Halophilic bacterium

Gene, 1997 Jun 3, 191(2), 225 - 32
Porin from the halophilic species Ectothiorhodospira vacuolata: cloning, structure of the gene and comparison with other porins; Wolf E et al.; The gene coding for the anion-specific porin of the halophilic eubacterium Ectothiorhodospira (Ect.) vacuolata was cloned and sequenced, the first such gene so analyzed from a purple sulfur bacterium . It encodes a precursor protein consisting of 374 amino acid (aa)-residues including a signal peptide of 22-aa residues . Comparison with aa sequences of porins from several other members of the Proteobacteria revealed little homology . Only two regions showed local homology with the previously sequenced porins of Neisseria species, Comamonas acidovorans, Bordetella pertussis, Alcaligenes eutrophus, and Burkholderia cepacia . Genomic Southern blot hybridization studies were carried out with a probe derived from the 5' end of the gene coding for the porin of Ect . vacuolata . Two related species, Ect . haloalkaliphila and Ect . shaposhnikovii, exhibited a clear signal, while the extremely halophilic bacterium Halorhodospira (Hlr.) halophila (formerly Ect . halophila) did not show any cross-hybridization even at low stringency . This result is in good accordance with a recently proposed reassignment within the family Ectothiorhodospiraceae, which included the separation of the extremely halophilic species into the new genus Halorhodospira.

FEMS Microbiol Rev, 1997 Jun, 20(1-2), 25 - 46
Biochemistry of S-layers; Messner P et al.; During evolution prokaryotes have developed different envelope structures exterior to the cell wall proper . Among these surface components are regularly arranged S-layers and capsules . The structural characterization and the detailed chemical analysis of these surface molecules is a prerequisite to understand their biosynthesis and functional role(s) at the molecular level . Of particular interest are the glycosylated S-layer proteins which belong to the first prokaryotic glycoproteins ever described . Their characterization was performed on strains belonging to the thermophilic Bacillaceae and included structural studies and experiments to learn about the pathways for the glycan biosynthesis of S-layer glycoproteins . As an example for non-glycosylated S-layer proteins those of Lactobacillus helveticus strains are described in detail . Recently, a novel type of bacterial glycoconjugate was observed in the cell envelope of the extremely halophilic archaeon Natronococcus occultus which consists of a glycosylated polyglutamyl polymer . Beside the conventional biochemical techniques for the analysis new sophisticated instrumental methods such as X-ray photoelectron spectroscopy and matrix-assisted laser desorption ionization or electrospray ionization mass spectrometry have been introduced for the analysis of the protein and glycan portions of these cell surface macromolecules.

J Bacteriol, 1997 Jun, 179(11), 3632 - 8
3-hydroxy-3-methylglutaryl coenzyme A reductase of Sulfolobus solfataricus: DNA sequence, phylogeny, expression in Escherichia coli of the hmgA gene, and purification and kinetic characterization of the gene product; Bochar DA et al.; The gene (hmgA) for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) from the thermophilic archaeon Sulfolobus solfataricus P2 was cloned and sequenced . S . solfataricus HMG-CoA reductase exhibited a high degree of sequence identity (47%) to the HMG-CoA reductase of the halophilic archaeon Haloferax volcanii . Phylogenetic analyses of HMG-CoA reductase protein sequences suggested that the two archaeal genes are distant homologs of eukaryotic genes . The only known bacterial HMG-CoA reductase, a strictly biodegradative enzyme from Pseudomonas mevalonii, is highly diverged from archaeal and eukaryotic HMG-CoA reductases . The S . solfataricus hmgA gene encodes a true biosynthetic HMG-CoA reductase . Expression of hmgA in Escherichia coli generated a protein that both converted HMG-CoA to mevalonate and cross-reacted with antibodies raised against rat liver HMG-CoA reductase . S . solfataricus HMG-CoA reductase was purified in 40% yield to a specific activity of 17.5 microU per mg at 50 degrees C by a sequence of steps that included heat treatment, ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography . The final product was homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The substrate was (S)- not (R)-HMG-CoA; the reductant was NADPH not NADH . The Km values for HMG-CoA (17 microM) and NADPH (23 microM) were similar in magnitude to those of other biosynthetic HMG-CoA reductases . Unlike other HMG-CoA reductases, the enzyme was stable at 90 degrees C and was optimally active at pH 5.5 and 85 degrees C.

