydrostatic pressure.

Appl Environ Microbiol, 1999 Jun, 65(6), 2748 - 53
Occurrence, diversity, and pathogenicity of halophilic Vibrio spp . and non-O1 Vibrio cholerae from estuarine waters along the Italian Adriatic coast; Barbieri E et al.; The occurrence, diversity, and pathogenicity of Vibrio spp . were investigated in two estuaries along the Italian Adriatic coast . Vibrio alginolyticus was the predominant species, followed by Vibrio parahaemolyticus, non-O1 Vibrio cholerae, and Vibrio vulnificus . By using a biochemical fingerprinting method, all isolates were grouped into nine phenotypes with similarity levels of 75 to 97.5% . The production of toxins capable of causing cytoskeleton-dependent changes was detected in a large number of Vibrio strains . These findings indicate a significant presence of potentially pathogenic Vibrio strains along the Adriatic coast.

J Food Prot, 1999 May, 62(5), 509 - 14
Halotolerant and halophilic histamine-forming bacteria isolated during the ripening of salted anchovies (Engraulis encrasicholus); Hernandez-Herrero MM et al.; This study was performed to investigate halotolerant and halophilic histamine-producing bacteria isolated during the ripening of salted anchovies . Of the isolates obtained during the ripening of anchovies, 1.37% showed histamine-forming activity, most of them (70%) belonging to the Staphylococcus genus . S . epidermidis showed a powerful histamine-forming activity, producing more than 1,000 microg/ml in the presence of 3% and 10% NaCl . Another powerful histamine-producing bacterium isolated during the ripening of salted anchovies was S . capitis . It was able to produce about 400 microg/ml of histamine in 10% NaCl under experimental conditions . Most of these species might be expected to be found as a result of contamination of fish during capture and subsequent unhygienic handling . However, no increase in histamine content was found in any batches through the ripening process . Histamine content always was acceptable in accordance with the maximum allowable levels of histamine fixed by the Spanish and European Union regulations.

J Bacteriol, 1999 May, 181(10), 3226 - 37
Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica; Hochuli M et al.; Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol . The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy . The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway . In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized . For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways . A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya.

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 705 - 24
Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp . nov; Venkateswaran K et al.; The genus Shewanella has been studied since 1931 with regard to a variety of topics of relevance to both applied and environmental microbiology . Recent years have seen the introduction of a large number of new Shewanella-like isolates, necessitating a coordinated review of the genus . In this work, the phylogenetic relationships among known shewanellae were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations . This polyphasic taxonomy takes into account all available phenotypic and genotypic data and integrates them into a consensus classification . Based on information generated from this study and obtained from the literature, a scheme for the identification of Shewanella species has been compiled . Key phenotypic characteristics were sulfur reduction and halophilicity . Fatty acid and quinone profiling were used to impart an additional layer of information . Molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in some cases . As a result, DNA-DNA hybridization and sequence analyses of a more rapidly evolving molecule (gyrB gene) were performed . Species-specific PCR probes were designed for the gyrB gene and used for the rapid screening of closely related strains . With this polyphasic approach, in addition to the ten described Shewanella species, two new species, Shewanella oneidensis and 'Shewanella pealeana', were recognized; Shewanella oneidensis sp . nov . is described here for the first time.

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 521 - 30
Bacillus marismortui sp . nov., a new moderately halophilic species from the Dead Sea; Arahal DR et al.; A group of 91 moderately halophilic, Gram-positive, rod-shaped strains were isolated from enrichments prepared from Dead Sea water samples collected 57 years ago . These strains were examined for 117 morphological, physiological, biochemical, nutritional and antibiotic susceptibility characteristics . All strains formed endospores and were motile, strictly aerobic and positive for catalase and oxidase . They grew in media containing 5-25% (w/v) total salts, showing optimal growth at 10% (w/v) . Eighteen strains were chosen as representative isolates and were studied in more detail . All these strains had mesodiaminopimelic acid in the cell wall and a DNA G + C content of 39.0-42.8 mol%; they constitute a group with levels of DNA-DNA similarity of 70-100% . The sequences of the 16S rRNA genes of three representative strains (strains 123T, 557 and 832) were almost identical (99.9%), and placed the strains in the low G + C content Gram-positive bacteria . On the basis of their features, these isolates should be regarded as members of a new species of the genus Bacillus, for which the name Bacillus marismortui sp . nov . is proposed . The type strain is strain 123T (= DSM 12325T = ATCC 700626T = CIP 105609T = CECT 5066T).

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 441 - 8
Fundibacter jadensis gen . nov., sp . nov., a new slightly halophilic bacterium, isolated from intertidal sediment; Bruns A et al.; A moderately halophilic hydrocarbon-degrading bacterium was isolated from continuous cultures containing a suspension of intertidal sediment from the German North Sea coast with hexadecane as the sole carbon source . On the basis of phenotypic characteristics, fatty acid analysis and 16S rDNA sequence analysis, it was considered to be a new species belonging to a new genus . It is a Gram-negative, aerobic, rod-shaped bacterium, whose cell size varies . It grows at concentrations of 0.5-15% (w/v) NaCl and utilizes a restricted spectrum of carbon sources . The G + C content of the DNA is 63.6 mol% . Comparative 16S rDNA studies show a clear affiliation of this bacterium to the gamma subclass of the class Proteobacteria . Comparison of phylogenetic data indicate that it is most closely related to Marinobacter hydrocarbonoclasticus (88.9% similarity in 16S rRNA gene sequence) . Since it is impossible to find a sufficiently closely related species, we propose the name Fundibacter jadensis gen . nov., sp . nov . for the bacteria . The type strain is T9T (= DSM 12178T).

Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 367 - 75
Marinobacter aquaeolei sp . nov., a halophilic bacterium isolated from a Vietnamese oil-producing well; Huu NB et al.; Several strains of moderately halophilic and mesophilic bacteria were isolated at the head of an oil-producing well on an offshore platform in southern Vietnam . Cells were Gram-negative, non-spore-forming, rod-shaped and motile by means of a polar flagellum . Growth occurred at NaCl concentrations between 0 and 20%; the optimum was 5% NaCl . One strain, which was designated VT8T, could degrade n-hexadecane, pristane and some crude oil components . It grew anaerobically in the presence of nitrate on succinate, citrate or acetate, but not on glucose . Several organic acids and amino acids were utilized as sole carbon and energy sources . The major components of its cellular fatty acids were C12:0 3-OH, C16:1, omega 9c, C16:0 and C18:1 omega 9c . The DNA G + C content was 55.7 mol% . 16S rDNA sequence analysis indicated that strain VT8T was closely related to Marinobacter sp . strain CAB (99.8% similarity) and Marinobaster hydrocarbonoclasticus (99.4% similarity) . Its antibiotic resistance, isoprenoid quinones and fatty acids were similar to those of Marinobacter hydrocarbonoclasticus and Pseudomonas nautica . However, the whole-cell protein pattern of VT8T differed from that of other halophilic marine isolates, including P . nautica . DNA-DNA hybridization indicated that the level of relatedness to Marinobacter hydrocarbonoclasticus was 65% and that to P . nautica was 75% . Further differences were apparent in Fourier-transformed IR spectra of cells and lipopolysaccharide composition . It is proposed that VT8T should be the type strain of a new species and should be named Marinobacter aquaeolei . P . nautica may have been misclassified, as suggested previously, and may also belong to the genus Marinobacter.

Novartis Found Symp, 1999, 221, 200 - 13; discussion 213-7
Bacterial energetics at high pH: what happens to the H+ cycle when the extracellular H+ concentration decreases?
Skulachev VP.
A decrease in the extracellular H+ concentration creates difficulties for membrane-linked energetics in bacteria employing H+ as the coupling ion . At high extracellular pH (pHo), H+ ions pumped from the cell by, say, the respiratory chain, are immediately neutralized by the alkaline extracellular medium . Under such conditions, the only driving force that might compel outer H+ ions to return to cytosol and perform their function is the electric potential difference across the cytoplasmic membrane (delta psi) . However, when delta pH in the opposite direction is equal to, e.g., 2 pH units (intracellular pH = 7.5 at pHo = 9.5), delta psi would be so high that the risk of membrane electric breakdown would increase . This is why some bacteria deal with high pH by, for example, replacing H+ by Na+ as the coupling ion rather than by increasing delta psi . It has been shown in several species of bacteria that the alkalinization of the growth medium induces primary Na+ pumps (e.g . Na(+)-motive respiratory chain enzymes and Na+ ATPase) . Electrogenic Na+ efflux via these pumps produces an electrochemical Na+ potential difference (delta mu Na+) composed of delta psi and delta pNa+ . delta mu Na+ can be used to perform various types of membrane-linked work . The delta psi constituent of delta mu Na+ may maintain electrophoretic influx of H+ such that the alkalinization of cytoplasm is prevented . The latter function may be supported by a mechanism based on the uphill influx of Cl- instead of Na+ . This seems to be the case for alkaliphilic and halophilic Natronobacter pharaonis . There is an indication that not only Na+ but also Ca2+ may substitute for H+ in Gleobacter violaceus growing at high pH.

J Bacteriol, 1999 Apr, 181(8), 2513 - 8
Saturation mutagenesis of the TATA box and upstream activator sequence in the haloarchaeal bop gene promoter; Baliga NS et al.; Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS) . The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeon Halobacterium sp . strain S9 . Active promoters were isolated by screening transformants of an orange (Pum- bop) Halobacterium mutant for purple (Pum+ bop+) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content . Sequence analysis yielded the consensus sequence 5'-tyT(T/a)Ta-3', corresponding to the promoter TATA box element 30 to 25 bp 5' of the transcription start site . A putative UAS, 5'-ACCcnactagTTnG-3', located 52 to 39 bp 5' of the transcription start site was found to be conserved in active promoters . This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity.

J Radiat Res (Tokyo), 1998 Dec, 39(4), 251 - 62
Protective roles of bacterioruberin and intracellular KCl in the resistance of Halobacterium salinarium against DNA-damaging agents; Shahmohammadi HR et al.; Halobacterium salinarium, a member of the extremely halophilic archaebacteria, contains a C50-carotenoid namely bacterioruberin . We have previously reported the high resistance of this organism against the lethal actions of DNA-damaging agents including ionizing radiation and ultraviolet light (UV) . In this study, we have examined whether bacterioruberin and the highly concentrated salts in this bacterium play protective roles against the lethal actions of ionizing radiation, UV, hydrogen peroxide, and mitomycin-C (MMC) . The colourless mutant of H . salinarium deficient in bacterioruberin was more sensitive than the red-pigmented wild-type to all tested DNA-damaging agents except MMC . Circular dichroism (CD) spectra of H . salinarium chromosomal DNA at various concentrations of KCl (0-3.5 M) were similar to that of B-DNA, indicating that no conformational changes occurred as a result of high salt concentrations . However, DNA strand-breaks induced by ionizing radiation were significantly reduced by the presence of either bacterioruberin or concentrated KCl, presumably due to scavenging of free radicals . These results suggest that bacterioruberin and intracellular KCl of H . salinarium protect this organism against the lethal effects of oxidative DNA-damaging agents.

Biosci Biotechnol Biochem, 1999 Feb, 63(2), 288 - 92
cAMP-mediated catabolite repression and electrochemical potential-dependent production of an extracellular amylase in Vibrio alginolyticus; Kim UO et al.; Vibrio alginolyticus, a halophilic marine bacterium, produced an extracellular amylase with a molecular mass of approximately 56,000, and the amylase appeared to be subject to catabolite repression mediated by cAMP . The production of amylase at pH 6.5, at which the respiratory chain-linked H+ pump functions, was inhibited about 75% at 24 hours following the addition of 2 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP), while the production at pH 8.5, at which the respiratory chain-linked Na+ pump functions, was only slightly inhibited by the addition of 2 microM CCCP . In contrast, the production of amylase in a mutant bacterium defective in the Na+ pump was almost completely inhibited even at pH 8.5 as well as pH 6.5 by the addition of 2 microM CCCP.

Syst Appl Microbiol, 1999 Feb, 22(1), 28 - 38
Phylogeny and diversity of Achromatium oxaliferum; Glockner FO et al.; Achromatium oxaliferum was first described in 1893 by Schewiakoff as an unusually large bacterium living in freshwater sediments . Up to now no pure culture is available . Physical enrichments of achromatia collected from the acidic Lake Fuchskuhle, which houses a peculiar, smaller variety, and the neutral Lake Stechlin were investigated by the cultivation-independent rRNA approach . PCR in combination with cloning and sequencing was used for the retrieval of 24 partial and 4 nearly full-length 16S rRNA sequences that formed two distinct phylogenetic clusters . Fluorescence-in-situ-hybridization (FISH) with four 16S rRNA-targeted oligonucleotide probes unambiguously assigned the different sequences to either regular, large A . oxaliferum cells or to the smaller Lake Fuchskuhle population, tentatively named "A . minus" . The two Achromatium sp . 16S rRNA sequence clusters form a stable deep branch in the gamma subclass of the class Proteobacteria . The closest cultivated relatives are Chromatium vinosum, Rhabdochromatium marinum and Ectothiorhodospira halophila with 16S rRNA similarities of 86.2 to 90.5% . Profound differences in the population structure of achromatia were revealed in the two lakes by FISH . In one sample from Lake Stechlin three genotypes could be visualized, and 49% of the cells were assigned to A . oxaliferum clone AST01, 28% to Achromatium sp . genotype AFK192/AFK433 and 23% to Achromatium sp . genotype AFK192/AST433 . In contrast, a morphologically and phylogenetically homogeneous population of "A . minus" . was present in Lake Fuchskuhle.

Biotechnol Bioeng, 1998 Feb 5, 57(3), 306 - 13
Bacterial milking: A novel bioprocess for production of compatible solutes; Sauer T et al.; A novel biotechnological process called "bacterial milking" has been established for the production of compatible solutes using the Gram-negative bacterium Halomonas elongata . Following a high-cell-density fermentation which provided biomass up to 48 g cell dry weight per liter, we applied alternating osmotic shocks in combination with crossflow filtration techniques to harvest the compatible solutes ectoine and hydroxyectoine . H . elongata, like other halophilic or halotolerant microorganisms, produces compatible solutes in response to the salinity of the medium . When transferred to a low salinity medium (osmotic downshock), H . elongata cells rapidly released their solutes to achieve osmotic equilibrium . Subsequent reincubation in a medium of higher salt concentration resulted in resynthesis of these compatible solutes and-after a defined regeneration time-the procedure could be repeated . By repeatedly performing this "bacterial milking" process (at least nine times) we were able to produce large amounts of ectoines with a biomass productivity of 155 mg of ectoine per cycle per gram cell dry weight . Further purification of the products was achieved by a simple two-step procedure based on cation exchange chromatography and crystallization . The principles described in this article may also be useful for the production of other low-molecular-weight compounds .

J Photochem Photobiol B, 1998 Dec, 47(2-3), 148 - 54
Fluorescence and quenching comparative studies of halophilic and bovine glutamate dehydrogenase; Ferrer J et al.; Fluorescence techniques have been used to study the structural characteristics of many proteins . The halophilic enzyme NADP-glutamate dehydrogenase from Haloferax mediterranei is found to be a hexameric enzyme composed of identical subunits . Fluorescence spectra of native and denatured halophilic and bovine glutamate dehydrogenase (h-GDH and b-GDH) have been analysed . Native h-GDH presents the maximum emission at 338 nm, whereas for b-GDH the maximum appears at 332 nm . The denaturation process is accompanied by an exposure to the solvent of the tryptophan residues, as manifested by the red shift of the emission maximum in both cases . The unfolding of h-GDH is a gradual process, which is accompanied by a loss in enzyme activity . Fluorescence quenching by external quenchers, KI and acrylamide, has also been carried out . The tryptophan residues in the protein are more exposed to the solvent in h-GDH than in b-GDH . The total amount of tryptophan residues is nearly the same for both enzymes.

Extremophiles, 1999 Jan, 3(1), 45 - 53
Degradation of 3-phenylpropionic acid by Haloferax sp . D1227; Fu W et al.; Haloferax sp . D1227, isolated from soil contaminated with highly saline oil brine, is the first halophilic archaeon to demonstrate the utilization of aromatic compounds (i.e., benzoic acid, cinnamic acid, and 3-phenylpropionic acid) as sole carbon and energy sources for growth . The degradation of 3-phenylpropionic acid in this strain was studied to examine the strategies utilized by Archaea to metabolize aromatic compounds . Based on our findings of (1) the extracellular accumulation of cinnamic acid, benzoic acid, 3-hydroxybenzoic acid, and gentisic acid in cultures of Haloferax D 1227 grown on 3-phenylpropionic acid, (2) the presence of an 3-phenylpropionylCoA dehydrogenase, (3) the ATP, CoA, and NAD-dependent conversion of cinnamic acid to benzoylCoA, and (4) the presence of gentisate 1,2-dioxygenase, we propose that Haloferax D1227 metabolizes 3-phenylpropionic acid by initial 2-carbon shortening of the side chain to benzoylCoA via a mechanism similar to fatty acid beta-oxidation, followed by aromatic degradation using a gentisate pathway . The upper aliphatic pathway from 3-phenylpropionic acid to benzoic acid is regulated separately from the lower gentisate pathway.

Appl Environ Microbiol, 1999 Mar, 65(3), 1222 - 7
Interactions between nitrogen fixation and osmoregulation in the methanogenic archaeon methanosarcina barkeri 227
Brabban AD, Orcutt EN, Zinder SH.
The nitrogenase enzyme complex of Methanosarcina barkeri 227 was found to be more sensitive to NaCl than previously studied molybdenum nitrogenases are, with total inhibition of activity occurring at 190 mM NaCl, compared with >600 mM NaCl for Azotobacter vinelandii and Clostridium pasteurianum nitrogenases . Na+ and K+ had equivalent effects, whereas Mg2+ was more inhibitory than either monovalent cation, even on a per-charge basis . The anion Cl- was more inhibitory than acetate was . Because M . barkeri 227 is a facultative halophile, we examined the effects of external salt on growth and diazotrophy and found that inhibition of growth was not greater with N2 than with NH4+ . Cells grown with N2 and cells grown with NH4+ produced equal concentrations of alpha-glutamate at low salt concentrations and equal concentrations of Nepsilon-acetyl-beta-lysine at NaCl concentrations greater than 500 mM . Despite the high energetic cost of fixing nitrogen for these osmolytes, we obtained no evidence that there is a shift towards nonnitrogenous osmolytes during diazotrophic growth . In vitro nitrogenase enzyme assays showed that at a low concentration (approximately 100 mM) potassium glutamate enhanced activity but at higher concentrations this compound inhibited activity; 50% inhibition occurred at a potassium glutamate concentration of approximately 400 mM.

Int J Syst Bacteriol, 1999 Jan, 49 Pt 1, 193 - 200
Desulfocella halophila gen . nov., sp . nov., a halophilic, fatty-acid-oxidizing, sulfate-reducing bacterium isolated from sediments of the Great Salt Lake; Brandt KK et al.; A new halophilic sulfate-reducing bacterium, strain GSL-But2T, was isolated from surface sediment of the Southern arm of the Great Salt Lake, UT, USA . The organism grew with a number of straight-chain fatty acids (C4-C16), 2-methylbutyrate, L-alanine and pyruvate as electron donors . Butyrate was oxidized incompletely to acetate . Sulfate, but not sulfite or thiosulfate, served as an electron acceptor . Growth was observed between 2 and 19% (w/v) NaCl with an optimum at 4-5% (w/v) NaCl . The optimal temperature and pH for growth were around 34 degrees C and pH 6.5-7.3, respectively . The generation time under optimal conditions in defined medium was around 28 h, compared to 20 h in complex medium containing yeast extract . The G+C content was 35.0 mol% . 16S rRNA gene sequence analysis revealed that strain GSL-But2T belongs to the family Desulfobacteriaceae within the delta-subclass of the Proteobacteria and suggested that strain GSL-But2T represents a member of a new genus . The name Desulfocella halophila gen . nov., sp . nov . is proposed for this organism . The type strain of D . halophila is strain GSL-But2T (= DSM 11763T = ATCC 700426T).

Int J Syst Bacteriol, 1999 Jan, 49 Pt 1, 103 - 12
Haloanaerobacter salinarius sp . nov., a novel halophilic fermentative bacterium that reduces glycine-betaine to trimethylamine with hydrogen or serine as electron donors; emendation of the genus Haloanaerobacter; Moune S et al.; A novel halophilic fermentative bacterium has been isolated from the black sediment below a gypsum crust and a microbial mat in hypersaline ponds of Mediterranean salterns . Morphologically, physiologically and genetically this organism belongs to the genus Haloanaerobacter . Haloanaerobacter strain SG 3903T (T = type strain) is composed of non-sporulating long flexible rods with peritrichous flagella, able to grow in the salinity range of 5-30% NaCl, with an optimum at 14-15% . The strain grows by fermenting carbohydrates or by using the Stickland reaction with either serine or H2 as electron donors and glycine-betaine as acceptor, which is reduced to trimethylamine . The two species described so far in the genus Haloanaerobacter are not capable of Stickland reaction with glycine-betaine + serine; however, Haloanaerobacter chitinovorans can use glycine-betaine with H2 as electron donor . Strain SG 3903T thus represents the first described strain in the genus Haloanaerobacter capable of the Stickland reaction with two amino acids . Although strain SG 3903T showed 67% DNA-DNA relatedness to H . chitinovorans, it is physiologically sufficiently different from the two described species to be considered as a new species which has been named Haloanaerobacter salinarius sp . nov.

FEMS Microbiol Lett, 1999 Feb 1, 171(1), 27 - 35
Extensive proteolysis inhibits high-level production of eukaryal G protein-coupled receptors in the archaeon Haloferax volcanii; Patenge N et al.; In this study the usage of the halophilic archaeon Haloferax volcanii as a production system for eukaryal G protein-coupled receptors (GPCRs) was characterized . The genes of four GPCRs were fused to the dihydrofolate reductase gene of H . volcanii . In Northern blots both 5' fragments and full-length fusion transcripts were found . In contrast, only C-terminal fusion protein fragments could be detected in Western blot analyses . Ligand binding experiments revealed that a minor amount of correctly folded human beta 2 adrenergic receptor was inserted into the membrane . The introduction of different modifications at the 5' and the 3' end of the receptor genes did not significantly increase the production level . Determination of the subcellular localization showed that fusion protein fragments containing one or more receptor helices were located in the membrane . The results indicate that neither transcription, translation nor membrane translocation but the activity of one or more proteases limits the level of GPCR production in H . volcanii.

Appl Environ Microbiol, 1999 Feb, 65(2), 828 - 33
Regulatory factors associated with synthesis of the osmolyte glycine betaine in the halophilic methanoarchaeon methanohalophilus portucalensis; Lai MC et al.; The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize de novo and accumulate beta-glutamine, Nepsilon-acetyl-beta-lysine, and glycine betaine (betaine) as compatible solutes (osmolytes) when grown at elevated salt concentrations . Both in vivo and in vitro betaine formation assays in this study confirmed previous nuclear magnetic resonance 13C-labelling studies showing that the de novo synthesis of betaine proceeded from glycine, sarcosine, and dimethylglycine to form betaine through threefold methylation . Exogenous sarcosine (1 mM) effectively suppressed the intracellular accumulation of betaine, and a higher level of sarcosine accumulation was accompanied by a lower level of betaine synthesis . Exogenous dimethylglycine has an effect similar to that of betaine addition, which increased the intracellular pool of betaine and suppressed the levels of Nepsilon-acetyl-beta-lysine and beta-glutamine . Both in vivo and in vitro betaine formation assays with glycine as the substrate showed only sarcosine and betaine, but no dimethylglycine . Dimethylglycine was detected only when it was added as a substrate in in vitro assays . A high level of potassium (400 mM and above) was necessary for betaine formation in vitro . Interestingly, no methylamines were detected without the addition of KCl . Also, high levels of NaCl and LiCl (800 mM) favored sarcosine accumulation, while a lower level (400 mM) favored betaine synthesis . The above observations indicate that a high sarcosine level suppressed multiple methylation while dimethylglycine was rapidly converted to betaine . Also, high levels of potassium led to greater amounts of betaine, while lower levels of potassium led to greater amounts of sarcosine . This finding suggests that the intracellular levels of both sarcosine and potassium are associated with the regulation of betaine synthesis in M . portucalensis.

Biochimie, 1998 Dec, 80(12), 1003 - 11
The protein sequence of an archaeal catalase-peroxidase; Cannac-Caffrey V et al.; The gene encoding a catalase-peroxidase of archaeal origin, the halophilic catalase-peroxidase from Haloarcula marismortui, was sequenced . The primary structure proposed was confirmed by Edman degradation and mass spectrometry analyses of proteolytic fragments of the purified protein . The open reading frame in the gene corresponds to 731 amino acids and the calculated mass of the mature protein (deleted of the N-terminal methionine) is 81,253.65 Da, in reasonable agreement with the value of 81,292 +/- 9 Da previously measured by mass spectrometry . Southern and Northern blot analyses showed that the protein is encoded by a single gene as a monocistronic transcript . The protein sequence shows a high level of identity with bacterial catalase-peroxidases, with strongly conserved regions around the heme binding histidines . Similarly to other soluble halophilic proteins, it shows the excess of acidic residues that has been associated with solvation in halophilic adaptation.

Syst Appl Microbiol, 1998 Dec, 21(4), 487 - 97
Characterization of the genes for the biosynthesis of the compatible solute ectoine in the moderately halophilic bacterium Halomonas elongata DSM 3043; Canovas D et al.; The ectoine synthesis genes of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been precisely located in a 2.8-kb EcoRI region of a cosmid clone previously isolated (CANOVAS, D., VARGAS, C., IGLESIAS-GUERRA, F., CSONKA, L . N., RHODES, D., VENTOSA, A., NIETO, J . J.: Isolation and characterization of salt-sensitive mutants of the moderate halophile Halomonas elongata and cloning of the ectoine synthesis genes . J . Biol . Chem . 272, 25794-25801, 1997) . This region was sequenced and three open reading frames were found corresponding to the genes ectA (encoding the diaminobutyric acid acetyl transferase), ectB (encoding the diaminobutyric acid aminotransferase) and ectC (encoding the ectoine synthase) . These three genes were able to restore the salt tolerance of two H . elongata mutants defective in the synthesis of ectoine (strains CHR62 and CHR63) . However, the H . elongata ectoine synthesis genes did not confer to Escherichia coli the ability to synthesize ectoine . Transposon insertion in the salt-sensitive mutant strain CHR63 was exactly mapped within the ectC gene . Moreover, sequences homologous to the H . elongata ect region have been found in a number of moderately halophilic bacteria belonging to the genera Halomonas and Chromohalobacter.

J Bacteriol, 1999 Feb, 181(3), 957 - 64
NolL of Rhizobium sp . strain NGR234 is required for O-acetyltransferase activity; Berck S et al.; Following (iso)flavonoid induction, nodulation genes of the symbiotic nitrogen-fixing bacterium Rhizobium sp . strain NGR234 elaborate a large family of lipooligosaccharidic Nod factors (NodNGR factors) . When secreted into the rhizosphere of compatible legumes, these signal molecules initiate root hair deformation and nodule development . The nonreducing glucosamine residue of NodNGR factors are N acylated, N methylated, and mono- or biscarbamoylated, while position C-6 of the reducing extremity is fucosylated . This fucose residue is normally 2-O methylated and either sulfated or acetylated . Here we present an analysis of all acetylated NodNGR factors, which clearly shows that the acetate group may occupy position C-3 or C-4 of the fucose moiety . Disruption of the flavonoid-inducible nolL gene, which is preceded by a nod box, results in the synthesis of NodNGR factors that lack the 3-O- or 4-O-acetate groups . Interestingly, the nodulation capacity of the mutant NGROmeganolL is not impaired, whereas introduction of the nod box::nolL construct into the related strain Rhizobium fredii USDA257 extends the host range of this bacterium to Calopogonium caeruleum, Leucaena leucocephala, and Lotus halophilus . Nod factors produced by a USDA257(pnolL) transconjugant were also acetylated . The nod box::nolL construct was also introduced into ANU265 (NGR234 cured of its symbiotic plasmid), along with extra copies of the nodD1 gene . When permeabilized, these cells possessed acetyltransferase activity, although crude extracts did not.

J Mol Biol, 1999 Jan 8, 285(1), 163 - 74
Evolution of the archaeal rhodopsins: evolution rate changes by gene duplication and functional differentiation; Ihara K et al.; The amino acid sequences of 25 archaeal retinal proteins from 13 different strains of extreme halophiles were analyzed to establish their molecular phylogenetic relationship . On the basis of amino acid sequence similarity, these proteins apparently formed a distinct family designated as the archaeal rhodopsin family (ARF), which was not related to other known proteins, including G protein-coupled receptors . The archaeal rhodopsin family was further divided into four clusters with different functions; H+ pump (bacteriorhodopsin), Cl- pump (halorhodopsin), and two kinds of sensor (sensory rhodopsin and phoborhodopsin) . These four rhodopsin clusters seemed to have occurred by gene duplication(s) before the generic speciation of halophilic archaea, based on phylogenetic analysis . Therefore, the degrees of differences in amino acid sequences within each cluster simply reflected the divergent evolution of halophilic archaea . By comparing the branch lengths after speciation points of the reconstituted tree, we calculated the relative evolution rates of the four archaeal rhodopsins bacteriorhodopsin:halorhodopsin:sensory rhodopsin: phoborhodopsin to be 5:4:3:10 . From these values, the degrees of functional and structural restriction of each protein can be inferred . The branching topology of four clusters grouped bacteriorhodopsin and halorhodopsin versus sensory rhodopsin and phoborhodopsin by likelihood mapping . Using bacteriorhodopsin (and halorhodopsin) as an outgroup, the gene duplication point of sensory rhodopsin/phoborhodopsin was determined . By calculating the branch lengths between the gene duplication point and each halophilic archaea speciation point, we could speculate upon the relative evolution rate of pre-sensory rhodopsin and pre-phoborhodopsin . The evolution rate of pre-sensory rhodopsin was fivefold faster than that of pre-phoborhodopsin, which suggests that the original function of the ancestral sensor was similar to that of phoborhodopsin, and that sensory rhodopsin evolved from pre-sensory rhodopsin by the accumulation of mutations . The changes in evolution rate by gene duplication and functional differentiation were demonstrated in the archaeal rhodopsin family using the gene duplication date and halobacterial speciation date as common time stamps .

J Bacteriol, 1999 Jan, 181(1), 91 - 9
Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata; Ono H et al.; 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant . The biosynthetic pathway of ectoine from aspartic beta-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved . 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with L-glutamate . This enzyme required pyridoxal 5'-phosphate and potassium ions for its activity and stability . The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa . This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25 degreesC and had Kms of 9.1 mM for L-glutamate and 4.5 mM for DL-ASA . DABA acetyltransferase catalyzed acetylation of DABA to gamma-N-acetyl-alpha,gamma-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20 degreesC in the presence of 0.4 M NaCl . The molecular mass was 45 kDa by gel filtration . Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15 degreesC in the presence of 0.5 M NaCl . This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0 . 77 M NaCl . DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30 degreesC.

Genome Res, 1998 Nov, 8(11), 1131 - 41
Snapshot of a large dynamic replicon in a halophilic archaeon: megaplasmid or minichromosome?
Ng WV, Ciufo SA, Smith TM, Bumgarner RE, Baskin D, Faust J, Hall B, Loretz C, Seto J, Slagel J, Hood L, DasSarma S.
Extremely halophilic archaea, which flourish in hypersaline environments, are known to contain a variety of large dynamic replicons . Previously, the analysis of one such replicon, pNRC100, in Halobacterium sp . strain NRC-1, showed that it undergoes high-frequency insertion sequence (IS) element-mediated insertions and deletions, as well as inversions via recombination between 39-kb-long inverted repeats (IRs) . Now, the complete sequencing of pNRC100, a 191,346-bp circle, has shown the presence of 27 IS elements representing eight families . A total of 176 ORFs or likely genes of 850-bp average size were found, 39 of which were repeated within the large IRs . More than one-half of the ORFs are likely to represent novel genes that have no known homologs in the databases . Among ORFs with previously characterized homologs, three different copies of putative plasmid replication and four copies of partitioning genes were found, suggesting that pNRC100 evolved from IS element-mediated fusions of several smaller plasmids . Consistent with this idea, putative genes typically found on plasmids, including those encoding a restriction-modification system and arsenic resistance, as well as buoyant gas-filled vesicles and a two-component regulatory system, were found on pNRC100 . However, additional putative genes not expected on an extrachromosomal element, such as those encoding an electron transport chain cytochrome d oxidase, DNA nucleotide synthesis enzymes thioredoxin and thioredoxin reductase, and eukaryotic-like TATA-binding protein transcription factors and a chromosomal replication initiator protein were also found . A multi-step IS element-mediated process is proposed to account for the acquisition of these chromosomal genes . The finding of essential genes on pNRC100 and its property of resistance to curing suggest that this replicon may be evolving into a new chromosome.

Biosci Biotechnol Biochem, 1998 Oct, 62(10), 2062 - 4
Selective fermentation of xylose by a mutant of Tetragenococcus halophila defective in phosphoenolpyruvate:mannose phosphotransferase, phosphofructokinase, and glucokinase; Abe K et al.; Tetragenococcus halophila is a Gram-positive halophilic lactic acid bacterium used for soy sauce fermentation . We isolated a mutant, T . halophila 3E4, triply defective in phosphoenolpyruvate:mannose phosphotransferase, phosphofructokinase, and glucokinase . 3E4 selectively metabolized pentoses such as xylose and arabinose in the presence of hexoses such as glucose and galactose . We present here an example of the metabolic engineering of catabolite control.

Appl Environ Microbiol, 1998 Dec, 64(12), 4720 - 8
Molecular cloning and functional expression in lactobacillus plantarum 80 of xylT, encoding the D-xylose-H+ symporter of Lactobacillus brevis; Chaillou S et al.; A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (encoding D-xylose isomerase), was cloned in Escherichia coli TG1 . The sequence revealed two open reading frames which could code for the D-xylulose kinase gene (xylB) and another gene (xylT) encoding a protein of 457 amino acids with significant similarity to the D-xylose-H+ symporters of E . coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the D-xylose-Na+ symporter of Tetragenococcus halophila, XylE (57%), and to the L-arabinose-H+ symporter of E . coli, AraE (60%) . The L . brevis xylABT genes showed an arrangement similar to that of the B . megaterium xylABT operon and the T . halophila xylABE operon . Southern hybridization performed with the Lactobacillus pentosus xylR gene (encoding the D-xylose repressor protein) as a probe revealed the existence of a xylR homologue in L . brevis which is not located with the xyABT locus . The existence of a functional XylR was further suggested by the presence of xylO sequences upstream of xylA and xylT and by the requirement of D-xylose for the induction of D-xylose isomerase, D-xylulose kinase, and D-xylose transport activities in L . brevis . When L . brevis was cultivated in a mixture of D-glucose and D-xylose, the D-xylose isomerase and D-xylulose kinase activities were reduced fourfold and the D-xylose transport activity was reduced by sixfold, suggesting catabolite repression by D-glucose of D-xylose assimilation . The xylT gene was functionally expressed in Lactobacillus plantarum 80, a strain which lacks proton motive force-linked D-xylose transport activity . The role of the XylT protein was confirmed by the accumulation of D-xylose in L . plantarum 80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of D-glucose . The apparent affinity constant of XylT for D-xylose was approximately 215 microM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight) . Furthermore, of a number of sugars tested, only 6-deoxy-D-glucose inhibited the transport of D-xylose by XylT competitively, with a Ki of 220 microM.

Int J Syst Bacteriol, 1998 Oct, 48 Pt 4, 1341 - 8
Marinospirillum gen . nov., with descriptions of Marinospirillum megaterium sp . nov., isolated from kusaya gravy, and transfer of Oceanospirillum minutulum to Marinospirillum minutulum comb . nov; Satomi M et al.; Two strains of helical, halophilic, Gram-negative, heterotrophic bacteria were isolated from kusaya gravy which is a traditional Japanese fermented brine . These strains were motile by means of a single polar or bipolar tuft flagellum . They had a large cell size, were helical, formed coccoid bodies, were microaerophilic and had quinone type Q-8 . The DNA G + C content of the strains was 44-45 mol % . A detailed investigation of the phenotypic, chemotaxonomic and phylogenetic characteristics of the strains revealed that they represent a new species of halophilic helical bacteria . The sequence of the 16S rRNA gene of strain H7T, designated the type strain of the new isolates, and all of the Oceanospirillum species except for Oceanospirillum linum were determined . Phylogenetic analysis indicated that these strains were closely related to Oceanospirillum minutulum, with enough distance to separate the O . minutulum/new isolate H7T cluster from Oceanospirillum sensu stricto on the genus level . It is proposed that a new genus, Marinospirillum, be created; this genus should include Marinospirillum minutulum ATCC 19193T (formerly Oceanospirillum minutulum) as the type species, as well as Marinospirillum megaterium JCM 10129T (= H7T).

Int J Syst Bacteriol, 1998 Oct, 48 Pt 4, 1305 - 12
Halogeometricum borinquense gen . nov., sp . nov., a novel halophilic archaeon from Puerto Rico; Montalvo-Rodriguez R et al.; A novel extremely halophilic archaeon was isolated from the solar salterns of Cabo Rojo, Puerto Rico . The organism is very pleomorphic, motile and requires at least 8% (w/v) NaCl to grow . Polar lipid composition revealed the presence of a novel non-sulfate-containing glycolipid and the absence of the glycerol diether analogue of phosphatidylglycerosulfate . The G + C content of the DNA is 59 mol% . On the basis of 16S rRNA sequence data, the new isolate cannot be classified in one of the recognized genera, but occupies a position that is distantly related to the genus Haloferax . All these features justify the creation of a new genus and a new species for the family Halobacteriaceae, order Halobacteriales . The name Halogeometricum borinquense gen . nov., sp . nov . is proposed . The type strain is ATCC 700274T.

Int J Syst Bacteriol, 1998 Oct, 48 Pt 4, 1129 - 43
Phylogenetic relationships among the Chromatiaceae, their taxonomic reclassification and description of the new genera Allochromatium, Halochromatium, Isochromatium, Marichromatium, Thiococcus, Thiohalocapsa and Thermochromatium; Imhoff JF et al.; Sequences of the 16S rDNA from all available type strains of Chromatium species have been determined and were compared to those of other Chromatiaceae, a few selected Ectothiorhodospiraceae and Escherichia coli . The clear separation of Ectothiorhodospiraceae and Chromatiaceae is confirmed . Most significantly the sequence comparison revealed a genetic divergence between Chromatium species originated from freshwater sources and those of truly marine and halophilic nature . Major phylogenetic branches of the Chromatiaceae contain (i) marine and halophilic species, (ii) freshwater Chromatium species together with Thiocystis species and (iii) species of the genera Thiocapsa and Amoebobacter as recently reclassified {Guyoneaud, R . & 6 other authors (1988) . Int J Syst Bacteriol 48, 957-964}, namely Thiocapsa roseopersicina, Thiocapsa pendens (formerly Amoebobacter pendens), Thiocapsa rosea (formerly Amoebobacter roseus), Amoebobacter purpureus and Thiolamprovum pedioforme (formerly Amoebobacter pedioformis) . The genetic relationships between the species and groups are not in congruence with the current classification of the Chromatiaceae and a reclassification is proposed on the basis of 16S rDNA sequence similarity supported by selected phenotypic properties . The proposed changes include the transfers of Chromatium minus and Chromatium violascens to Thiocystis minor comb . nov . and Thiocystis violascens comb . nov., of Chromatium vinosum, Chromatium minutissimum and Chromatium warmingii to the new genus Allochromatium as Allochromatium vinosum comb . nov., Allochromatium minutissimum comb . nov., and Allochromatium warmingii comb . nov., of Chromatium tepidum to the new genus Thermochromatium as Thermochromatium tepidum comb . nov., of Chromatium salexigens and Chromatium glycolicum to the new genus Halochromatium as Halochromatium salexigens comb . nov . and Halochromatium glycolicum comb . nov., of Chromatium gracile and Chromatium purpuratum to the new genus as Marichromatium gracile comb . nov . and Marichromatium purpuratum comb . nov., of Thiocapsa pfennigii to Thiococcus pfennigii gen . nom . rev., of Thiocapsa halophila to the new genus Thiohalocapsa as Thiohalocapsa halophila comb . nov., and of Chromatium buderi to the new genus Isochromatium as Isochromatium buderi comb . nov.