Arch Biochem Biophys, 1997 May 15, 341(2), 267 - 72
Identification of proteolipid from an extremely halophilic archaeon Halobacterium salinarum as an N,N'-dicyclohexyl-carbodiimide binding subunit of ATP synthase; Ihara K et al.; ATP synthesis in an extremely halophilic archaeon, Halobacterium salinarum, was inhibited by N-cyclohexyl-N'-{4-(dimethylamino)-alpha-naphthyl}carbodiimide (NCD-4), a fluorescent analog of N,N'-dicyclohexylcarbodiimide (DCCD) . By tracing the fluorescent signal, a hydrophobic 8-kDa protein (proteolipid) was purified from the halobacterial membrane as one of the most DCCD-reactive proteins and its N-terminal amino acid sequence was determined . The gene encoding the proteolipid was found in the region upstream of the genes encoding the two major subunits of halobacterial A-type ATPase {K . Ihara and Y.Mukohata (1991) Arch . Biochem . Biophys . 286, 111-116} . Halobacterial proteolipid was more similar in size to the proteolipid of F-type ATPase than that of V-type ATPase . However, multiple amino acid sequence alignment of proteolipids showed a higher degree of relatedness between V-type and A-type ATPase proteolipids . Together with the recent finding of a triplicate proteolipid encoding gene from the methanogenic archaeon Methanococcus jannaschii {C . J . Bult et al . (1996) Science 273, 1058-1073}, proteolipids from archaea seem to have diverse characteristics in comparison with those from eubacteria or from eukaryotes.

J Biochem (Tokyo), 1997 May, 121(5), 876 - 80
Functional expression and site-directed mutagenesis of photoactive yellow protein; Mihara K et al.; The gene encoding photoactive yellow protein (PYP) was isolated from Ectothiorhodospira halophila, and a high-level expression system for PYP was constructed in Escherichia coli . The molecular weight and the absorption spectrum of PYP expressed in E . coli were identical with those of the native PYP isolated from E . halophila . The amino acid residues which might interact with the chromophore (Tyr42, Glu46, Thr50, Arg52, and Cys69) were mutated by site-directed mutagenesis and the absorption spectra of these mutants were examined to study the chromophore/protein interaction in PYP . The former three substitutions (Y42F, E46Q, and T50V) brought about red-shifts of the absorption spectra, but the substitution of Arg52 (R52Q) brought about no change and that of Cys69 (C69S) led to no formation of pigments . These results suggest that Tyr42, Glu46, and Thr50 strongly interact with the chromophore, while Arg52 does not contribute the color tuning of PYP.

J Appl Microbiol, 1997 May, 82(5), 557 - 66
Pril-ampicillin-dextrin-ethanol agar for the isolation and quantification of Aeromonas spp . from polluted environmental waters; Imziln B et al.; Several selective media were evaluated for their suitability for the isolation and quantification of mesophilic Aeromonas species from naturally polluted samples . Satisfactory recoveries were obtained with most of them but only when densities of background microflora were low . When analysed samples were from highly polluted waters, results were inconsistent because they did not give quantitative recovery of mesophilic aeromonads or they did not permit ready differentiation of Aeromonas species from the competitive bacteria . A new medium was developed on the basis of the combination of some positive aspects of several published media, pril-ampicillin-dextrin-ethanol (PADE) agar . The medium employs dextrin (Merck 3006) as a fermentable carbohydrate and pril, ampicillin and ethanol as inhibitory substances . Recovery on PADE agar from suspensions of 15 tested strains of Aeromonas prepared from pure cultures was excellent . The confirmation rate of typical colonies designated Aeromonas spp . isolated from polluted samples exceeded 90% . Recoveries of stressed aeromonad strains on both PADE agar and a non-selective medium (TSA) did not show any significant difference (P > 0.05) . PADE agar was more reliable for quantitative recovery of mesophilic aeromonads than the other selective media because of its characteristics: (i) inhibition of the swarming of Proteus, (ii) good reduction of the background, (iii) inhibition of the over growth of Klebsiella spp., (iv) absence of NaCl makes it unfavourable for the growth of halophilic vibrios, (v) combination of two pH indicators permitted a very easy differentiation between Aeromonas colonies and the competitive microflora . The medium can also be used for isolation of aeromonads from various sources by membrane filtration.