Extremophiles, 1998 Nov, 2(4), 439 - 46
Gentisate 1,2-dioxygenase from Haloferax sp . D1227; Fu W et al.; Gentisate 1,2-dioxygenase from the extreme halophile Haloferax sp . D1227 (Hf . D1227) was purified using a three-step procedure . The enzyme was found to be a homotetramer of 42,000 +/- 1,000 Da subunits, with a native molecular weight of 174,000 +/- 6,000 Da . The optimal salt concentration, temperature, and pH for enzyme activity were 2 M KCl or NaCl, 45 degrees C, and pH 7.2, respectively . The gene encoding Hf . D1227 gentisate 1,2-dioxygenase was cloned, sequenced, and expressed in Haloferax volcanii . The deduced amino acid sequence exhibited a 9.2% excess acidic over basic amino acids typical of halophilic enzymes . Four novel histidine clusters and a possible extradiol dioxygenase fingerprint region were identified.

Extremophiles, 1998 Nov, 2(4), 435 - 8
Analysis of the genome of the gram-negative moderate halophiles Halomonas and Chromohalobacter by using pulsed-field gel electrophoresis; Mellado E et al.; The genomes of 11 moderately halophilic bacteria belonging to the genera Halomonas and Chromohalobacter have been analyzed by restriction endonuclease digestion and pulsed-field gel electrophoresis (PFGE) . By using the infrequently cutting restriction endonucleases SpeI and SwaI, highly characteristic fingerprintings were obtained for each of the isolates studied . On the basis of the lengths of the SpeI and SwaI fragments, separated by PFGE, the genome size of the 11 strains studied was estimated . The genome size for 8 Halomonas strains ranged from 1450 to 2830 kb, whereas for the 3 Chromohalobacter strains studied it ranged from 1770 to 2295 kb . Finally, we show that macrorestriction fingerprints could be a useful tool to elucidate the taxonomic position of bacteria belonging to the Halomonas-Deleya complex.

Res Microbiol, 1998 Oct, 149(9), 675 - 9
Influence of salt concentration on the cellular fatty acid composition of the moderately halophilic bacterium Halomonas salina; Valderrama MJ et al.; The cellular fatty acid composition of Halomonas salina, a moderately halophilic bacterium grown at different salt concentrations, is reported . Fatty acids C16:0 and C18:1 were major components and significant amounts of C16:1, C18:0 and cyc-C19:0 were also detected . The results showed clear chemotaxonomic relationships with recognized members of the genus Halomonas . The salt concentration greatly influenced the fatty acid composition, suggesting activation of cyclopropane synthetase at high levels of salt, since increases in cyclopropane fatty acids with decreases in monounsaturated fatty acids were observed as the salt concentration in the medium rose.

FEBS Lett, 1998 Oct 30, 438(1-2), 124 - 6
F420H2:NADP oxidoreductase from Methanobacterium thermoautotrophicum: identification of the encoding gene via functional overexpression in Escherichia coli; Berk H et al.; F420H2:NADP oxidoreductase is found in methanogenic, sulfate-reducing and halophilic archaea and also in some bacteria . The putative gene encoding the enzyme was cloned from Methanobacterium thermoautotrophicum (strain Marburg) and heterologously expressed in Escherichia coli . The overproduced active enzyme was purified, characterized and crystallized.

Biochem Mol Biol Int, 1998 Oct, 46(3), 495 - 507
Cloning and sequencing of the homologues of both the bacterial and eukaryotic initiation factor genes (hIF-2 and heIF-2 gamma) from archaeal Halobacterium halobium; Hasegawa Y et al.; The cloning and sequencing of the genes encoding the translational initiation factors (hIF-2 and heIF-2 gamma) was performed by screening the halophilic archaeon Halobacterium halobium genomic library with a probe constructed from the peptide IGHVDHGK that is conserved in archaeal GTP-binding elongation factors . The codon usage by the hIF-2 and heIF-2 gamma genes showed a preference for triplets ending in G or C . This characteristic is almost identical to that of other H . halobium genes . The translated protein of hIF-2 and heIF-2 gamma genes is made of 414 and 583 amino acid residues, respectively, and contains the sequence motif for the binding of GTP . The sequence of hIF-2 shows a strong similarity to the initiation factor IF-2 from Bacteria whereas heIF-2 gamma shows a strong similarity to the initiation factor eIF-2 gamma from Eucarya.

FEMS Microbiol Lett, 1998 Oct 15, 167(2), 287 - 93
Functional expression of green fluorescent protein derivatives in Halobacterium salinarum; Nomura S et al.; We investigated the applicability of the green fluorescent protein (GFP) of Aequorea victoria as a reporter for gene expression in an extremely halophilic organism: Halobacterium salinarum . Two recombinant GFPs were fused with bacteriorhodopsin, a typical membrane protein of H . salinarum . These fusion proteins preserved the intrinsic functions of each component, bacteriorhodopsin and GFP, were expressed in H . salinarum under conditions with an extremely high salt concentration, and were proved to be properly localized in its plasma membrane . These results suggest that GFP could be used as a versatile reporter of gene expression in H . salinarum for investigations of various halophilic membrane proteins, such as sensory rhodopsin or phoborhodopsin.

Biosci Biotechnol Biochem, 1998 Sep, 62(9), 1676 - 83
Xylose transport insensitivity to catabolite inhibition by phosphoenolpyruvate:sugar phosphotransferase system in Tetragenococcus halophila; Abe K et al.; Tetragenococcus halophila accumulates glucose and 2-deoxyglucose (dGlc) via the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and pentoses, maltose, and glycerol via non-PTS carriers . Based on the discovery that xylose metabolism in T . halophila was subject to catabolite repression but not to catabolite inhibition, we designed a selection protocol for thermosensitive mutants pleiotropically unable to use sugars . One such mutant was a ptsI mutant with a thermosensitive enzyme I (EI) of the PTS (leaky at 30 degrees C) . Using this ptsI mutant, catabolite inhibitions was studied . dGlc was more strongly inhibitory of glycerol uptake in the mutant than in the parent because of the leaky ptsI mutation . Thermoinactivation of EI at 42 degrees C resulted in the total loss of uptake of PTS sugars and in the virtual abolishment of glycerol uptake . However, xylose uptake of the ptsI mutant was scarcely inhibited by dGlc even after thermoinactivation of EI . These results suggest that sensitivities of non-PTS uptakes to PTS-mediated inhibition vary among non-PTS sugars.

Arch Microbiol, 1998 Nov, 170(6), 435 - 41
The respiratory chain of the halophilic anoxygenic purple bacterium Rhodospirillum sodomense; Bonora P et al.; The halophilic purple nonsulfur bacterium Rhodospirillum sodomense has been previously described as an obligate phototroph that requires yeast extract and a limited number of organic compounds for photoheterotrophic growth . In this work, we report on chemoheterotrophic growth of R . sodomense in media containing either acetate or succinate supplemented with 0.3-0.5% yeast extract . Plasma membranes isolated from cells grown aerobically in the dark contained three b-type and three c-type membrane-bound cytochromes with Em,7 of +171 +/- 10, +62 +/- 10 and -45 +/- 13 mV (561-575 nm), and +268 +/- 6, +137 +/- 10 and -43 +/- 12 mV (551-540 nm) . A small amount of a soluble c-type cytochrome with a mol . mass of 15 kDa (Em, 7 >/= +150 mV) was identified . Spectroscopic and immunological methods excluded the presence of cytochrome of the c2 class and high-potential iron-sulfur proteins . Inhibitory studies indicated that only 60-70% of the respiratory activity was blocked by low concentrations of cyanide, antimycin A, and myxothiazol (10, 0.1, and 0.2 microM, respectively) . These results were interpreted to show that the oxidative electron transport chain of R . sodomense is branched, leads to a quinol oxidase that is fully blocked by 1 mM cyanide and that is involved in light-dependent oxygen reduction, and leads to a cytochrome c oxidase that is inhibited by 10 microM cyanide . These features taken together suggest that R . sodomense differs from the closely related species Rhodospirillum salinarum and from other species of the genus Rhodospirillum in that it contains multiple membrane-bound cytochromes c.

J Mol Evol, 1998 Nov, 47(5), 565 - 77
Heat shock protein 70 family: multiple sequence comparisons, function, and evolution; Karlin S et al.; The heat shock protein 70 kDa sequences (HSP70) are of great importance as molecular chaperones in protein folding and transport . They are abundant under conditions of cellular stress . They are highly conserved in all domains of life: Archaea, eubacteria, eukaryotes, and organelles (mitochondria, chloroplasts) . A multiple alignment of a large collection of these sequences was obtained employing our symmetric-iterative ITERALIGN program (Brocchieri and Karlin 1998) . Assessments of conservation are interpreted in evolutionary terms and with respect to functional implications . Many archaeal sequences (methanogens and halophiles) tend to align best with the Gram-positive sequences . These two groups also miss a signature segment {about 25 amino acids (aa) long} present in all other HSP70 species (Gupta and Golding 1993) . We observed a second signature sequence of about 4 aa absent from all eukaryotic homologues, significantly aligned in all prokaryotic sequences . Consensus sequences were developed for eight groups {Archaea, Gram-positive, proteobacterial Gram-negative, singular bacteria, mitochondria, plastids, eukaryotic endoplasmic reticulum (ER) isoforms, eukaryotic cytoplasmic isoforms} . All group consensus comparisons tend to summarize better the alignments than do the individual sequence comparisons . The global individual consensus "matches" 87% with the consensus of consensuses sequence . A functional analysis of the global consensus identifies a (new) highly significant mixed charge cluster proximal to the carboxyl terminus of the sequence highlighting the hypercharge run EEDKKRRER (one-letter aa code used) . The individual Archaea and Gram-positive sequences contain a corresponding significant mixed charge cluster in the location of the charge cluster of the consensus sequence . In contrast, the four Gram-negative proteobacterial sequences of the alignment do not have a charge cluster (even at the 5% significance level) . All eukaryotic HSP70 sequences have the analogous charge cluster . Strikingly, several of the eukaryotic isoforms show multiple mixed charged clusters . These clusters were interpreted with supporting data related to HSP70 activity in facilitating chaperone, transport, and secretion function . We observed that the consensus contains only a single tryptophan residue and a single conserved cysteine . This is interpreted with respect to the target rule for disaggregating misfolded proteins . The mitochondrial HSP70 connections to bacterial HSP70 are analyzed, suggesting a polyphyletic split of Trypanosoma and Leishmania protist mitochondrial (Mt) homologues separated from Mt-animal/fungal/plant homologues . Moreover, the HSP70 sequences from the amitochondrial Entamoeba histolytica and Trichomonas vaginalis species were analyzed . The E . histolytica HSP70 is most similar to the higher eukaryotic cytoplasmic sequences, with significantly weaker alignments to ER sequences and much diminished matching to all eubacterial, mitochondrial, and chloroplast sequences . This appears to be at variance with the hypothesis that E . histolytica rather recently lost its mitochondrial organelle . T . vaginalis contains two HSP70 sequences, one Mt-like and the second similar to eukaryotic cytoplasmic sequences suggesting two diverse origins.

Prog Biophys Mol Biol, 1998, 70(2), 95 - 136
The structure of iron-sulfur proteins; Sticht H et al.; Ferredoxins are a group of iron-sulfur proteins for which a wealth of structural and mutational data have recently become available . Previously unknown structures of ferredoxins which are adapted to halophilic, acidophilic or hyperthermophilic environments and new cysteine patterns for cluster ligation and non-cysteine cluster ligation have been described . Site-directed mutagenesis experiments have given insight into factors that influence the geometry, stability, redox potential, electronic properties and electron-transfer reactivity of iron-sulfur clusters.

FEMS Microbiol Lett, 1998 Oct 1, 167(1), 57 - 61
Presence of Na(+)-stimulated V-type ATPase in the membrane of a facultatively anaerobic and halophilic alkaliphile; Kaieda N et al.; It was found that a facultatively anaerobic and halophilic alkaliphile, M-12 (Amphibacillus sp.), possesses a Na(+)-stimulated ATPase in the membrane . The ATPase activity was inhibited by NO3- and SCN- which are the inhibitors of V-type ATPase, but not by azide and vanadate, inhibitors of F-type ATPase and P-type ATPase, respectively . Upon the incubation of the membrane in buffer containing ATP and MgCl2, several polypeptides were released from the membrane . Among them, two major polypeptides with apparent molecular masses of 79 and 55 kDa crossreacted with an antiserum against the catalytic units (subunits A and B) of V-type ATPase from Enterococcus hirae . The N-terminal amino acid sequences of the 79 and 55 kDa polypeptides showed high similarity to those of subunits A and B of V-type ATPase from Enterococcus hirae, respectively . M-12 is likely to possess a V-type Na(+)-ATPase.

Extremophiles, 1998 Aug, 2(3), 321 - 31
Distribution and diversity of halophilic bacteria in a subsurface salt formation; Vreeland RH et al.; The Waste Isolation Pilot Plant (WIPP) is a salt mine constructed 650 meters below the ground surface by the United States Department of Energy . The facility will be used for permanent disposal of transuranic wastes . This underground repository has been constructed in the geologically stable Permian age Salado salt formation . Of the wastes to be placed into the facility, 85% will be biodegradable cellulose . A 3-year survey of the bacterial populations existing within the facility was conducted . Bacterial populations were found to be heterogeneously distributed throughout the mine . Populations in some mine areas reached as high as 1.0 x 10(4) colony-forming units per gram of NaCl . The heterogeneous distribution of bacteria within the mine did not follow any recognizable pattern related to either age of the workings or to human activity . A biochemical comparison between ten known species of halophilic bacteria, and strains isolated from both the mine and nearby surface hypersaline lakes, showed the presence of extreme halophiles with wide biochemical diversity, some of which could prove to represent previously undescribed groups . The halophilic bacteria isolated from the mine were found to degrade cellulose and a wide variety of other carbon compounds . When exposed to two types of common laboratory paper, the cellulose-degrading halophiles attached to the substrate within 30 minutes of inoculation . Cultures enriched directly from a brine seep in the mine easily destroyed both papers and produced detectable amounts of oxalacetic and pyruvic acids . The combination of heterogeneity in the distribution of organisms, the presence of a physiologically diverse community, and the relatively slow metabolism of cellulose may explain several long-standing debates about the existence of microorganisms in ancient underground salt formations.

Can J Microbiol, 1998 Jul, 44(7), 637 - 45
Alterococcus agarolyticus, gen.nov., sp.nov., a halophilic thermophilic bacterium capable of agar degradation; Shieh WY et al.; Five strains of facultatively anaerobic moderately thermophilic bacteria were isolated from two hot springs in the intertidal zone of Lutao, Taiwan . They produced extracellular agarase on agar medium, yielding reducing sugars and organic acids as the end products under either aerobic or anaerobic conditions . The growth temperature range was approximately 38-58 degrees C with an optimal temperature of about 48 degrees C . The five strains tolerated a relatively narrow pH range from 7.0 to 8.5 . They were Gram-negative halophiles growing optimally at 2.0-2.5% NaCl (ca . 0.34-0.43 M) . They were capable of anaerobic growth by fermenting glucose and producing various organic acids such as butyrate, propionate, formate, lactate, and acetate . Cells grown in liquid medium were motile monotrichous cocci, normally 0.8-0.9 micron in diameter . They possessed saturated anteiso-15-carbon acid (anteiso-C15:0) as the most abundant cellular fatty acid (46.0-51.3 mo1%) and had G + C contents ranging from 65.5 to 67.0 mo1% . They are the first thermophiles found to degrade agar and also the first halophilic thermophilic bacteria known to be capable of both aerobic and anaerobic fermentative growth . These bacteria are considered to represent a new genus that we named Alterococcus, and Alterococcus agarolyticus is the type species.

Extremophiles, 1998 Aug, 2(3), 297 - 304
Moderately halophilic gram-positive bacterial diversity in hypersaline environments; Ventosa A et al.; Moderately halophilic bacteria are microorganisms that grow optimally in media containing 3%-15% (w/v) salt . They are represented by a heterogeneous group of microorganisms included in many different genera . Gram-negative moderately halophilic bacteria have been studied in more detail, but studies on gram-positive species are more scarce . Recent studies carried out by our research group on gram-positive moderate halophiles have permitted clarifying their taxonomic and phylogenetic position and describing new species . Thus, we have isolated six strains from ponds of salterns that show phenotypic and genotypic characteristics similar to those of Nesterenkonia halobia (formerly Micrococcus halobius), a moderately halophilic gram-positive coccus that was described on the basis of a single strain . Our data demonstrate quite clearly that they are members of this species and contribute to a better description of these moderately halophilic cocci . Similarly, a study of a large number of gram-positive moderately halophilic rods that were able to produce endospores led us to describe a new species, designated Bacillus salexigens . Further, isolates grouped in other three phenons, obtained by numerical taxonomy analysis and showing phenotypic features quite similar to those of this species, represent different genomovars, with very low DNA-DNA homology . Although they might represent additional new species, it will be necessary to determine new phenotypic features to differentiate them from previously described Bacillus species . We have also studied the viability of some old enrichments provided by B.E . Volcani, which were set up in 1936 . We isolated 31 gram-positive motile endospore-forming rods that, according to their phenotypic characteristics, could represent a new species of the genus Bacillus.

Extremophiles, 1998 Aug, 2(3), 289 - 95
Diversity of extremely halophilic bacteria; Kamekura M; In this review, the history of the classification of the family Halobacteriaceae, the extremely halophilic aerobic Archaea, is reviewed with some emphasis on the recently described new genera Halobaculum, Halorubrum, Natrialba, Natronomonas, and "Haloterrigena." Speculation is made about the evolutionary relationship between members of the Halobacteriaceae and the extremely halophilic, anaerobic methanogens of the genera Methanohalobium and Methanohalophilus . Efforts to find missing links between the two groups are also reviewed.

Extremophiles, 1998 Aug, 2(3), 279 - 87
Halobacteria: the evidence for longevity; Grant WD et al.; Subterranean salt deposits are the remains of ancient hypersaline waters that presumably supported dense populations of halophilic microorganisms including representatives of the haloarchaea (halobacteria) . Ancient subterranean salt deposits (evaporites) are common throughout the world, and the majority sampled to date appear to support diverse populations of halobacteria . The inaccessibility of deep subsurface deposits, and the special requirements of these organisms for survival, make contamination by halobacteria from surface sites unlikely . It is conceivable that these subterranean halobacteria are autochthonous, presumably relict populations derived from ancient hypersaline seas that have been revived from a state of dormancy . One would predict that halobacteria that have been insulated and isolated inside ancient evaporites would be different from comparable bacteria from surface environments, and that it might be possible to use a molecular chronometer to establish if the evolutionary position of the subsurface isolates correlated with the geological age of the evaporite . Extensive comparisons have been made between the 16S rRNA genes of surface and subsurface halobacteria without showing any conclusive differences between the two groups . A further phylogenetic comparison exploits an unusual feature of one particular group of halobacteria that possess at least two heterogeneous copies of the 16S rRNA gene, the sequences of which may have been converging or diverging over geological time . However, results to date have yet to show any gene sequence differences between surface and evaporite-derived halobacteria that might arguably be an indication of long-term dormancy.

Biochim Biophys Acta, 1998 Oct 14, 1388(1), 209 - 14
Sodium chloride enhances markedly the thermal stability of thermolysin as well as its catalytic activity; Inouye K et al.; Thermolysin, a thermophilic metalloproteinase, is markedly activated in the presence of high concentrations (1-5 M) of neutral salts . The activity increases in an exponential fashion with increasing salt concentration, and is enhanced 13-15 times with 4 M NaCl at pH 7.0 and 25 degreesC (K . Inouye, Effects of salts on thermolysin: activation of hydrolysis and synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester, and a unique change in the absorption spectrum of thermolysin, J . Biochem . 112 (1992) 335-340) . In this study, the effect of NaCl on the thermal stability of thermolysin has been examined at 60-85 degreesC . The activation energy, Ea, for the thermal inactivation is 15 kcal/mol at 0 M NaCl, and increases up to 30-33 kcal/mol by the addition of 0 . 5-1.5 M NaCl . Further increase in {NaCl} decreases the Ea value, and at 4 M NaCl it is almost the same as that at 0 M NaCl . Thermolysin at 0.5-1.5 M NaCl is twice as heat-stable as in the absence of NaCl . The NaCl dependence of the stability is different from that of the activity, suggesting that the effects of NaCl on activity and stability are independent . Thermolysin has been demonstrated to be not only a thermophilic enzyme but also a highly halophilic one.

Curr Microbiol, 1998 Nov, 37(5), 347 - 51
Transfer and expression of a multiple antibiotic resistance plasmid in marine bacteria; Chandrasekaran S et al.; Conjugal transfer of a multiresistance plasmid from Pseudomonas fluorescens to halophilic and halotolerant bacteria was studied under in vitro and in situ conditions . Mating conducted in broth as well as on plates yielded a plasmid transfer frequency of as high as 10(-3) . Among these two, plate mating facilitated conjugal transfer of plasmid, because the cell-to-cell contact is more in plate mating . When P . fluorescens was incubated in seawater, the organism progressively lost its colony forming activity within 15 days . Microscopic examination revealed the presence of very short rods, indicating that the cells have become viable but nonculturable (VNC) . Mating conducted in natural seawater without any added nutrients revealed that the conjugal transfer is influenced by the physical state of the donor and the recipients as well as the availability of nutrients . But a plasmid transfer frequency of 10(-7) was obtained even after the donor cells have become VNC suggesting that the nonculturable state and nutrient deprived condition may not limit plasmid transfer . The results suggest that the terrestrial bacteria entering into the seawaters with antibiotic resistance plasmids may be responsible for the prevalence of resistance genes in the marine environment.

Res Microbiol, 1998 Apr, 149(4), 277 - 87
Biomineralization of carbonates by Halomonas eurihalina in solid and liquid media with different salinities: crystal formation sequence; Rivadeneyra MA et al.; Carbonate precipitation by 20 strains of the moderately halophilic species Halomonas eurihalina in both solid and liquid media was studied . The influence of salinity and temperature on the quantity and type of crystals precipitated was also investigated . Some strains of H . eurihalina formed crystals in all conditions tested . The mineral phases precipitated were magnesium calcite, aragonite and monohydrocalcite in variable proportions depending on various factors such as the type of growth medium employed and its salinity . Scanning electron microscopy and X-ray dispersive energy microanalysis were used to investigate the crystal formation sequence . The process of biolith formation was sequential . It started with chains or filaments of bacteria, giving way to discs which finally produced spherical forms of approximately 50 microns in diameter . We suggest a mechanism of carbonate crystal formation by H . eurihalina.

J Virol, 1998 Nov, 72(11), 9392 - 5
His1, an archaeal virus of the Fuselloviridae family that infects Haloarcula hispanica; Bath C et al.; A novel archaeal virus, His1, was isolated from hypersaline waters in southeastern Australia . It was lytic, grew only on Haloarcula hispanica (titers of up to 10(11) PFU/ml), and displayed a lemon-shaped morphology (74 by 44 nm) previously reported only for a virus of the extreme thermophiles (SSV1) . The density of His1 was approximately 1.28 g/ml, similar to that of SSV1 (1.24 g/ml) . Purified particles were resistant to low salt concentrations . The genome was linear, double-stranded DNA of 14.9 kb, similar to the genome of SSV1 (15.5 kb) . Morphologically, this isolate clearly belongs to the recently proposed Fuselloviridae family of archaeal viruses . It is the first member of this family from the extremely halophilic archaea, and its host, H . hispanica, can be readily manipulated genetically.

Appl Environ Microbiol, 1998 Oct, 64(10), 4095 - 7
Synthesis of glycine betaine from exogenous choline in the moderately halophilic bacterium halomonas elongata
Canovas D, Vargas C, Csonka LN, Ventosa A, Nieto JJ.
The role of choline in osmoprotection in the moderate halophile Halomonas elongata has been examined . Transport and conversion of choline to betaine began immediately after addition of choline to the growth medium . Intracellular accumulation of betaine synthesized from choline was salt dependent up to 2.5 M NaCl . Oxidation of choline was enhanced at 2.0 M NaCl in the presence or absence of externally provided betaine . This indicates that the NaCl concentration in the growth medium has major effects on the choline-betaine pathway of H . elongata.

Appl Environ Microbiol, 1998 Oct, 64(10), 3813 - 7
Quantitative and physiological analyses of chloride dependence of growth of halobacillus halophilus
Roessler M, Muller V V.
A quantitative analysis of the Cl- dependence of growth of Halobacillus halophilus was performed . Optimal growth rates were obtained at Cl- concentrations of between 0.5 and 2.0 M, and the final yield was also strictly dependent on the Cl- concentration . Br- but not I-, SO42-, NO2-, SO2-, OCN-, SCN-, BO2-, or BrO3- could substitute for Cl- . To analyze the function of chloride, chloride concentration was determined . At low external Cl- (Cle-) concentrations, the growth rate was low and Cl- was excluded from the cytoplasm; increasing the Cle- concentration led to an increase in the growth rate and an energy-dependent uptake of Cl-, thus decreasing the Cle-/internal Cli- gradient from >/=10 at 0.1 M Cle- to a nearly constant value of 2 at Cle- concentrations which allowed optimal growth . Two membrane proteins with apparent molecular masses of 31 and 16 kDa which were identified to be specific for Cl--grown cultures are possible candidates for a chloride uptake system.

J Biomol NMR, 1998 Aug, 12(2), 307 - 18
Identification of slow motions in the reduced recombinant high-potential iron sulfur protein I (HiPIP I) from Ectothiorhodospira halophila via 15N rotating-frame NMR relaxation measurements; Banci L et al.; Rotating-frame 15N relaxation rate (R1 rho) NMR experiments have been performed in order to study the dynamic behavior of the reduced recombinant high-potential iron-sulfur protein iso I (HiPIP I) from Ectothiorhodospira halophila, in the microsecond to ms time range . Measurements of R1 rho were performed as a function of the effective spinlock magnetic field amplitude by using both on and off-resonance radio frequency irradiation . The two data sets provided consistent results and were fit globally in order to identify possible exchange processes in an external loop of the reduced HiPIP I . The loop consists of residues 43-45 and the correlation time of the exchange process was determined to be 50 +/- 8 microseconds for the backbone nitrogen of Gln 44.

Kansenshogaku Zasshi, 1998 Jul, 72(7), 720 - 6
{A number of Vibrio cholerae non-O1 isolated from aquatic environments}; Uchiyama H; To estimated existence of Vibrio cholerae non-O1 in aquatic environments, the organisms isolated from river, estuary and sea water . V . cholerae non-O1 isolated form midstream and estuary water could be counted from 1.6 to 2400 CFU/100 ml by the membrane filtrated method (MF) . V . cholerae non-O1 existed in midstream water more than in estuary water . However, the isolated organisms from estuary rate by MF (37.5%) was lower than it by alkaline peptone enrichment medium method (AP) (75.0%), as a result of halophilic bacteria grow an selected medium of MF . And the number of V . cholerae non-O1 isolated from aquatic environment did not correlate environmental parameters . The number of V . cholerae non-O1 isolated from river water varied, it suggested that the organism collectively adhere a floating matter . V . cholerae non-O1 was not detected in 500 ml sea water by AP and MF method . These results conclude that V . cholerae non-O1 exist in river water more than in sea.

FEBS Lett, 1998 Aug 28, 434(1-2), 13 - 6
Operation of glyoxylate cycle in halophilic archaea: presence of malate synthase and isocitrate lyase in Haloferax volcanii; Serrano JA et al.; The occurrence of the glyoxylate cycle has not previously been demonstrated in any of the Archaea . In halophilic archaea, only isocitrate lyase activity has been detected . The halophilic archaeon Haloferax volcanii was tested for the presence of the other key enzyme of this pathway, malate synthase . High activities of this enzyme were detected when the carbon source was acetate . Both glyoxylate cycle key enzymes, isocitrate lyase and malate synthase, from Hf . volcanii were purified and characterized.

Biochemistry, 1998 Sep 15, 37(37), 12689 - 99
Solution structure and backbone dynamics of the photoactive yellow protein; Dux P et al.; The solution structure of photoactive yellow protein (PYP), a photosensory protein from Ectothiorhodospira halophila, has been determined by multidimensional NMR spectroscopy . The structure consists of an open, twisted, 6-stranded, antiparallel beta-sheet, which is flanked by four alpha-helices on both sides . The final set of 26 selected structures is well-defined for the regions spanning residues Phe6-Ala16, Asp24-Ala112, and Tyr118-Val125 and displays a root-mean-square deviation, versus the average, of 0.45 A for the backbone and 0.88 A for all heavy atoms . Comparison of the solution structure with an earlier published 1.4 A crystal structure (Borgstahl, G . E . O., Williams, D . R., and Getzoff, E . D . (1995) Biochemistry 34, 6278-6287) reveals a similarity with a root-mean-square deviation of 1.77 A for the backbone for the well-defined regions . The most distinct difference in the backbone with the crystal structure is found near the N-terminus, for residues Asp19-Leu23, which corresponds to an alpha-helix in the crystal structure and to one of the poorest defined regions in the solution structure . To characterize the dynamic behavior of PYP in solution, we undertook a 15N relaxation study and measurements of hydrogen/deuterium exchange . Determination of order parameters through the model-free Lipari-Szabo approach enabled the identification of several regions of enhanced dynamics . The comparison of atomic displacements in the backbone traces of the ensemble structures, with mobility measurements from NMR, show that the poorly defined regions feature fast internal motions in the nanosecond to picosecond time scale.

Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 973 - 82
Proposal of six new species in the genus Microbacterium and transfer of Flavobacterium marinotypicum ZoBell and Upham to the genus Microbacterium as Microbacterium maritypicum comb . nov; Takeuchi M et al.; Reference strains, including two mis-named organisms, 'Chromobacterium chocolatum' and Flavobacterium marinotypicum, isolates from soil and clinical specimens, all previously recognized as Aureobacterium or Microbacterium, were characterized taxonomically . On the basis of morphological, physiological and chemotaxonomic characteristics, as well as DNA-DNA hybridization data, six new species and one new combination are proposed in the genus Microbacterium: Microbacterium ketosireducens sp . nov . (type strain IFO 14548T), Microbacterium chocolatum sp . nov . (type strain IFO 3758T), Microbacterium aurantiacum sp . nov . (type strain IFO 15234T), Microbacterium hominis sp . nov . (type strain IFO 15708T), Microbacterium thalassium sp . nov . (type strain IFO 16060T), Microbacterium halophilum sp . nov . (type strain IFO 16062T) and Microbacterium maritypicum comb . nov . (type strain IFO 15779T).

Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 957 - 64
Taxonomic rearrangements of the genera Thiocapsa and Amoebobacter on the basis of 16S rDNA sequence analyses, and description of Thiolamprovum gen . nov; Guyoneaud R et al.; Complete nucleotide sequences of the 16S rDNAs were determined from Thiocapsa and Amoebobacter species, including all available type strains and some additional isolates . The distance-matrix analysis and the dendrogram for estimating the genetic relationships revealed that the investigated strains were found in two major clusters within the Chromatiaceae . One cluster comprises all Amoebobacter species, Thiocapsa roseopersicina and several isolates related to Thiocapsa roseopersicina . Representatives of the species Amoebobacter roseus, Amoebobacter pendens and Thiocapsa roseopersicina, the so called 'Thiocapsa roseopersicina group', are very closely related, justifying their inclusion into one genus, Thiocapsa, for which an emended description is presented . Amoebobacter purpureus and Amoebobacter pedioformis formed two separate lines of descent with less than 93% (89.6-92.9%) similarity to strains of the 'Thiocapsa roseopersicina group' . Therefore, they will be considered as two separate genera . As a consequence, an emended description is presented for the genus Amoebobacter, with Amoebobacter purpureus as the new type species and A . pedioformis is transferred to Thiolamprovum pedioforme gen . nov., comb . nov . Two species, Thiocapsa pfennigii and Thiocapsa halophila, which have been classified with the genus Thiocapsa because of their morphological properties, were found within another major cluster of the Chromatiaceae and are only distantly phylogenetically related to the first cluster with 88.4-90.6% and 90.4-92.2% sequence similarity, respectively.

Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 821 - 8
Methanocalculus halotolerans gen . nov., sp . nov., isolated from an oil-producing well; Ollivier B et al.; Two irregular coccoid methanogens designated SEBR 4845T and FR1T were isolated from an oilfield in Alsace, France . Strain SEBR 4845T (T = type strain) is a hydrogenotrophic halotolerant methanogen, which grows optimally at 5% NaCI (w/v) and tolerates up to 12% NaCI . It does not use methylated compounds and therefore cannot be ascribed to any of the known genera of the halophilic methylotrophic methanogens . It differs from hydrogenotrophic members of the orders Methanococcales and Methanomicrobia les in the NaCI growth range (0-12% NaCI), which is the widest reported to data for any hydrogenotrophic methanogen . 16S rRNA gene sequence analysis indicated that strain SEBR 4845T is a novel isolate for which a new genus is proposed, Methanocalculus halotolerans gen . nov., sp . nov . (= OCM470T) that might be indigenous to the oilfield ecosystem . Strain FR1T (=OCM 471) is a moderately halophilic methanogen which growths optimally at 10% NaCI and tolerates up to 20% NaCI . It grows on trimethylamine and methanol as carbon and energy sources . The G+C content of its DNA is 43 mol% . It is therefore phenotypically and genotypically related to members of the genus Methanohalophilus . This report provides evidence that methylotrophic and hydrogenotrophic, but not aceticlastic methanogens are present in a saline subsurface oilfield environment, as already observed in surface saline to hypersaline environments.

J Bacteriol, 1998 Sep, 180(18), 4804 - 13
Transcription analysis of two disparate rRNA operons in the halophilic archaeon Haloarcula marismortui; Dennis PP et al.; The genome of the halophilic archaeon Haloarcula marismortui contains two rRNA operons designated rrnA and rrnB . Genomic clones of the two operons and their flanking regions have been sequenced, and primary transcripts and processing intermediates derived from each operon have been characterized . The 16S, 23S, and 5S genes from the two operons were found to differ at 74 of 1,472 positions, 39 of 2,922 positions, and 2 of 122 positions, respectively . This degree of sequence divergence for multicopy (paralogous) rRNA genes was 10- to 50-fold or more higher than anticipated . The two operons exhibit other profound differences that include (i) the presence in rrnA and the absence in rrnB of tRNAAla and tRNACys genes in the intergenic and distal regions, respectively, (ii) divergent 5' flanking sequences, and (iii) distinct pathways for processing and maturation of 16S rRNA . Processing and maturation of 16S and 23S rRNA from rrnA operon transcripts and of 23S rRNA from rrnB operon transcripts follow the canonical halophilic pathway, whereas maturation of 16S rRNA from rrnB operon transcripts follows an unusual and different pathway that is apparently devoid of any 5' processing intermediate.

Int J Syst Bacteriol, 1998 Apr, 48 Pt 2, 333 - 8
Desulfotomaculum halophilum sp . nov., a halophilic sulfate-reducing bacterium isolated from oil production facilities; Tardy-Jacquenod C et al.; A halophilic endospore-forming, sulfate-reducing bacterium was isolated from an oilfield brine in France . The strain, designated SEBR 3139, was composed of long, straight to curved rods . It grew in 1-14% NaCl with an optimum at 6% . On the basis of morphological, physiological and phylogenetical characteristics, strain SEBR 3139 should be classified in the genus Desulfotomaculum . However, it is sufficiently different from the hitherto described Desulfotomaculum species to be considered as a new species . Strain SEBR 3139T (= DSM 11559T) represents the first moderate halophilic species of the genus Desulfotomaculum . The name Desulfotomaculum halophilum sp . nov . is proposed.

Curr Opin Struct Biol, 1998 Aug, 8(4), 489 - 500
The structure and mechanism of the family of retinal proteins from halophilic archaea; Oesterhelt D; Retinal proteins from halophilic archaea provide a unique opportunity to analyze vectorial ion translocation . Studies on its structure, conformational changes, proton conduction and electrogenic steps have helped to elucidate the catalytic cycle of bacteriorhodopsin in increasing detail . Experimental modulation of the vectoriality and ion specificity by altering the substrate availability, point mutations and light conditions for the different retinal proteins allows the proposal of a general model of ion transport for this protein family.

Proc R Soc Lond B Biol Sci, 1998 Aug 7, 265(1404), 1461 - 5
Fungal life in the extremely hypersaline water of the Dead Sea: first records; Buchalo AS et al.; The first report, to our knowledge, on the occurrence of filamentous fungi in the hypersaline (340 g salt l-1) Dead Sea is presented . Three species of filamentous fungi from surface water samples of the Dead Sea were isolated: Gymnascella marismortui (Ascomycota), which is described as a new species, Ulocladium chlamydosporum and Penicillium westlingii (Deuteromycota) . G . marismortui and U . chlamydosporum grew on media containing up to 50% Dead Sea water . G . marismortui was found to be an obligate halophile growing optimally in the presence of 0.5-2 M NaCl or 10 30% (by volume) of Dead Sea water . Isolated cultures did not grow on agar media without salt, but grew on agar prepared with up to 50% Dead Sea water . This suggests that they may be adapted to life in the extremely stressful hypersaline Dead Sea.

Biochemistry, 1998 Aug 18, 37(33), 11563 - 8
New insights into the photocycle of Ectothiorhodospira halophila photoactive yellow protein: photorecovery of the long-lived photobleached intermediate in the Met100Ala mutant; Devanathan S et al.; There are previously two known intermediates (I1 and I2) in the room-temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila . The three-dimensional structures of ground-state PYP and of I2 have shown that light-induced conformational changes are localized to the active site . Previous site-specific mutagenesis studies of PYP in our laboratories have characterized two active site mutants (Glu46Gln and Arg52Ala) . We now report the construction and characterization of a mutant at a third active site position (Met100Ala) in order to establish the role of this residue in the photocycle . Met100Ala PYP has an absorption spectrum which is very similar to wild-type (WT) PYP, but exhibits very different kinetic properties . At pH 7.0, the light-induced bleaching reaction (I2 formation) has a half-life <1 microseconds and the recovery in the dark has a half-life of 5.5 min, as compared with half-lives of 100 microseconds and 140 ms for the same reactions in WT PYP . The slow rate of recovery from I2 for Met100Ala results in the accumulation of the bleached intermediate even under room light illumination . These results are qualitatively similar to what has been observed with the Arg52Ala mutant of PYP, and with WT PYP in the presence of alcohols or urea, and suggest that Met100 acts to stabilize the ground state of the protein . The midpoint for guanidine denaturation confirms this . The slow recovery of I2 in the Met100Ala mutant has allowed us to obtain direct evidence that this intermediate species is also photoactive and can be returned to the ground state by a 365 nm laser flash, with kinetics (half-life = 160 microseconds; k = 6300 s-1) which are 6 orders of magnitude faster than dark recovery . This implies that chromophore reisomerization limits the rate of conversion of I2 to the ground state in PYP . Met100 is in van der Waals contact with the chromophore in the I2 state, and we suggest that the sulfur atom catalyzes cis-trans isomerization in WT PYP.