J Bacteriol, 1997 May, 179(10), 3146 - 53
2-Sulfotrehalose, a novel osmolyte in haloalkaliphilic archaea; Desmarais D et al.; A novel 1-->1 alpha-linked glucose disaccharide with sulfate at C-2 of one of the glucose moieties, 1-(2-O-sulfo-alpha-D-glucopyranosyl)-alpha-D-glycopyranose, was found to be the major organic solute accumulated by a Natronococcus sp . and several Natronobacterium species . The concentration of this novel disaccharide, termed sulfotrehalose, increased with increasing concentrations of external NaCl, behavior consistent with its identity as an osmolyte . A variety of noncharged disaccharides (trehalose, sucrose, cellobiose, and maltose) were added to the growth medium to see if they could suppress synthesis and accumulation of sulfotrehalose . Sucrose was the most effective in suppressing biosynthesis and accumulation of sulfotrehalose, with levels as low as 0.1 mM being able to significantly replace the novel charged osmolyte . Other common osmolytes (glycine betaine, glutamate, and proline) were not accumulated or used for osmotic balance in place of the sulfotrehalose by the halophilic archaeons.

Arch Microbiol, 1997 May, 167(5), 259 - 68
Gas vesicle formation in halophilic Archaea; Pfeifer F et al.; Gas vesicles are intracellular, microbial flotation devices that consist of mainly one protein, GvpA . The formation of halobacterial gas vesicles occurs along a complex pathway involving 14 different gvp genes that are clustered in a genomic region termed the "vac region" . Various vac regions found in Halobacterium salinarum (p-vac and c-vac), Haloferax mediterranei (mc-vac), and Natronobacterium vacuolatum (nv-vac) have been investigated . Except for the latter vac region, the arrangement of the gvp genes is identical . Single gvp genes have been mutated to study the effect on gas vesicle synthesis in transformants and to determine their possible function . Each vac region exhibits a characteristic transcription pattern, and regulatory steps have been observed at the DNA, RNA, and protein level, indicating a complex regulatory network acting during gas vesicle gene expression.

Microbiology, 1997 Apr, 143 ( Pt 4), 1141 - 9
Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli; Louis P et al.; The genes of the biosynthetic pathway of ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) from the Gram-positive moderate halophile Marinococcus halophilus were cloned by functional expression in Escherichia coli . These genes were not only expressed, but also osmoregulated in E . coli, as demonstrated by increasing cytoplasmic ectoine concentration in response to medium salinity . Sequencing of a 4.4 kb fragment revealed four major ORFs, which were designated ectA, ectB, ectC and orfA . The significance of three of these genes for ectoine synthesis was proved by sequence comparison with known proteins and by physiological experiments . Several deletion derivatives of the sequenced fragment were introduced into E . coli and the resulting clones were investigated for their ability to synthesize ectoine or one of the intermediates in its biosynthetic pathway . It was demonstrated that ectA codes for L-2,4-diaminobutyric acid acetyltransferase, ectB for L-2,4-diaminobutyric acid transaminase and ectC for L-ectoine synthase . A DNA region upstream of ectA was shown to be necessary for the regulated expression of ectoine synthesis in response to the osmolarity of the medium.

Int J Syst Bacteriol, 1997 Apr, 47(2), 515 - 21
Desulfovibrio profundus sp . nov., a novel barophilic sulfate-reducing bacterium from deep sediment layers in the Japan Sea; Bale SJ et al.; Several strains of a strictly anaerobic, vibrio-shaped or sigmoid, sulfate-reducing bacterium were isolated from deep marine sediments (depth, 80 and 500 m) obtained from the Japan Sea (Ocean Drilling Program Leg 128, site 798B) . This bacterium was identified as a member of the genus Desulfovibrio on the basis of the presence of desulfoviridin and characteristic phospholipid fatty acids (iso 17:1 omega 7 and iso 15:0), the small number of growth substrates utilized (lactate, pyruvate, and hydrogen), and 16S rRNA gene sequence analysis data . Based on data for 16S rRNA sequences (1,369 bp), all of the Japan Sea strains were identical to each other and were most closely related to Desulfovibrio salexigens and less closely related to Desulfovibrio desulfuricans (levels of similarity, 91 and 89.6%, respectively) . There were, however, considerable phenotypic differences (in temperatures, pressures, and salinities tolerated, growth substrates, and electron donors) between the Japan Sea isolates and the type strains of previously described desulfovibrios, as well as important differences among the Japan Sea isolates . The Japan Sea isolates were active (with sulfide production) over a wide temperature range (15 to 65 degrees C) and a wide sodium chloride concentration range (0.2 to 10%) (moderate halophile), and they were barophiles that were active at pressures up to about 40 MPa (400 atm) . The optimum pressures for activity corresponded to the calculated pressures at the depths from which the organisms were isolated (for isolates obtained at depths of 80 and 500 m the optimum activities occurred at 10 and 15 MPa, respectively {100 and 150 atm, respectively}) . This confirms that the organisms came from deep sediments and indicates that they are well-adapted for deep sediment conditions, which is consistent with other characteristics (utilization of hydrogen, fermentation, and utilization of ferric iron and organic sulfonates as electron acceptors) . We propose that Japan Sea isolate 500-1 is the type strain of a new species, Desulfovibrio profundus.