Arch Microbiol, 1998 Aug, 170(2), 138 - 40
Confirmation that Thiobacillus halophilus and Thiobacillus hydrothermalis are distinct species within the gamma-subclass of the Proteobacteria; Kelly DP et al.; Thiobacillus halophilus and Thiobacillus hydrothermalis share 98.7% similarity in 16S rRNA sequence, possess similar gross DNA composition (64.2 and 67.4 mol% G+C values, respectively), and have similar physiological properties . While this might have indicated that they were strains of a single species, DNA-DNA hybridization between the type strains of the two species showed only 59% hybridization, indicating the organisms to be different at the species level . Thiobacillus neapolitanus is the phylogenetically nearest neighbour of T . halophilus and T . hydrothermalis (91.6-92.1% similarity in 16S rRNA sequence) and is the only other Thiobacillus in the gamma-subclass of the Proteobacteria that can be regarded as exclusively related to these two species . The 16S rRNA gene sequences of these three species are so different from those of the other thiobacilli in the gamma-subclass that they justify recognition as a distinct phyletic group . Their comparative properties are summarized.

FEMS Microbiol Lett, 1998 Jul 15, 164(2), 237 - 42
The treA gene of Bacillus subtilis is a suitable reporter gene for the archaeon Methanococcus voltae; Sniezko I et al.; The similarity of the transcriptional apparatus of Archaea with that of Eucarya makes studies of their transcriptional regulation especially interesting . Such investigations are greatly facilitated by reporter genes . The concomitant analysis of several promoters for investigations of regulatory patterns requires different reporter genes . The archaeon Methanococcus voltae is a moderately halophilic mesophile . The treA gene from Bacillus subtilis appeared to be a good candidate for a reporter, since its product trehalase is salt-resistant . We show that it is indeed expressed under the control of a M . voltae promoter and that the enzyme is easily testable in cell lysates.

Extremophiles, 1997 Feb, 1(1), 52 - 60
Glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: molecular characterization and phylogenetic implications; Kort R et al.; The hyperthermophilic bacterium Thermotoga maritima, which grows at up to 90 degrees C, contains an L-glutamate dehydrogenase (GDH) . Activity of this enzyme could be detected in T . maritima crude extracts, and appeared to be associated with a 47-kDa protein which cross-reacted with antibodies against purified GDH from the hyperthermophilic archaeon Pyrococcus woesei . The single-copy T . maritima gdh gene was cloned by complementation in a glutamate auxotrophic Escherichia coli strain . The nucleotide sequence of the gdh gene predicts a 416-residue protein with a calculated molecular weight of 45,852 . The gdh gene was inserted in an expression vector and expressed in E . coli as an active enzyme . The T . maritima GDH was purified to homogeneity . The NH2-terminal sequence of the purified enzyme was PEKSLYEMAVEQ, which is identical to positions 2-13 of the peptide sequence derived from the gdh gene . The purified native enzyme has a size of 265 kDa and a subunit size of 47kDa, indicating that GDH is a homohexamer . Maximum activity of the enzyme was measured at 75 degrees C and the pH optima are 8.3 and 8.8 for the anabolic and catabolic reaction, respectively . The enzyme was found to be very stable at 80 degrees C, but appeared to lose activity quickly at higher temperatures . The T . maritima GDH shows the highest rate of activity with NADH (Vmax of 172 U/mg protein), but also utilizes NADPH (Vmax of 12 U/mg protein) . Sequence comparisons showed that the T . maritima GDH is a member of the family II of hexameric GDHs which includes all the GDHs isolated so far from hyperthermophiles . Remarkably, phylogenetic analysis positions all these hyperthermophilic GDHs in the middle of the GDH family II tree, with the bacterial T . maritima GDH located between that of halophilic and thermophilic euryarchaeota.

Extremophiles, 1997 Feb, 1(1), 29 - 35
Cloning and sequencing of the gene encoding the cell surface glycoprotein of Haloarcula japonica strain TR-1; Wakai H et al.; The triangular disk-shaped halophilic archaeon Haloarcula japonica strain TR-1 has a glycoprotein on its cell surface . The complete gene encoding the cell surface glycoprotein (CSG) was cloned and sequenced . The gene has an open reading frame of 2586 bp, and a potential archaeal promoter sequence approximately 150 bp upstream of the ATG initiation codon . The mature CSG is composed of 828 amino acids and is preceded by a signal sequence of 34 amino acid residues . A hydropathy analysis showed a hydrophobic stretch at the C-terminus, that probably serves as a transmembrane domain . The amino acid sequence of the Ha . japonica CSG showed 52.1% and 43.2% identities to those from the Halobacterium halobium and Haloferax volcanii CSGs, respectively . Five potential N-glycosylation sites were found in the mature Ha . japonica CSG, sites that were distinctly different from those in Hb . halobium and Hf . volcanii . The Ha . japonica CSG gene was expressed in Escherichia coli.

Extremophiles, 1997 Aug, 1(3), 143 - 9
Occurrence of virus-like particles in the Dead Sea; Oren A et al.; Electron-microscopic examination of water samples from the hypersaline Dead Sea showed the presence of high numbers of virus-like particles . Between 0.9 and 7.3 x 10(7) virus-like particles ml(-1) were enumerated in October 1994 in the upper 20 m of the water column during the decline of a bloom of halophilic Archaea . Virus-like particles outnumbered bacteria by a factor of 0.9-9.5 (average 4.4) . A variety of viral morphologies were detected, the most often encountered being spindle-shaped, followed by polyhedral and tailed phages . In addition, other types of particles were frequently found, such as unidentified algal scales, and virus-sized star-shaped particles . Water samples collected during 1995 contained low numbers of both bacteria and virus-like particles (1.9-2.6 x 10(6) and 0.8-4.6 x 10(7) ml(-1) in April 1995), with viral numbers sharply declining afterwards (less than 10(4) ml(-1) in November 1995-January 1996) . It is suggested that viruses may play a major role in the decline of halophilic archaeal communities in the Dead Sea . an environment in which protozoa and other predators are absent.

Extremophiles, 1997 May, 1(2), 94 - 9
Silicibacter lacuscaerulensis gen . nov., sp . nov., a mesophilic moderately halophilic bacterium characteristic of the Blue Lagoon geothermal lake in Iceland; Petursdottir SK et al.; Mesophilic, moderately halophilic bacteria were isolated from a silica-rich geothermal lake, the Blue Lagoon in Iceland . The isolates are strictly aerobic, but reduce nitrate to nitrite, and are oxidase- and catalase-positive . The nonsporeforming and nonmotile Gram negative rods are 0.6-0.8 microm in diameter and variable in length (9-18 microm), and contain gas vacuoles . The GC content in their DNA is 66.15% . The minimum, optimum, and maximum temperatures for growth are 22 degrees C, 45 degrees C, and 50 degrees C, respectively . The isolates do not grow without added salt in the medium and can grow at up to 7% NaCl (w/v) . The optimal salinity for growth is 3.5%-4% NaCl . The pH range for growth is 6.5-8.5, with the optimal pH at 7.0 . At optimal conditions the bacterium has a doubling time of 80 min . The main cytochrome is a membrane-bound cytochrome c with an alpha-peak at 549nm . Sequencing of 16S rRNA from the type strain ITI-1157 revealed it to be a proteobacterium of the alpha-subclass with the closest relatives being Roseobacter litoralis and Paracoccuss kocuri . The new isolates do not contain bacteriochlorophyll a and are considered to represent a new genus and a new species, Silicibacter lacuscaerulensis.

Mol Microbiol, 1998 Jun, 28(6), 1051 - 8
Variations on a molecular switch: transport and sensory signalling by archaeal rhodopsins; Spudich JL; The archaeal rhodopsins are a family of seven-transmembrane-helix, visual pigment-like proteins found in Halobacterium salinarum and related halophilic Archaea . Two, bacteriorhodopsin (BR) and halorhodopsin (HR), are transport rhodopsins that carry out light-driven electrogenic translocation of protons and chloride, respectively, across the cell membrane . The other two, sensory rhodopsins I and II (SRI and SRII), are phototaxis receptors that send signals to tightly bound transducer proteins that in turn control a phosphorylation cascade modulating the cell's flagellar motors . Recent progress has cast light on how nature has modified the common design of these proteins to carry out their distinctly different functions: electrogenic ion transport and non-electrogenic signal transduction . A key shared mechanism between BR and SRII appears to be an interhelical salt bridge locked conformational switch that is released by photoisomerization of retinal . In BR disruption of the lock opens a cytoplasmic half-channel that ensures uptake of the transported proton from the cytoplasmic side of the membrane at a critical time in the pumping cycle . Transducer-free SRI uses the same mechanism to carry out light-driven proton transport, but interaction with its transducer blocks the cytoplasmic half-channel thereby interrupting the transport cycle . In SRI, transducer interaction also disrupts the salt bridge in the dark, poising the receptor in an intermediate conformation able to produce opposite signals depending on the colour of the stimulus light . A model for signalling is proposed in which the salt bridge-controlled half-channel is used to modulate interaction with the Htr proteins when the receptor signalling states are formed.

J Mol Biol, 1998 Jul 24, 280(4), 731 - 48
Electrostatic contributions to the stability of halophilic proteins; Elcock AH et al.; Examination of the first crystal structures of proteins from a halophilic organism suggests that an abundance of acidic residues distributed over the protein surface is a key determinant of adaptation to high-salt conditions . Although one extant theory suggests that acidic residues are favored because of their superior water-binding capacity, it is clear that extensive repulsive electrostatic interactions will also be present in such proteins at physiological pH . To investigate the magnitude and importance of such electrostatic interactions, we conducted a theoretical analysis of their contributions to the salt and pH-dependence of stability of two halophilic proteins . Our approach centers on use of the Poisson-Boltzmann equation of classical electrostatics, applied at an atomic level of detail to crystal structures of the proteins . We first show that in using the method, it is important to account for the fact that the dielectric constant of water decreases at high salt concentrations, in order to reproduce experimental changes in pKa values of small acids and bases . We then conduct a comparison of salt and pH effects on the stability of 2Fe-2S ferredoxins from the halophile Haloarcula marismortui and the non-halophile anabaena . In both proteins, substantial upward shifts in pKa accompany protein folding, though shifts are considerably larger, on average, in the halophile . Upward shifts for basic residues occur because of favorable salt-bridge interactions, whilst upward shifts for acidic residues result from unfavorable electrostatic interactions with other acidic groups . Our calculations suggest that at pH 7 the stability of the halophilic protein is decreased by 18.2 kcal/mol on lowering the salt concentration from 5 M to 100 mM, a result that is in line with the fact that halophilic proteins generally unfold at low salt concentrations . For comparison, the non-halophilic ferredoxin is calculated to be destabilized by only 5.1 kcal/mol over the same range . Analysis of the pH stability curve suggests that lowering the pH should increase the intrinsic stability of the halophilic protein at low salt concentrations, although in practice this is not observed because of aggregation effects . We report the results of a similar analysis carried out on the tetrameric malate dehydrogenase from H . marismortui . In this case, we investigated the salt and pH dependence of the various monomer-monomer interactions present in the tetramer . All monomer-monomer interactions are found to make substantial contributions to the salt-dependence of stability of the tetramer . Excellent agreement is obtained between our calculated results for the stability of the tetramer and experimental results . In particular, the finding that at 4 M NaCl, the tetramer is stable only between pH 4.8 and 10 is accurately reproduced . Taken together, our results suggest that repulsive electrostatic interactions between acidic residues are a major factor in the destabilization of halophilic proteins in low-salt conditions, and that these interactions remain destabilizing even at high salt concentrations . As a consequence, the role of acidic residues in halophilic proteins may be more to prevent aggregation than to make a positive contribution to intrinsic protein stability .

Extremophiles, 1998 Jan, 2(1), 33 - 9
Detection and expression of a gene encoding a new bacteriorhodopsin from an extreme halophile strain HT (JCM 9743) which does not possess bacteriorhodopsin activity; Kamekura M et al.; Membrane vesicles prepared from an extreme halophile strain, HT (JCM 9743), showed no bacteriorhodopsin activity . However, a DNA fragment, amplified by polymerase chain reaction (PCR), appeared to encode the C to G helices of a bacteriorhodopsin (bR)-like protein . With the PCR product as a probe, the gene coding for a novel bacteriorhodopsin was cloned from the genomic DNA of the strain HT . The open reading frame of the gene was ligated with the promoter region of the bop gene of Halobacterium salinarum bR, and expressed in a bR-deficient host strain, L33, using the plasmid vector pXLNov-R . The purplish membrane fraction purified from cells of a transformant exhibited a cyclic photoreaction characteristic of bacteriorhodopsin.

Adv Biochem Eng Biotechnol, 1998, 61, 117 - 53
An overview of the role and diversity of compatible solutes in Bacteria and Archaea; da Costa MS et al.; The accumulation of compatible solutes is a prerequisite for the adaptation of microorganisms to osmotic stress imposed by salt or organic solutes . Two types of strategies exist to cope with high external solute concentrations; one strategy is found in the extremely halophilic Archaea of the family Halobacteriaceae and the Bacteria of the order Haloanaerobiales involving the accumulation of inorganic ions . The other strategy of osmoadaptation involves the accumulation of specific organic solutes and is found in the vast majority of microorganisms . The organic osmolytes range from sugars, polyols, amino acids and their respective derivatives, ectoines and betaines . The diversity of these organic solutes has increased in the past few years as more organisms, especially thermophilic and hyperthermophilic Bacteria and Archaea, have been examined . The term compatible solute can also be applied to solutes that protect macromolecules and cells against stresses such as high temperature, desiccation and freezing . The mechanisms by which compatible solutes protect enzymes, cell components and cells are still a long way from being thoroughly elucidated, but there is a growing interest in the utilization of these solutes to protect macromolecules and cells from heating, freezing and desiccation.

Biophys J, 1998 Jul, 75(1), 406 - 12
New photocycle intermediates in the photoactive yellow protein from Ectothiorhodospira halophila: picosecond transient absorption spectroscopy; Ujj L et al.; Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al . 1997 . Biochemistry . 36:8-14) . Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously . The first new intermediate (I0) exhibits maximum absorption at approximately 510 nm and appears in </=3 ps after 452 nm excitation (5 ps pulse width) of PYP . Kinetic analysis shows that I0 decays with a 220 +/- 20 ps lifetime, forming another intermediate (Idouble dagger0) that has a similar difference wavelength maximum, but with lower absorptivity . Idouble dagger0 decays with a 3 +/- 0.15 ns time constant to form I1 . Stimulated emission from an excited electronic state of PYP is observed both within the 4-6-ps cross-correlation times used in this work, and with a 16-ps delay for all probe wavelengths throughout the 426-525-nm region studied . These transient absorption and emission data provide a more detailed understanding of the mechanistic dynamics occurring during the PYP photocycle.

Appl Environ Microbiol, 1998 Jul, 64(7), 2513 - 9
Sequencing and characterization of the xyl operon of a gram-positive bacterium, Tetragenococcus halophila; Takeda Y et al.; The xyl operon of a gram-positive bacterium, Tetragenococcus halophila (previously called Pediococcus halophilus), was cloned and sequenced . The DNA was about 7.7 kb long and contained genes for a ribose binding protein and part of a ribose transporter, xylR (a putative regulatory gene), and the xyl operon, along with its regulatory region and transcription termination signal, in this order . The DNA was AT rich, the GC content being 35.8%, consistent with the GC content of this gram-positive bacterium . The xyl operon consisted of three genes, xylA, encoding a xylose isomerase, xylB, encoding a xylulose kinase, and xylE, encoding a xylose transporter, with predicted molecular weights of 49,400, 56,400, and 51,600, respectively . The deduced amino acid sequences of the XylR, XylA, XylB, and XylE proteins were similar to those of the corresponding proteins in other gram-positive and -negative bacteria, the similarities being 37 to 64% . Each polypeptide of XylB and XylE was expressed functionally in Escherichia coli . XylE transported D-xylose in a sodium ion-dependent manner, suggesting that it is the first described xylose/Na+ symporter . The XylR protein contained a consensus sequence for binding catabolites of glucose, such as glucose-6-phosphate, which has been discovered in glucose and fructose kinases in bacteria . Correspondingly, the regulatory region of this operon contained a putative binding site of XylR with a palindromic structure . Furthermore, it contained a consensus sequence, CRE (catabolite-responsive element), for binding CcpA (catabolite control protein A) . We speculate that the transcriptional regulation of this operon resembles the regulation of catabolite-repressible operons such as the amy, lev, xyl, and gnt operons in various gram-positive bacteria . We discuss the significance of the regulation of gene expression of this operon in T . halophila.

J Biochem (Tokyo), 1998 Jul, 124(1), 72 - 8
Effects of nitration and amination of tyrosyl residues in thermolysin on its hydrolytic activity and its remarkable activation by salts; Inouye K et al.; Thermolysin is remarkably activated in the presence of high concentrations (1-5 M) of neutral salts and its activity is enhanced 15 times by 4 M NaCl at pH 7.0 and 25 degrees C {Inouye, K . (1992) J . Biochem . 112, 335-340} . In this study, the effects of nitration and amination of tyrosyl residues in thermolysin on its halophilic properties were examined . Nitration and successive amination inactivate thermolysin progressively as the degree of modification increases . When 16 tyrosyl residues were nitrated, the activity decreased to 10% of that of the native enzyme, whereas it recovered to 30% when they were aminated . The decrease in the activity by the nitration and amination was shown to be brought about only by a decrease in the molecular activity, kcat; the Michaelis constant, Km, was unaltered . When 14 tyrosyl residues of thermolysin were nitrated, the degree of activation by 4 M NaCl at pH 7.0 decreased from 15 to 10, and this decreased further to 5 when the pH of the reaction medium was raised to 8.5 . However, when the nitrated tyrosyl residues were reduced to aminotyrosyl residues, the degree of activation was restored to that of the native enzyme . The change in the degree of activation by nitration and amination of thermolysin could be due to the change in the ionization of tyrosyl residues, and it was suggested that removing negative charges from tyrosyl residues of thermolysin enhances its halophilicity.

J Mol Biol, 1998 Jun 19, 279(4), 761 - 71
The transcriptional activator GvpE for the halobacterial gas vesicle genes resembles a basic region leucine-zipper regulatory protein; Kruger K et al.; The GvpE protein involved in the regulation of gas vesicles synthesis in halophilic archaea has been identified as the transcriptional activator for the promoter located upstream of the gvpA gene encoding the major gas vesicle structural protein GvpA . A closer inspection of the GvpE protein sequence revealed that GvpE resembles basic leucine-zipper proteins typically involved in the gene regulation of eukarya . A molecular modelling study of the C-terminal part implied a cluster of basic amino acid residues constituting the DNA-binding site (DNAB) followed by an amphiphilic helix, suitable for the formation of a leucine-zipper structure within a GvpE dimer . The model of a GvpE dimer docked onto DNA indicated that the side-chains of the basic residues could perfectly interact with the negatively charged phosphate groups of the DNA backbone . Substitution of three basic amino acid residues of this putative DNAB by alanine and/or glutamate generated mutated GvpE proteins . None of these was able to activate the c-gvpA promoter in vivo, indicating that these basic residues are required for GvpE activity . This identification of an archaeal gene regulator displaying similarity to eukaryal regulatory proteins implies that the basic transcription machinery of eukarya and archaea are closely related, and that the regulatory proteins have evolved according to common principles .

Microbiology, 1998 Jun, 144 ( Pt 6), 1601 - 9
Psychroflexus torquis gen . nov., sp . nov., a psychrophilic species from Antarctic sea ice, and reclassification of Flavobacterium gondwanense (Dobson et al . 1993) as Psychroflexus gondwanense gen . nov., comb . nov; Bowman JP et al.; A group of sea-ice-derived psychrophilic bacterial strains possessing the unusual ability to synthesize the polyunsaturated fatty acids eicosapentaenoic acid (20:5 omega 3) and arachidonic acid (20:4 omega 6) belong to the Family Flavobacteriaceae (Flexibacter-Bacteroides-Flavobacterium phylum), according to 16S rRNA sequence analysis . Surprisingly, the isolates were also found to cluster closely to the moderately halophilic and psychrotrophic species {Flavobacterium} gondwanense (sequence similarity 97.8-98.1%) . The whole-cell fatty acid profiles of this group and {Flavobacterium} gondwanense were very similar and distinct from other related flavobacteria . The sea ice strains and {Flavobacterium} gondwanense differed substantially in terms of ecophysiology, possibly representing divergent adaptations to sympagic and planktonic marine habitats, respectively . Evidence based on phylogeny and fatty acid profiles supports the conclusion that the taxa are close relatives distinct from other bacterial groups . It is thus proposed that the sea ice strains represent a novel taxon designated Psychroflexus torquis gen . nov., sp . nov . (type strain ACAM 623T) while {Flavobacterium} gondwanense becomes Psychroflexus gondwanense gen . nov., comb . nov.