Presse Med, 1997 Mar 8, 26(7), 316 - 8
{Primary Vibrio vulnificus septicemia . 1st documented case in the French West Indies}; Lamaury I et al.; BACKGROUND: Vibrio vulnificus is a non-choleric halophilic vibrion widely distributed in marine environments . Contamination in humans is uncommon except in coastal areas of the United States and Asia . We report the first documented case in the French West Indies . CASE REPORT: A 57-year-old native with alcoholic cirrhosis was hospitalized for septic shock . The infectious syndrome began suddenly a few hours earlier with fever, diarrhea, and intense pain in the calf muscles . In the absence of a suspected agent, a wide spectrum antibiotic was prescribed . On day 3, bullae developed over the legs and progressed, despite early surgical debridement, to bilateral rapidly extensive necrosing cellulitis . An above the knee amputation was required but did not prevent death on day 9 due to irreversible multiple organ failure . Blood cultures were positive for V . vulnificus . DISCUSSION: Primary septicemia due to V . vulnificus is mainly observed in subjects with an underlying liver disease and usually occurs after ingestion of contamined raw halieutic products such as oysters . The clinical presentation is characteristic with secondary necrotic ulcerations on the lower limbs . Improvement in the extremely poor prognosis of these infections depends on early initiation of an effective antibiotic with wide exeresis of necrotic tissue . Physicians should be aware of this severe infection despite its low frequency.

J Biol Chem, 1997 Mar 7, 272(10), 6261 - 9
Characterization and subunit structure of the ATP synthase of the halophilic archaeon Haloferax volcanii and organization of the ATP synthase genes; Steinert K et al.; The archaeal ATPase of the halophile Haloferax volcanii synthesizes ATP at the expense of a proton gradient, as shown by sensitivity to the uncoupler carboxyl cyanide p-trifluoromethoxyphenylhydrazone, to the ionophore nigericin, and to the proton channel-modifying reagent N,N'-dicyclohexylcarbodiimide . The conditions for an optimally active ATP synthase have been determined . We were able to purify the enzyme complex and to identify the larger subunits with antisera raised against synthetic peptides . To identify additional subunits of this enzyme complex, we cloned and sequenced a gene cluster encoding five hydrophilic subunits of the A1 part of the proton-translocating archaeal ATP synthase . Initiation, termination, and ribosome-binding sequences as well as the result of a single transcript suggest that the ATPase genes are organized in an operon . The calculated molecular masses of the deduced gene products are 22 . 0 kDa (subunit D), 38.7 kDa (subunit C), 11.6 kDa (subunit E), 52.0 kDa (subunit B), and 64.5 kDa (subunit A) . The described operon contains genes in the order D, C, E, B, and A; it contains no gene for the hydrophobic, so-called proteolipid (subunit c, the proton-conducting subunit of the A0 part) . This subunit has been isolated and purified; its molecular mass as deduced by SDS-polyacrylamide gel electrophoresis is 9.7 kDa.