Infect Immun, 1998 Jul, 66(7), 3134 - 41
Cloning and characterization of an outer membrane protein of Vibrio vulnificus required for heme utilization: regulation of expression and determination of the gene sequence; Litwin CM et al.; Vibrio vulnificus is a halophilic, marine pathogen that has been associated with septicemia and serious wound infections in patients with iron overload and preexisting liver disease . For V . vulnificus, the ability to acquire iron from the host has been shown to correlate with virulence . V . vulnificus is able to use host iron sources such as hemoglobin and heme . We previously constructed a fur mutant of V . vulnificus which constitutively expresses at least two iron-regulated outer membrane proteins, of 72 and 77 kDa . The N-terminal amino acid sequence of the 77-kDa protein purified from the V . vulnificus fur mutant had 67% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae heme receptor, HutA . In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for HupA, the heme receptor of V . vulnificus . DNA sequencing of hupA demonstrated a single open reading frame of 712 amino acids that was 50% identical and 66% similar to the sequence of V . cholerae HutA and similar to those of other TonB-dependent outer membrane receptors . Primer extension analysis localized one promoter for the V . vulnificus hupA gene . Analysis of the promoter region of V . vulnificus hupA showed a sequence homologous to the consensus Fur box . Northern blot analysis showed that the transcript was strongly regulated by iron . An internal deletion in the V . vulnificus hupA gene, done by using marker exchange, resulted in the loss of expression of the 77-kDa protein and the loss of the ability to use hemin or hemoglobin as a source of iron . The hupA deletion mutant of V . vulnificus will be helpful in future studies of the role of heme iron in V . vulnificus pathogenesis.

J Biol Chem, 1998 May 15, 273(20), 12116 - 9
Mechanosensitive ion channels of the archaeon Haloferax volcanii; Le Dain AC et al.; Mechanosensitive (MS) ion channels have been documented in a variety of cells belonging to Eukarya and Eubacteria . We report the novel finding of two types of MS ion channels in the cell membrane of the halophilic archaeon Haloferax volcanii, a member of the Archaea that comprise the third phylogenetic domain . The two channels, MscA1 and MscA2, differed in their kinetic properties with MscA1 exhibiting more frequent open-closed transitions than MscA2 . Both channels have large conductances that rectify between -40 mV and +40 mV where the conductance of MscA1 ranged from 380 to 680 picosiemens, whereas MscA2 ranged from 850 to 490 picosiemens . Both channels were blocked by submillimolar gadolinium . In addition, the channels of either membrane vesicles or detergent-solubilized membrane proteins remained functional upon reconstitution into artificial liposomes, a result that indicates that these channels are activated by mechanical force transmitted via the lipid bilayer alone . Subsequently a 37-kDa protein corresponding to the MscA1 channel activity was purified . With the possible functional similarity to bacterial MS channels, our finding of MS channels in Archaea emphasizes the ubiquity and importance of these channels in all domains of the evolutionary tree.

FEMS Microbiol Lett, 1998 Jun 1, 163(1), 91 - 7
Typing of halophilic Archaea and characterization of their cell surface carbohydrates by use of lectins; Gilboa-Garber N et al.; Lectins are important tools for cell typing and for the study of cell surface components . They have been widely used for the analysis of carbohydrates on the surface of many eukaryotic and prokaryotic cells, but they have not yet been exploited in the study of the halophilic Archaea (family Halobacteriaceae), because of the high salinity required for the structural integrity of these microorganisms . We have defined the salt concentration threshold high enough for survival of the Archaea, but sufficiently low for lectins to bind to them . Under these conditions we studied the interactions of a series of lectins, exhibiting different sugar specificities, with diverse halophilic Archaea . Concanavalin A was the most reactive by virtue of its glucose (and mannose) binding . The other lectins varied in their interactions . The results indicate that lectins might be useful probes for both archaeal typing and analysis of their cell surface carbohydrates.

Biochim Biophys Acta, 1998 Jun 11, 1385(1), 1 - 6
Sequence, chromophore extraction and 3-D model of the photoactive yellow protein from Rhodobacter sphaeroides; Kort R et al.; The photoactive yellow protein (pyp) gene has been isolated from Rhodobacter sphaeroides by probing with a homologous PCR-product . A sequence analysis shows that this pyp gene encodes a 124 AA protein with 48% identity to the three known PYPs . Downstream from pyp, a number of adjacent open reading frames were identified, including a gene encoding a CoA-ligase homologue (pCL) . This latter protein is proposed to be involved in PYP chromophore activation, required for attachment to the apoprotein . We have demonstrated the presence of the chromophoric group, previously identified in PYP from Ectothiorhodospira halophila as trans 4-hydroxy cinnamic acid, in phototrophically cultured R . sphaeroides cells by capillary zone electrophoresis . The basic structure of the chromophore binding pocket in PYP has been conserved, as shown by a 3D model of R . sphaeroides PYP, constructed by homology-based molecular modelling . In addition, this model shows that R . sphaeroides PYP contains a characteristic, positively charged patch .

Microbiol Mol Biol Rev, 1998 Jun, 62(2), 504 - 44
Biology of moderately halophilic aerobic bacteria; Ventosa A et al.; The moderately halophilic heterotrophic aerobic bacteria form a diverse group of microorganisms . The property of halophilism is widespread within the bacterial domain . Bacterial halophiles are abundant in environments such as salt lakes, saline soils, and salted food products . Most species keep their intracellular ionic concentrations at low levels while synthesizing or accumulating organic solutes to provide osmotic equilibrium of the cytoplasm with the surrounding medium . Complex mechanisms of adjustment of the intracellular environments and the properties of the cytoplasmic membrane enable rapid adaptation to changes in the salt concentration of the environment . Approaches to the study of genetic processes have recently been developed for several moderate halophiles, opening the way toward an understanding of haloadaptation at the molecular level . The new information obtained is also expected to contribute to the development of novel biotechnological uses for these organisms.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 4970 - 5
Dynamics of different functional parts of bacteriorhodopsin: H-2H labeling and neutron scattering; Reat V et al.; We show that dynamics of specific amino acids within a protein can be characterized by neutron spectroscopy and hydrogen-deuterium labeling, and we present data on the motions of a selected set of groups within bacteriorhodopsin (BR), the retinal-based proton pump in the purple membrane of halophilic Archaea . Elastic incoherent neutron scattering experiments allow the definition of motions in the nano- to picosecond time scale and have revealed a dynamical transition from a harmonic to a softer, anharmonic atomic fluctuation regime in the global behavior of proteins . Biological activity in proteins is correlated with this transition, suggesting that flexibility is required for function . Elastic incoherent neutron scattering is dominated by H atom scattering, and to study the dynamics of a selected part of BR, fully deuterated purple membrane with BR containing H-retinal, H-tryptophan, and H-methionine was prepared biosynthetically in Halobacterium salinarum . These amino acids cluster in the functional center of the protein . In contrast to the protein globally, the thermal motions of the labeled atoms were found to be shielded from solvent melting effects at 260 K . Above this temperature, the labeled groups appear as more rigid than the rest of the protein, with a significantly smaller mean square amplitude of motion . These experimental results quantify the dynamical heterogeneity of BR (which meets the functional requirements of global flexibility), on the one hand, to allow large conformational changes in the molecule and of a more rigid region in the protein, on the other, to control stereo-specific selection of retinal conformations.

Protein Eng, 1998 Mar, 11(3), 185 - 98
Homology model of the quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni; Jongejan A et al.; A molecular model of QH-ADH, the quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni, has been built by homology modelling . Sequence similarity of N-terminal residues 1-570 with the alpha-subunit of quinoprotein methanol dehydrogenases (MDHs) from Methylophilus methylotrophus W3A1 and Methylobacterium extorquens provided a basis for the design of the PQQ-binding domain of QH-ADH . Minimal sequence similarity with cytochrome c551 from Ectothiorhodospira halophila and cytochrome c5 from Azotobacter vinelandii has been used to model the C-terminal haem c-binding domain, residues 571-677, absent in MDHs . Distance constraints inferred from 19F-NMR relaxation studies of trifluoromethylphenylhydrazine-derivatized PQQ bound to QH-ADH apoenzyme as well as theoretical relations for optimal electron transfer have been employed to position the haem- and PQQ-binding domains relative to each other . The homology model obtained shows overall topological similarity with the crystal structure of cd1-nitrite reductase from Thiosphera pantotropha . The proposed model accounts for the following: (i) the site that is sensitive to in vivo proteolytic attack; (ii) the substrate specificity in comparison with MDHs; (iii) changes of the spectral properties of the haem c upon reconstitution of apo-enzyme with PQQ; (iv) electronic interaction between haem and PQQ; and (v) enantioselectivity in the conversion of a chiral sec alcohol.

Microbiology, 1998 May, 144 ( Pt 5), 1331 - 42
Structural characteristics of halobacterial gas vesicles; Offner S et al.; Gas vesicle formation in halophilic archaea is encoded by a DNA region (the vac region) containing 14 different genes: gvpACNO and gvpDEFGHIJKLM . In Halobacterium salinarum PHH1 (which expresses the p-vac region from plasmid pHH1), gas vesicles are spindle shaped, whereas predominantly cylindrical gas vesicles are synthesized by the chromosomal c-vac region of H . salinarum PHH4 and the single chromosomal mc-vac region of Haloferax mediterranei . Homologous complementation of gvp gene clusters derived from the chromosomal c-vac region led to cylindrical gas vesicles in transformants and proved that the activity of the c-gvpA promoter depended on a gene product from the c-gvpE-M DNA region . Heterologous complementation experiments with transcription units of different vac regions demonstrated that the formation of chimeric gas vesicles was possible . Comparison of micrographs of wild-type and chimeric gas vesicles indicated that the shape was not exclusively determined by GvpA, the major structural protein of the gas vesicle wall . More likely, a dynamic equilibrium of several gvp gene products was responsible for determination of the shape . Transmission electron microscopy of frozen hydrated, wild-type gas vesicles showed moire patterns due to the superposition of the front and back parts of the ribbed gas vesicle envelope . Comparison of projections of model helices with the moire pattern seen on the cylindrical part of the gas vesicles provided evidence that the ribs formed a helix of low pitch and not a stack of hoops.

NMR Biomed, 1998 Apr, 11(2), 80 - 6
Nuclear magnetic resonance studies of cesium-133 in the halophilic halotolerant bacterium Ba1 . Chemical shift and transport studies; Sakhnini A et al.; Ba1 bacteria (Halomonas israelensis) were grown on different salt concentrations 0.2-4 M . When the cells were transferred to a medium containing 25 mM CsCl without potassium there was an uptake of cesium by the cells . The intracellular cesium signal was shifted from the cesium signal in the medium without the use of a shift reagent . The shift was depended on the salt concentration in the growth medium . The intracellular cesium shift showed a much smaller dependence on the concentration of salts in the medium than the extracellular cesium; the same results were detected for cells grown on a medium containing 25 mM CsCl . The cesium transport through the cell membrane was mostly by active transport . The cesium concentration in the cell was higher than that of the medium, approximately 100 mM intracellular concentration compared to 25 mM in the medium . The first order constants for influx or efflux of cesium were from 2 x 10(-4) and up to 24 x 10(-4)/min for the different medium concentrations.

Microbiologia, 1997 Dec, 13(4), 489 - 92
Comparison between the polypeptide profile of halophilic bacteria and salt tolerant plants; Munoz G et al.; Changes in the polypeptide profile induced by salt stress in halotolerant and halophilic bacteria, isolated from the Atacama desert (northern Chile), were compared with those in the cotyledons of Prosopis chilensis (Leguminoseae) seedlings, a salt tolerant plant . SDS-PAGE analyses show the presence of four predominant polypeptides, with molecular weights around 78, 70, 60 and 44 kDa respectively, both in bacteria and in cotyledons from P . chilensis seedlings raised under salt stress conditions . Moreover, the 60 and 44 kDa polypeptides seem to be salt responsive, since their concentration increases with increasing NaCl in the growth medium . Our results suggest a common mechanism for salt tolerance in prokaryotes and in eukaryotes.

Nucleic Acids Symp Ser, 1997, (37), 163 - 4
High-salt effects on the structure and damage of chromosomal DNA in Halobacterium salinarium, an extremely halophilic bacterium; Shahmohammadi HR et al.; High concentration salt effects on the structure and radiation-induced damages of DNA were studied to elucidate the biochemical mechanism of the resistance of halophilic H . salinarium against DNA damaging agents . High concentration of KCl did not induce significant conformational changes in H . salinarium chromosomal DNA, but exhibited an extensive protective effect on the radiation-induced single-strand breaks of plasmid DNA.

Nucleic Acids Symp Ser, 1997, (37), 111 - 12
Primary structure of the novel bacterial rhodopsin from extremely halophilic archaeon Haloarcula japonica strain TR-1; Yatsunami R et al.; A novel bacterial rhodopsin was identified in Haloarcula japonica strain TR-1 . The gene encoding the bacterial rhodopsin was cloned and sequenced . The structural gene consisted of an open reading frame of 750 nucleotides encoding 250 amino acids . The deduced amino acid sequence of the Ha . japonica bacterial rhodopsin showed the highest homology to those of cruxrhodopsins.

Nucleic Acids Symp Ser, 1997, (37), 109 - 10
Molecular cloning of the gene encoding a 2Fe-2S ferredoxin from extremely halophilic archaeon Haloarcula japonica strain TR-1; Ikeda A et al.; A ferredoxin was purified from extremely halophilic archaeon Haloarcula japonica strain TR-1, and then characterized . The Ha . japonica ferredoxin proved to contain a 2Fe-2S cluster . A part of the gene encoding the ferredoxin was amplified by PCR . Subsequently, the entire ferredoxin gene was cloned from chromosomal DNA of Ha . japonica using the PCR product as a probe . The structural gene consisted of an open reading frame of 387 nucleotides . The deduced amino acid sequence showed 89-98% identities with those of the ferredoxins from other extremely halophilic archaea.

J Biol Chem, 1998 Apr 10, 273(15), 9023 - 30
Insights into the molecular basis of salt tolerance from the study of glutamate dehydrogenase from Halobacterium salinarum; Britton KL et al.; A homology-based modeling study on the extremely halophilic glutamate dehydrogenase from Halobacterium salinarum has been used to provide insights into the molecular basis of salt tolerance . The modeling reveals two significant differences in the characteristics of the surface of the halophilic enzyme that may contribute to its stability in high salt . The first of these is that the surface is decorated with acidic residues, a feature previously seen in structures of halophilic enzymes . The second is that the surface displays a significant reduction in exposed hydrophobic character . The latter arises not from a loss of surface-exposed hydrophobic residues, as has previously been proposed, but from a reduction in surface-exposed lysine residues . This is the first report of such an observation.

J Bacteriol, 1998 May, 180(9), 2450 - 8
Gas vesicle genes identified in Bacillus megaterium and functional expression in Escherichia coli; Li N et al.; Gas vesicles are intracellular, protein-coated, and hollow organelles found in cyanobacteria and halophilic archaea . They are permeable to ambient gases by diffusion and provide buoyancy, enabling cells to move upwards in liquid to access oxygen and/or light . In halobacteria, gas vesicle production is encoded in a 9-kb cluster of 14 genes (4 of known function) . In cyanobacteria, the number of genes involved has not been determined . We now report the cloning and sequence analysis of an 8,142-bp cluster of 15 putative gas vesicle genes (gvp) from Bacillus megaterium VT1660 and their functional expression in Escherichia coli . Evidence includes homologies by sequence analysis to known gas vesicle genes, the buoyancy phenotype of E . coli strains that carry this gvp gene cluster, the presence of pressure-sensitive, refractile bodies in phase-contrast microscopy, structural details in phase-contrast microscopy, structural details in direct interference-contrast microscopy, and shape and size revealed by transmission electron microscopy . In B . megaterium, the gvp region carries a cluster of 15 putative genes arranged in one orientation; they are open reading frame 1 and gvpA, -P, -Q, -B, -R, -N, -F, -G, -L, -S, -K, -J, -T, and -U, of which the last 11 genes, in a 5.7-kb gene cluster, are the maximum required for gas vesicle synthesis and function in E . coli . To our knowledge, this is the first example of a functional gas vesicle gene cluster in nonaquatic bacteria and the first example of the interspecies transfer of genes resulting in the synthesis of a functional organelle.

FEMS Microbiol Lett, 1998 Apr 15, 161(2), 293 - 300
Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis; Goller K et al.; Using transposon mutagenesis we generated a salt-sensitive mutant of the halophilic eubacterium Halomonas elongata impaired in the biosynthesis of the compatible solute ectoine . HPLC determinations of the cytoplasmic solute content showed the accumulation of a biosynthetic precursor of ectoine, L-2,4-diaminobutyric acid . Ectoine and hydroxyectoine were not detectable . This mutant failed to grow in minimal medium with NaCl concentrations exceeding 4% . However, when supplemented with organic osmolytes, the ability to grow in high-salinity medium (15% and higher) was regained . We cloned and sequenced the regions flanking the transposon insertion in the H . elongata chromosome . Sequence comparisons with known proteins revealed significant similarity of the mutated gene to the L-2,4-diaminobutyric acid acetyltransferase from the ectoine biosynthetic pathway in Marinococcus halophilus . Analysis of a PCR product demonstrated that the ectoine biosynthetic genes (ectABC) follow the same order as in M . halophilus.

Biochim Biophys Acta, 1998 Mar 2, 1369(2), 213 - 20
A new Na+/H+ antiporter, NhaD, of Vibrio parahaemolyticus; Nozaki K et al.; A gene encoding an Na+/H+ antiporter was cloned from chromosomal DNA of Vibrio parahaemolyticus, a slightly halophilic bacterium, and expressed in Escherichia coli cells . The gene enabled mutant E . coli cells, which were unable to grow in the presence of 10 mM LiCl (or 0.2 M NaCl) because of the lack of major Na+(Li+)/H+ antiporters, to grow under such conditions . We detected Na+/H+ antiport activity due to the gene in membrane vesicles prepared from E . coli cells that harbored the plasmid carrying the gene . Li+ was also a substrate for this antiporter . Activity of this antiporter was pH-dependent with highest activity at pH 8.5 to 9 and no activity at 7.0 to 7.5 . Restriction mapping and a Southern blot analysis revealed that the cloned gene was different from the nhaA and the nhaB of V . parahaemolyticus . We designated the gene nhaD . The gene was sequenced, and the amino acid sequence of the NhaD protein was deduced . The NhaD is a unique Na+/H+ antiporter with respect to the primary structure compared with known Na+/H+ antiporters .

Biochim Biophys Acta, 1998 Mar 9, 1396(2), 148 - 52
Nucleotide sequence of the sspE genes coding for gamma-type small, acid-soluble spore proteins from the round-spore-forming bacteria Bacillus aminovorans, Sporosarcina halophila and S . ureae; Loshon CA et al.; The single sspE genes coding for gamma-type small, acid-soluble spore proteins (SASP) of three round-spore-forming bacteria, Bacillus aminovorans, Sporosarcina halophila and S . ureae, have been cloned and sequenced . While the deduced amino acid sequences of these three gamma-type SASP show clear homology to those from six Bacillus species that do not form round spores, there are no residues conserved completely among the 9 sequences known . In addition, the 139 residue B . aminovorans protein is 35 residues larger than any other while the 60 residue S . halophila protein is one of the smallest . These data suggest that the sspE genes have been under little selective pressure in recent evolutionary time.

Arch Microbiol, 1998 Mar, 169(3), 231 - 8
Psychromonas antarcticus gen . nov., sp . nov., A new aerotolerant anaerobic, halophilic psychrophile isolated from pond sediment of the McMurdo ice shelf, antarctica
Mountfort DO, Rainey FA, Burghardt J, Kaspar HF, Stackebrandt E.
A gram-negative, rod- to oval-shaped, aerotolerant anaerobic bacterium was isolated from an anaerobic enrichment inoculated with sediment taken from below the cyanobacterial mat of a high-salinity pond near Bratina Island on the McMurdo Ice Shelf, Antarctica . The organism was positive for terminal oxidase and catalase and was motile by means of a polar flagellum . Optimal growth of anaerobic cultures occurred at 12 degrees C, at pH 6.5, and at an NaCl concentration of 3% (w/v) . Of a variety of polysaccharides tested, only starch and glycogen supported growth . No growth was observed on cellulosic substrates and xylan, and the organism was unable to attack esculin . Monosaccharides and disaccharides, including the cyanobacterial cell-wall constituent N-acetyl glucosamine, were fermented . Per 100 mol of hexose, the following products (in mol) were formed: acetate, 60; formate, 130; ethanol, 56; lactate, 73; CO2, 15; and butyrate, 2 . Propionate, ethanol, n-propanol, n-butanol and succinate were not detectable in the culture medium (< 1 mol per 100 mol of monomer) . Hydrogen was not detected in the head space (detection limit < 10(-5) atm) . Growth yields in aerobic static liquid cultures were slightly higher than those in anaerobic culture, and fermentation favoured acetate at the expense of electron sink products . Growth was inhibited in aerobic shaking cultures, and the organism did not utilize nitrate or sulfate as electron acceptors . The G+C content of the DNA from the bacterium was 42.8 mol% . A phylogenetic analysis indicated that the organism is a member of the gamma-subgroup of Proteobacteria, but that it is distinct from other members of this group based on the sequence of its 16S rRNA gene, mol% G+C, morphology, and physiological and biochemical characteristics . It is designated as a new genus and species; the type strain is star-1 (DSM 10704).

Nephron, 1998, 78(2), 221 - 4
Fatal sepsis from Vibrio vulnificus in a hemodialyzed patient; Stabellini N et al.; Vibrio vulnificus, a particularly virulent halophilic vibrio, has been isolated from the blood and skin necrotic lesion of a hemodialyzed patient with sepsis . The patient has had exposure of the skin to seawater . Various chronic conditions including renal failure have a great risk for developing septicemia due to V vulnificus . It is necessary to inform persons with liver diseases or immunocompromising conditions of hazards associated with the consumption of undercooked seafood and seawater exposure.

J Bacteriol, 1998 Mar, 180(5), 1119 - 28
Helicobacter pylori porCDAB and oorDABC genes encode distinct pyruvate:flavodoxin and 2-oxoglutarate:acceptor oxidoreductases which mediate electron transport to NADP; Hughes NJ et al.; Helicobacter pylori, a major cause of human gastric disease, is a microaerophilic bacterium that contains neither pyruvate nor 2-oxoglutarate dehydrogenase activity . Previous studies (N . J . Hughes, P . A . Chalk, C . L . Clayton, and D . J . Kelly, J . Bacteriol . 177:3953-3959, 1995) have indicated that the major routes for the generation of acetyl coenzyme A (acetyl-CoA) and succinyl-CoA are via pyruvate:flavodoxin oxidoreductase (POR) and 2-oxoglutarate:acceptor oxidoreductase (OOR), respectively . The purified POR is a heterotetrameric protein, with subunits of 48 (PorA), 36 (PorB), 24 (PorC), and 14 (PorD) kDa . In this study OOR has been purified, and it is similarly composed of polypeptides of 43 (OorA), 33 (OorB), 24 (OorC), and 10 (OorD) kDa . Both POR and OOR are oxygen labile and are likely to be major contributors to the microaerophilic phenotype of H . pylori . Unlike POR, OOR was unable to use a previously identified flavodoxin (FldA) as an electron acceptor . Although the purified enzymes were unable to reduce NAD(P), electrons from both pyruvate and 2-oxoglutarate could reduce NADP in cell extracts, consistent with a role for these oxidoreductases in the provision of NADPH as a respiratory electron donor . The H . pylori por, oor, and fldA genes were cloned and sequenced . The deduced por gene products showed significant sequence similarity to archaeal four-subunit 2-oxoacid:acceptor oxidoreductases . However, the amino acid sequences of OorA and -B were more closely related to that of the two-subunit POR of the aerobic halophile Halobacterium halobium . Both porD and oorD encode integral ferredoxin-like subunits . POR and OOR are probably essential enzymes in H . pylori, as insertion inactivation of porB and oorA appeared to be lethal.

Structure, 1998 Jan 15, 6(1), 75 - 88
Structural features of halophilicity derived from the crystal structure of dihydrofolate reductase from the Dead Sea halophilic archaeon, Haloferax volcanii; Pieper U et al.; BACKGROUND: The proteins of halophilic archaea require high salt concentrations both for stability and for activity, whereas they denature at low ionic strength . The structural basis for this phenomenon is not yet well understood . The crystal structure of dihydrofolate reductase (DHFR) from Haloferax volcanii (hv-DHFR) reported here provides the third example of a structure of a protein from a halophilic organism . The enzyme is considered moderately halophilic, as it retains activity and secondary structure at monovalent salt concentrations as low as 0.5 M . RESULTS: The crystal structure of hv-DHFR has been determined at 2.6 A resolution and reveals the same overall fold as that of other DHFRs . The structure is in the apo state, with an open conformation of the active-site gully different from the open conformation seen in other DHFR structures . The unique feature of hv-DHFR is a shift of the alpha helix encompassing residues 46-51 and an accompanied altered conformation of the ensuing loop relative to other DHFRs . Analysis of the charge distribution, amino acid composition, packing and hydrogen-bonding pattern in hv-DHFR and its non-halophilic homologs has been performed . CONCLUSIONS: The moderately halophilic behavior of hv-DHFR is consistent with the lack of striking structural features expected to occur in extremely halophilic proteins . The most notable feature of halophilicity is the presence of clusters of non-interacting negatively charged residues . Such clusters are associated with unfavorable electrostatic energy at low salt concentrations, and may account for the instability of hv-DHFR at salt concentrations lower than 0.5 M . With respect to catalysis, the open conformation seen here is indicative of a conformational transition not reported previously . The impact of this conformation on function and/or halophilicity is unknown.

Rocz Panstw Zakl Hig, 1997, 48(2), 139 - 43
{The role of halophilic bacteria in decarboxylation of histidine in salted fish}; Peconek J et al.; The purpose of this study was to determine frequency of occurrence of halotolerant and halophilic histamine-forming bacteria under laboratory conditions in salted herring stored at 20 degrees C for 2 months . The other aim was to isolate these bacteria both from herring bought in a retail shop and examine the ability of decarboxylation of histidine by these microorganisms using Karnop's method modified by the authors . The amount of histamine formed was determined by fluorometric method . The level of histamine in media containing homogenates of herring was in a range from 5 to 1950 micrograms/100 ml of a nutrient medium . All isolated bacteria belong to halotolerant and halophilic strains and produce histamine both at 8% and 20% content of NaCl in a nutrient medium . The obtained results indicate that during storage of salted herring at ambient temperature the increase of histamine content in their flesh can occur.

J Bacteriol, 1998 Feb, 180(3), 457 - 63
Purine salvage in two halophilic archaea: characterization of salvage pathways and isolation of mutants resistant to purine analogs; Stuer-Lauridsen B et al.; In exponentially growing cultures of the extreme halophile Halobacterium halobium and the moderate halophile Haloferax volcanii, growth characteristics including intracellular protein levels, RNA content, and nucleotide pool sizes were analyzed . This is the first report on pool sizes of nucleoside triphosphates, NAD, and PRPP (5-phosphoribosyl-alpha-1-pyrophosphate) in archaea . The presence of a number of salvage and interconversion enzymes was determined by enzymatic assays . The levels varied significantly between the two organisms . The most significant difference was the absence of GMP reductase activity in H . halobium . The metabolism of exogenous purines was investigated in growing cultures . Both purine bases and nucleosides were readily taken up and were incorporated into nucleic acids . Growth of both organisms was affected by a number of inhibitors of nucleotide synthesis . H . volcanii was more sensitive than H . halobium, and purine base analogs were more toxic than nucleoside analogs . Growth of H . volcanii was inhibited by trimethoprim and sulfathiazole, while these compounds had no effect on the growth of H . halobium . Spontaneous mutants resistant to purine analogs were isolated . The most frequent cause of resistance was a defect in purine phosphoribosyltransferase activity coupled with reduced purine uptake . A single phosphoribosyltransferase seemed to convert guanine as well as hypoxanthine to nucleoside monophosphates, and another phosphoribosyltransferase had specificity towards adenine . The differences in the metabolism of purine bases and nucleosides and the sensitivity to purine analogs between the two halobacteria were reflected in differences in purine enzyme levels . Based on our results, we conclude that purine salvage and interconversion pathways differ just as much between the two archaeal species as among archaea, bacteria, and eukarya.

Gene, 1997 Dec 19, 204(1-2), 139 - 44
Gene for a cyclophilin-type peptidyl-prolyl cis-trans isomerase from a halophilic archaeum, Halobacterium cutirubrum; Iida T et al.; A gene encoding a cyclophilin (CyP)-type peptidyl prolyl cis-trans isomerase (PPIase) was cloned from a halophilic archaeum, Halobacterium cutirubrum DSM 669, and sequenced . Although amino-acid residues common to CyPs were conserved, an insertion that showed no homology to other CyPs was found in its N-terminal region . Sequence analysis revealed that the amino-acid sequence of this CyP was 40-45% identical to those of eukaryotes and Bacillus subtilis with high cyclosporin A sensitivity, but 27% identical to those of cyclosporin A-insensitive PPIases of Escherichia coli . The gene was also expressed in E . coli . The activity of purified recombinant CyP-type PPIase was stimulated by the addition of KCl, and was suppressed by cyclosporin A.

Biochim Biophys Acta, 1997 Nov 14, 1343(1), 23 - 30
Iron superoxide dismutase from the archaeon Sulfolobus solfataricus: average hydrophobicity and amino acid weight are involved in the adaptation of proteins to extreme environments; Dello Russo A et al.; The iron-superoxide dismutase in the thermoacidophilic archaeon Sulfolobus solfataricus has a homodimeric structure with a metal content of 0.7 atom of iron per subunit . The enzyme is insensitive to cyanide inhibition, sensitive to inactivation by H2O2 and is the most heat resistant SOD known so far being its half-life 2 h at 100 degrees C . Its primary structure was determined by a profitable combination of advanced mass spectrometry and automated sequence analysis of peptides obtained after cleavage of the purified protein . The enzyme subunit is composed of 210 amino acid residues accounting for a relative molecular mass of 24,112 . It does not contain cysteine residues and has a high average of both hydrophobicity and amino acid weight . Vice versa, the hydrophobicity is lower in halophilic SODs . Therefore, it seems that the average hydrophobicity is involved in the adaptation of proteins to extreme environments . The multiple alignment of the primary structure of archaeal and thermophilic eubacterial SODs indicated that archaeal SODs evolved separately from the thermophilic eubacterial SODs and that halophiles originated from a gene different from that of thermophilic archaea.

Appl Environ Microbiol, 1997 Dec, 63(12), 4729 - 33
Phylogenetic diversity of Archaea in sediment samples from a coastal salt marsh; Munson MA et al.; The Archaea present in salt marsh sediment samples from a tidal creek and from an adjacent area of vegetative marshland, both of which showed active methanogenesis and sulfate reduction, were sampled by using 16S rRNA gene libraries created with Archaea-specific primers . None of the sequences were the same as reference sequences from cultured taxa, although some were closely related to sequences from methanogens previously isolated from marine sediments . A wide range of Euryarchaeota sequences were recovered, but no sequences from Methanococcus, Methanobacterium, or the Crenarchaeota were recovered . Clusters of closely related sequences were common and generally contained sequences from both sites, suggesting that some related organisms were present in both samples . Recovery of sequences closely related to those of methanogens such as Methanococcoides and Methanolobus, which can use substrates other than hydrogen, provides support for published hypotheses that such methanogens are probably important in sulfate-rich sediments and identifies some likely candidates . Sequences closely related to those of methanogens such as Methanoculleus and Methanogenium, which are capable of using hydrogen, were also discovered, in agreement with previous inhibitor and process measurements suggesting that these taxa are present at low levels of activity . More surprisingly, we recovered a variety of sequences closely related to those from different halophilic Archaea and a cluster of divergent sequences specifically related to the marine group II archaeal sequences recently shown by PCR and probing to have a cosmopolitan distribution in marine samples.

Antonie Van Leeuwenhoek, 1997 Oct, 72(3), 261 - 70
Soil mycoflora from the Dead Sea Oases of Ein Gedi and Einot Zuqim (Israel); Steiman R et al.; Samples were taken from the top 10 cm of soils from 24 points in the Ein Gedi area . Among 329 isolates, 142 species were identified: 11 genera of ascomycetes, one genus of coelomycetes, 28 genera of hyphomycetes, 7 genera of zygomycetes and one yeast, in addition to some unidentified basidiomycetes . The hyphomycetes were represented by 17 dematiaceous, 9 mucedinaceous and two tuberculariaceous . Melanconiaceous and stilbellaceous genera were not found . Two new varieties of Microascus recently described were reisolated . No strict thermophiles or halophiles were obtained . There is apparently no very characteristic or specific fungal flora of the Dead Sea Oases although it was different from that found in the desert soil surrounding this area.

Antonie Van Leeuwenhoek, 1997 Oct, 72(3), 239 - 43
Growth, maintenance and fermentation pattern of the salt-tolerant lactic acid bacterium Tetragenococcus halophila in anaerobic glucose limited retention cultures; Roling WF et al.; The homofermentative lactic acid bacterium Tetragenococcus halophila showed mixed acid fermentation at low growth-rates under glucose limiting conditions and in the presence of 10% NaCl . Maximum growth yields in fermentors with cell retention were not affected by pH, but maintenance requirement was at pH 5.2 four times higher than at pH 7.0 . Despite the high salt-concentration of the medium, maintenance requirements were low compared to other lactic acid bacteria . The possible causes of the observed differences in maintenance requirements are discussed.

Eur J Biochem, 1997 Nov 1, 249(3), 905 - 11
The cell wall polymer of the extremely halophilic archaeon Natronococcus occultus; Niemetz R et al.; The cell wall polymer of Natronococcus occultus (DSM 3396) consists of L-glutamate, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-galacturonic acid, D-glucuronic acid and D-glucose in a molar ratio of 5:7:1:8:0.5:0.3 . Partial acid hydrolysis of the cell wall polymer produced soluble fragments that could be separated by HPLC . A gamma-glutamyl dipeptide was isolated . In the intact cell wall polymer, the glutamate residues form a poly-(gamma-glutamine) chain with a length of about 60 monomers, which corresponds to a relative molecular mass of approximately 7700 Da . Two other soluble dimeric fragments, composed of glutamate and either glucosamine or galactosamine in a molar ratio of 1:1, were purified from the hydrolysate, suggesting the presence of two different oligosaccharides linked to the poly-(gamma-glutamine) chain of the intact polymer . The analysis of additional fragments, which were composed of an amino sugar and galacturonic acid or glucose indicated that one oligosaccharide consisted of a glucosamine pentamer in an alpha-1,3 linkage at the reducing end and an oligomer with at least five beta-1,4-linked galacturonic acid residues at the non-reducing end . The second oligosaccharide was comprised of a galactosamine dimer in a beta-1,3 linkage at the reducing end and a maltose unit at the non-reducing end . Both oligosaccharides were linked to the alpha-amide group of the glutamine residues of the poly-(gamma-glutamine) chain . The whole cell wall polymer, which represents a novel type of natural glycoconjugate, has a relative molecular mass of 54 kDa.

Carbohydr Res, 1997 Sep 26, 303(3), 333 - 8
Structure of a phosphorylated polysaccharide from Shewanella putrefaciens strain S29; Shashkov AS et al.; A phosphorylated polysaccharide was isolated from the aqueous layer of the phenol-water extract of a non-halophilic bacterium Shewanella putrefaciens strain S29 . The glycosyl phosphate linkage in the polysaccharide was split under mild acid conditions to give, after borohydride reduction, a phosphorylated oligosaccharide-alditol . On the basis of sugar analysis and 1H, 13C and 31P NMR spectroscopy, including 2D COSY, relayed COSY, rotating-frame NOE spectroscopy (ROESY), heteronuclear 13C,1H COSY, and H-detected heteronuclear 1H,31P multiple-quantum coherence (HMQC), it was concluded that the polysaccharide is built up of tetrasaccharide-phosphate repeating units having the following structure: {sequence: see text} where QuiNAc and Qui4NAc are 2-acetamido-2,6-dideoxyglucose and 4-acetamido-4,6-dideoxyglucose, respectively.

Eur J Biochem, 1997 Oct 15, 249(2), 607 - 11
Stabilisation of halophilic malate dehydrogenase from Haloarcula marismortui by divalent cations -- effects of temperature, water isotope, cofactor and pH; Madern D et al.; Halophilic malate dehydrogenase is stable in a limited concentration range of MgCl2 or CaCl2 . Thermal deactivation of the protein at low concentrations of these divalent salts is very different from that occurring at high concentrations . In low salt, stability always increases as the temperature is lowered . In high salt, stability shows bell-shaped behaviour as a function of temperature: increasing to a maximum at 4 degrees C, and subsequently decreasing as the temperature is lowered . This is in contrast to other salts, for which the deactivation behaviour depends on the salt type but not on its concentration . Cofactor addition or replacement of H2O by D2O modify only the deactivation at low MgCl2 or CaCl2 concentrations . A pH transition between pH 7 and pH 8, however, modified enzyme deactivation at both low and high MgCl2 or CaCl2 concentrations . The pH effect on stability was also observed in other salts . By comparing the effect of CaCl2, MgCl2, and NaCl, a strong correlation was found between the minimum salt concentration required for the stabilisation of halophilic malate dehydrogenase and the hydration of the cation.

Glycobiology, 1997 Oct, 7(7), 897 - 904
Isolation and characterization of dolichol-linked oligosaccharides from Haloferax volcanii; Kuntz C et al.; Biosynthesis of procaryotic glycoproteins has been studied in some detail only in the extreme halophile Halobacterium halobium . To extend these studies for a moderate halophile, dolichol phosphate-linked oligosaccharides were isolated and characterized from Haloferax volcanii . Mannosyl (beta 1-->4)galactosyl phosphodolichol could be characterized as a main component by GC/MS permethylation analysis, mass spectrometry and 1H-NMR-spectroscopy . Furthermore, two additional components, present in lower quantities, were partially characterized and identified as a tetrasaccharyl phosphodolichol containing mannose, galactose, and rhamnose in the ratio of 2:1:1 as well as another dihexosyl phosphodolichol . Both the latter compounds contain an additional charged group . All these oligosaccharides were found to be linked to a dolichol consisting of 11 or 12 isoprene units including a saturated omega-terminal isoprene residue.

Eur J Biochem, 1997 Sep 1, 248(2), 362 - 8
Site-directed mutagenesis and halophilicity of dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii; Jolley KA et al.; A homology-modelled structure of dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii, has been generated using the crystal structure of the enzyme from Pseudomonas fluorescens . Analysis of the halophilic enzyme structure identified a potential K+-binding site comprising four co-ordinated glutamate residues (E423 and E426 from each monomer) at the subunit interface of the dimeric protein . Whilst E426 is conserved throughout non-halophilic dihydrolipoamide dehydrogenases, E423 is only present in the halophilic enzyme . Four site-directed mutations of the Haloferax dihydrolipoamide dehydrogenase have been made (E423D, E423Q, E423S, and E423A) and the recombinant mutants expressed and characterised . From an analysis of their kinetic properties, salt-dependent activities and thermal stabilities, it is concluded that this site has an important influence on the halophilicity of the enzyme . The findings support the view that the arrangement and interaction of the negatively charged amino acids are as important as the total net charge in determining the adaptation of proteins to high salt concentrations.

Appl Environ Microbiol, 1997 Oct, 63(10), 4005 - 9
Physiological basis for the high salt tolerance of Debaryomyces hansenii; Prista C et al.; The effects of KCl, NaCl, and LiCl on the growth of Debaryomyces hansenii, usually considered a halotolerant yeast, and Saccharomyces cerevisiae were compared . KCl and NaCl had similar effects on D . hansenii, indicating that NaCl created only osmotic stress, while LiCl had a specific inhibitory effect, although relatively weaker than in S . cerevisiae . In media with low K+, Na+ was able to substitute for K+, restoring the specific growth rate and the final biomass of the culture . The intracellular concentration of Na+ reached values up to 800 mM, suggesting that metabolism is not affected by rather high concentrations of salt . The ability of D . hansenii to extrude Na+ and Li+ was similar to that described for S . cerevisiae, suggesting that this mechanism is not responsible for the increased halotolerance . Also, the kinetic parameters of Rb+ uptake in D . hansenii (Vmax, 4.2 nmol mg {dry weight}-1 min-1; K(m), 7.4 mM) indicate that the transport system was not more efficient than in S . cerevisiae . Sodium (50 mM) activated the transport of Rb+ by increasing the affinity for the substrate in D . hansenii, while the effect was opposite in S . cerevisiae . Lithium inhibited Rb+ uptake in D . hansenii . We propose that the metabolism of D . hansenii is less sensitive to intracellular Na+ than is that of S . cerevisiae, that Na+ substitutes for K+ when K+ is scarce, and that the transport of K+ is favored by the presence of Na+ . In low K+ environments, D . hansenii behaved as a halophilic yeast.

J Biol Chem, 1997 Oct 10, 272(41), 25794 - 801
Isolation and characterization of salt-sensitive mutants of the moderate halophile Halomonas elongata and cloning of the ectoine synthesis genes; Canovas D et al.; The moderate halophile Halomonas elongata Deustche Sammlung fur Mikroorganismen 3043 accumulated ectoine, hydroxyectoine, glutamate, and glutamine in response to osmotic stress (3 M NaCl) . Two Tn1732-induced mutants, CHR62 and CHR63, that were severely affected in their salt tolerance were isolated . Mutant CHR62 could not grow above 0.75 M NaCl, and CHR63 did not grow above 1.5 M NaCl . These mutants did not synthesize ectoine but accumulated ectoine precursors, as shown by 13C NMR and mass spectroscopy . Mutant CHR62 accumulated low levels of diaminobutyric acid, and mutant CHR63 accumulated high concentrations of N-gamma-acetyldiaminobutyric acid . These results suggest that strain CHR62 could be defective in the gene for diaminobutyric acid acetyltransferase (ectB), and strain CHR63 could be defective in the gene for the ectoine synthase (ectC) . Salt sensitivity of the mutants at 1.5-2.5 M NaCl could be partially corrected by cytoplasmic extracts of the wild-type strain, containing ectoine, and salt sensitivity of strain CHR62 could be partially repaired by the addition of extracts of strain CHR63, which contained N-gamma-acetyldiaminobutyric acid . This is the first evidence for the role of N-gamma-acetyldiaminobutyric acid as osmoprotectant . Finally, a cosmid from the H . elongata genomic library was isolated which complemented the Ect- phenotype of both mutants, indicating that it carried at least the genes ectB and ectC of the biosynthetic pathway of ectoine.

Plasmid, 1997, 38(2), 107 - 14
Identification of plasmids in the genus Marinococcus and complete nucleotide sequence of plasmid pPL1 from Marinococcus halophilus; Louis P et al.; Several plasmids were detected in the Gram-positive halophilic eubacterium Marinococcus halophilus and in the related strain M52 . The complete nucleotide sequence (3874 bp) of one of these plasmids, pPL1, was determined . Four major open reading frames were identified . Whereas orf3 and orf4 showed no sequence similarities to known proteins, rep displayed a high sequence similarity to replication proteins of rolling circle plasmids . Upstream of this ORF, a sequence resembling the double-strand origin was detected . A region probably constituting the single-strand origin was identified . The ORF mob showed sequence similarity with Mob proteins of rolling circle plasmids . The observed characteristics suggest that pPL1 replicates according to the rolling circle mechanism.

Int J Syst Bacteriol, 1997 Oct, 47(4), 1231 - 5
Polyphasic taxonomy of Nesterenkonia halobia; Mota RR et al.; A phenotypic study has been carried out on six moderately halophilic gram-positive nonmotile cocci isolated from ponds of a saltern located in Huelva, Spain . These strains were examined for 150 morphological, physiological, biochemical, and nutritional traits and showed phenotypic characteristics similar to those of Nesterenkonia halobia (formerly Micrococcus halobius) . The guanine-plus-cytosine (G + C) content of their DNA ranged between 70 and 72 mol%, values quite similar to those described for N . halobia (71.5 mol%) . The 16S rDNA sequence analysis of one representative isolate showed that it is phylogenetically quite close to N . halobia, within the high-G + C-content gram-positive branch . DNA-DNA hybridization experiments showed a high degree of homology (72 to 100%) among the six isolates and the type strain N . halobia ATCC 21727 . All data demonstrate quite clearly that the six isolates are members of the species N . halobia . Since this species was described on the basis of a single strain isolated from unrefined solar salt, and its description is not complete (especially in the utilization of different compounds), our study contributes to a better description of the moderate halophile N . halobia.

Int J Syst Bacteriol, 1997 Oct, 47(4), 1068 - 72
Methanogenium frigidum sp . nov., a psychrophilic, H2-using methanogen from Ace Lake, Antarctica; Franzmann PD et al.; Methanogenium frigidum sp . nov . was isolated from the perennially cold, anoxic hypolimnion of Ace Lake in the Vesfold Hills of Antarctica . The cells were psychrophilic, exhibiting most rapid growth at 15 degrees C and no growth at temperatures above 18 to 20 degrees C . The cells were irregular, nonmotile coccoids (diameter, 1.2 to 2.5 microns) that occurred singly and grew by CO2 reduction by using H2 as a reductant . Formate could replace H2, but growth was slower . Acetate, methanol, and trimethylamine were not catabolized . Cells grew with acetate as the only organic compounds in the culture medium, but growth was much faster in medium also supplemented with peptones and yeast extract . The cells were slightly halophilic; good growth occurred in medium supplemented with 350 to 600 mM Na+, but no growth occurred with 100 or 850 mM Na+ . The pH range for growth was 6.5 to 7.9; no growth occurred at pH 6.0 or 8.5 . Growth was slow (maximum specific growth rate, 0.24 day-1; doubling time, 2.9 days) . This is the first report of a psychrophilic methanogen growing by CO2 reduction.

Proc Int Conf Intell Syst Mol Biol, 1997, 5, 131 - 9
RIFLE: rapid identification of microorganisms by fragment length evaluation; Hermjakob H et al.; Biological macromolecules represent a valuable source of information for the identification and phylogenetic classification of microorganisms . One of the most commonly used macromolecules for this task is the 16S rDNA . The WWW-based RIFLE system presented here supports large-scale identification tasks by comparing 16S rDNA restriction patterns to a database of restriction patterns derived from sequence databases . Computing efficiency and robustness against experimental errors are gained by employing a new distance measure for restriction patterns, the fragment length distance . Results from the application of the system to the identification of uncultured microorganisms associated with the seagrass halophila stipulacea show the reliability of the method.

Biosci Biotechnol Biochem, 1997 Aug, 61(8), 1388 - 90
Identification and NH2-terminal amino acid sequences of DnaK and groEL homologues in moderate eubacterial halophiles; Tokunaga M et al.; We have identified 2 DnaK and 3 GroEL homologues from moderately halophilic Acinetobacter, Pseudomonas, and Planococcus species by partial purification using an ATP-agarose column and by the analysis and similarity search of these NH2-terminal amino acid sequences . Although these bacteria required 1 to 2M NaCl for growth, these DnaK and GroEL homologues did not require high salt to bind to the ATP column, thus suggesting that these chaperones did not require high salts for their biochemically activities.

Biochem Biophys Res Commun, 1997 Aug 28, 237(3), 588 - 94
RNA splicing ligase activity in the archaeon Haloferax volcanii; Gomes I et al.; At least two separate enzymes, an endonuclease and a ligase, appear to be involved in tRNA splicing in halophilic archaea . We have identified and partially characterized a splicing ligase activity in cell extracts of Haloferax volcanii that can ligate deproteinized exon products generated in a separate endonuclease reaction . As in vitro transcribed partial intron-deleted derivative of H . volcanii elongator tRNA(Met) is used as substrate for the endonuclease . The ligase can also join the two exons that are independently eluted from the gels . This ligase activity is observed at a range (50 mM to 2.8 M) of monovalent cations in the assays, but is abolished when the enzyme preparations are depleted of the monovalent cations . In contrast, H . volcanii splicing endonuclease has been reported to require divalent cations and is inhibited by monovalent cations . Our endonuclease assays confirm these reports, and also show that the endonuclease is not permanently inactivated even in high monovalent cation containing extracts . The ligase activity in the extracts does not appear to require any divalent cation or exogenously added source of energy or phosphate.

FEMS Microbiol Lett, 1997 Sep 1, 154(1), 45 - 51
Identification of a promoter region on the Halomonas elongata cryptic plasmid pHE1 employing the inaZ reporter gene of Pseudomonas syringae; Tegos G et al.; A native promoter located on the cryptic plasmid pHE1 from the moderate halophile Halomonas elongata was identified employing a promoterless ice nucleation gene inaZ of Pseudomonas syringae by direct subcloning and assaying for ice nucleation activity . The presence of the promoter was verified by inserting the corresponding intact or deleted pHE1 fragment in the promoter analysis vector pKK232-8 upstream of the promoterless cat or inaZ gene . Only constructs carrying the intact pHE1 fragment gave CAT phenotype (chloramphenicol resistance) or ice nucleation activity, respectively . Comparative evaluation of the sequence analysis data of the intact and deleted fragment suggested the localization of an Escherichia coli-type promoter region.

Arch Microbiol, 1997 Oct, 168(4), 302 - 9
The electron transport system of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum . 1 . A functional and thermodynamic analysis of the respiratory chain in aerobically and photosynthetically grown cells; Moschettini G et al.; Plasma membranes isolated from cells of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum grown in light or in the dark were examined . Membranes isolated from cells grown aerobically in the dark contained three b-type and two c-type membrane-bound cytochromes with Em,7 of +180, +72 and -5 mV (561-575 nm), and +244 and +27 mV (551-540 nm), respectively . Conversely, membranes isolated from cells grown anaerobically in the light contained two b-type and five c-type haems with Em,7 of +60 and -45 mV and +290, +250, +135, -20 and -105 mV, respectively . In addition to haems of the b- and c-type, two haems of the a-type (Em,7 of +325 and +175 mV) were present only in cells grown in the dark . Four soluble cytochromes of the c type, but not cytochrome c2, along with two high-potential iron-sulfur proteins (HiPIP iso-1 and iso-2) were also identified in cells grown aerobically . Inhibitory studies showed that 85-90% of the respiratory activity was blocked by very low concentrations of cyanide, antimycin A and myxothiazol (50, 0.1 and 0.2 mM, respectively) . These results taken together were interpreted to show that the oxidative electron transport chain of Rsp . salinarum is linear, leading to a membrane-bound oxidase of the aa3 type in cells grown in the dark, while no significant cytochrome oxidase activity is catalyzed by photosynthetic membranes . These features suggest that this halophilic species is unique among the genus Rhodospirillum and that it also differs from other facultative phototrophs (e.g., Rhodobacter species) in that it does not contain either cytochrome c2 or a branched respiratory chain.

Mol Gen Genet, 1997 Aug, 255(5), 504 - 13
Molecular analysis of the replication region of the theta-replicating plasmid pUCL287 from Tetragenococcus (Pediococcus) halophilus ATCC33315; Benachour A et al.; The complete nucleotide sequence of the 8.7-kb theta-replicating plasmid pUCL287 from Tetragenococcus halophilus (formerly Pediococcus halophilus) ATCC33315 has been determined . The replication region was identified and analyzed . Its nucleotide sequence contains an untranslated region, the replication origin, followed by two open reading frames (ORFs) encoding two proteins of 311 (RepA287) and 168 (RepB287) amino acids, respectively . Evidence is presented to show that RepA287 represents the plasmid replication protein . RepB287, which is non-essential for replication, is involved in the plasmid copy-number control and segregational stability . The roles of lactococcal proteins homologous to RepB287 have not been defined so far . Nevertheless, the structural organization of the pUCL287 replication region is remarkably similar to those of well known theta-replicating lactococcal plasmids despite the absence of homology of the replication origin and of the replication protein, and this suggests that pUCL287 uses the same mechanism of replication . Nucleotide sequence comparisons show that pSMB74, a pediococcal plasmid encoding bacteriocin production, is a member of the pUCL287 replicon family.

J Bacteriol, 1997 Sep, 179(17), 5471 - 81
Osmotically induced response in representatives of halophilic prokaryotes: the bacterium Halomonas elongata and the archaeon Haloferax volcanii; Mojica FJ et al.; Haloferax volcanii and Halomonas elongata have been selected as representatives of halophilic Archaea and Bacteria, respectively, to analyze the responses to various osmolarities at the protein synthesis level . We have identified a set of high-salt-related proteins (39, 24, 20, and 15.5 kDa in H . elongata; 70, 68, 48, and 16 kDa in H . volcanii) whose synthesis rates increased with increasing salinities . A different set of proteins (60, 42, 15, and 6 kDa for H . elongata; 63, 44, 34, 18, 17, and 6 kDa for H . volcanii), some unique for low salinities, was induced under low-salt conditions . For both organisms, and especially for the haloarchaeon, adaptation to low-salt conditions involved a stronger and more specific response than adaptation to high-salt conditions, indicating that unique mechanisms may have evolved for low-salinity adaptation . In the case of H . volcanii, proteins with a typical transient response to osmotic shock, induced by both hypo- and hyperosmotic conditions, probably corresponding to described heat shock proteins and showing the characteristics of general stress proteins, have also been identified . Cell recovery after a shift to low salinities was immediate in both organisms . In contrast, adaptation to higher salinities in both cases involved a lag period during which growth and general protein synthesis were halted, although the high-salt-related proteins were induced rapidly . In H . volcanii, this lag period corresponded exactly to the time needed for cells to accumulate adequate intracellular potassium concentrations, while extrusion of potassium after the down-shift was immediate . Thus, reaching osmotic balance must be the main limiting factor for recovery of cell functions after the variation in salinity.

Biotechnol Appl Biochem, 1997 Aug, 26 ( Pt 1), 1 - 6
beta-Agarase from Pseudomonas sp . W7: purification of the recombinant enzyme from Escherichia coli and the effects of salt on its activity; Ha JC et al.; The recombinant plasmid (pJAI), harbouring the agarase gene (pjaA) of Pseudomonas sp . W7, was introduced and expressed in Escherichia coli JM83 . The agarase was purified using a combination of acetone precipitation and anion-exchange, gel-filtration and affinity chromatographies, with overall yield of 10% from the culture supernatant of E . coli JM83 (pJAI) . The purified agarase migrated as a single band (molecular mass 59 kDa) on SDS/PAGE and was found to be beta-agarase, which could hydrolyse the beta-1,4 linkage of agarose to yield neoagarotetraose as the main product . Optimal enzyme activity was at pH 7.8 and the temperature optimum spanned the broad range 20-40 degrees C . The recombinant agarase was halophilic, maximum activity being exhibited at 0.9 M NaCl . This halophilic property could improve the production of neoagaro-oligosaccharides available in a marine environment.

FEMS Microbiol Lett, 1997 Jul 15, 152(2), 321 - 6
Purification and characterization of a GroEL homologue from the moderately eubacterial halophile Pseudomonas sp . #43; Tokunaga M et al.; We have purified to apparent homogeneity and characterized a molecular chaperonin GroEL homologue (hpGroEL) from a moderately halophilic eubacterium, Pseudomonas sp . #43 . Although this halophilic bacterium requires 1-2 M NaCl for growth, hpGroEL did not require a high concentration of salt for its stability, ATPase activity and refold-promoting activity for denatured protein . The ATPase activity was even more halo-sensitive than that of GroEL from Escherichia coli . The hpGroEL protein promotes Mg(2+)-ATP-dependent refolding of urea-denatured alpha-glucosidase in the presence of E . coli-GroES, indicating that chaperonins 60 and 10 isolated from halophilic and nonhalophilic eubacteria, respectively, can cooperate with each other.

Int J Syst Bacteriol, 1997 Jul, 47(3), 832 - 6
Tetragenococcus muriaticus sp . nov., a new moderately halophilic lactic acid bacterium isolated from fermented squid liver sauce; Satomi M et al.; A total of 11 strains of moderately halophilic histamine-producing bacteria isolated from fermented squid liver sauce were studied phenotypically, genotypically, and phylogenetically . These strains are considered members of the genus Tetragenococcus based on their physiological, morphological, and chemotaxonomic characteristics . A 16S rRNA gene sequence analysis showed that these strains clustered with, but were separate from, Tetragenococcus halophilus . The results of DNA-DNA hybridization experiments indicated that the new isolates represent a new Tetragenococcus species, for which we propose the name Tetragenococcus muriaticus; strain X-1 (= JCM 10006) is the type strain of this species.

Int J Syst Bacteriol, 1997 Jul, 47(3), 818 - 24
Dethiosulfovibrio peptidovorans gen . nov., sp . nov., a new anaerobic, slightly halophilic, thiosulfate-reducing bacterium from corroding offshore oil wells; Magot M et al.; A strictly anaerobic thiosulfate-reducing bacterium was isolated from a corroding offshore oil well in Congo and was designated strain SEBR 4207T . Pure culture of the strain induced a very active pitting corrosion of mild steel, with penetration rates of up to 4 mm per year . This constitutes the first experimental evidence of the involvement of thiosulfate reduction in microbial corrosion of steel . Strain SEBR 4207T cells were vibrios (3 to 5 by 1 microns), stained gram negative, and possessed lateral flagella . Spores were not detected . Optimum growth occurred in the presence of 3% NaCl at pH 7.0 and 42 degrees C . Strain SEBR 4207T utilized peptides and amino acids, but not sugars or fatty acids . It fermented serine, histidine, and Casamino Acids, whereas arginine, glutamate, leucine, isoleucine, alanine, valine, methionine, and asparagine were only used in the presence of thiosulfate . Peptides were fermented to acetate, isobutyrate, isovalerate, 2-methylbutyrate, H2, and CO2 . The addition of either thiosulfate or sulfur but not sulfate increased peptide utilization, growth rate, and biomass; during growth, H2S was produced and a concomitant decrease in H2 was observed . The addition of either thiosulfate or sulfur also reversed H2 inhibition . 16S rRNA sequence analysis indicates that strain SEBR 4207T is distantly related to members of the genus Thermoanaerobacter (83% similarity) . Because the phenotypic and phylogenetic characteristics cannot be assigned to any described genus, strain SEBR 4207T is designated as a new species of a new genus, Dethiosulfovibrio peptidovorans gen . nov., sp . nov . Strain SEBR 4207T has been deposited in the Deutsche Sammlung von Mikroorganismen und zellkulturen GmbH (= DSM 11002).

Int J Syst Bacteriol, 1997 Jul, 47(3), 735 - 41
Bacillus salexigens sp . nov., a new moderately halophilic Bacillus species; Garabito MJ et al.; Bacillus salexigens sp . nov . is proposed based on the characteristics of six moderately halophilic, grampositive, rod-shaped strains isolated from salterns and hypersaline soils located in different geographical areas of Spain . These strains were motile, formed endospores, were strictly aerobic, were catalase and oxidase positive, and contained peptidoglycan of the meso-diamlnopimelic acid type in their vegetative cell walls . The DNA base compositions of these strains ranged from 36.3 to 39.5 mol%, and these organisms constitute a homology group with levels of DNA-DNA homology ranging from 73 to 100% . The 16S rRNA sequence of strain C-20MoT, which was used as the representative strain of these isolates, groups with the 16S rRNA sequences of members of the genus Bacillus, and the highest level of similarity is 95.4% . The type strain is strain C-20Mo (= ATCC 700290 = DSM 11483 = CCM 4646).

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