Zh Mikrobiol Epidemiol Immunobiol, 1997 Mar-Apr, (2), 11 - 4
{Phagovars of halophilic vibrios}; Khaitovich AB et al.; The results of the phage typing of 164 halophilic vibrios revealed the most frequent combinations of lysing monophages, denoted by numbers: 1 (A, B, C, D), 2 (B, C, D), 3 (B, C) and 4 (C) . In accordance with the proposed scheme, the phage typing of 398 cultures from different ecosystems was carried out . Phagovar was determined in 77.1% of the cultures . Most frequently occurred phagovars 1 (31.9%), 4 (15.8%), 3 (6%), and 2 (3.7%) . Their proportion was 61% . 11 other phage combination causing the lysis of the cultures constituted 16.1%; 22.9% of the cultures could not be types . The use of the proposed scheme of phage typing permitted the determination of the temporal, regional, ecosystemic features of the circulation of halophilic vibrios in different ecosystems and regions, which was important for epidemiological analysis.

Microbiol Mol Biol Rev, 1997 Mar, 61(1), 90 - 104
Evolutionary divergence and salinity-mediated selection in halophilic archaea; Dennis PP et al.; Halophilic (literally salt-loving) archaea are a highly evolved group of organisms that are uniquely able to survive in and exploit hypersaline environments . In this review, we examine the potential interplay between fluctuations in environmental salinity and the primary sequence and tertiary structure of halophilic proteins . The proteins of halophilic archaea are highly adapted and magnificently engineered to function in an intracellular milieu that is in ionic balance with an external environment containing between 2 and 5 M inorganic salt . To understand the nature of halophilic adaptation and to visualize this interplay, the sequences of genes encoding the L11, L1, L10, and L12 proteins of the large ribosome subunit and Mn/Fe superoxide dismutase proteins from three genera of halophilic archaea have been aligned and analyzed for the presence of synonymous and nonsynonymous nucleotide substitutions . Compared to homologous eubacterial genes, these halophilic genes exhibit an inordinately high proportion of nonsynonymous nucleotide substitutions that result in amino acid replacement in the encoded proteins . More than one-third of the replacements involve acidic amino acid residues . We suggest that fluctuations in environmental salinity provide the driving force for fixation of the excessive number of nonsynonymous substitutions . Tinkering with the number, location, and arrangement of acidic and other amino acid residues influences the fitness (i.e., hydrophobicity, surface hydration, and structural stability) of the halophilic protein . Tinkering is also evident at halophilic protein positions monomorphic or polymorphic for serine; more than one-third of these positions use both the TCN and the AGY serine codons, indicating that there have been multiple nonsynonymous substitutions at these positions . Our model suggests that fluctuating environmental salinity prevents optimization of fitness for many halophilic proteins and helps to explain the unusual evolutionary divergence of their encoding genes.

Biochim Biophys Acta, 1997 Feb 8, 1337(2), 276 - 86
Purification and analysis of an extremely halophilic beta-galactosidase from Haloferax alicantei; Holmes ML et al.; As a first step in the development of a reporter system for gene expression in halophilic archaea, a beta-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2) . An overproducing mutant was first isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate . Cytoplasmic extracts of the mutant contained 25-fold higher enzyme levels than the parent . Purification of the active enzyme was greatly facilitated by the ability of sorbitol to stabilise enzyme activity in the absence of salt, which allowed conventional purification methods (e.g., ion-exchange chromatography) to be utilised . The enzyme was optimally active at 4 M NaCl and was estimated to be 180 +/- 20 kDa in size, consisting of two monomers (each 78 +/- 3 kDa) . It cleaves several different beta-galactoside substrates such as ONP-Gal, X-Gal and lactulose, but not lactose, and also has beta-D-fucosidase activity . No beta-glucosidase, beta-arabinosidase or beta-xylosidase activity could be detected . The amino-acid sequence at the N-terminus and of four proteolytic products has been determined.

Mol Microbiol, 1997 Feb, 23(4), 791 - 7
Construction and analysis of a recombination-deficient (radA) mutant of Haloferax volcanii; Woods WG et al.; By deleting the radA open reading frame of an extreme halophile, Haloferax volcanii, we created and characterized a recombination-deficient archaeon . This strain, Hf . volcanii DS52, has no detectable DNA recombination, is more sensitive to DNA damage by UV light and ethylmethane sulfonate, and has a slower growth rate than the wild type . These characteristics are similar to those observed in recombination mutants of Eukarya and Bacteria, and show that the radA gene belongs in the recA/RAD51 family by function as well as sequence homology . In addition, strain DS52 was not transformable by plasmids pWL102 or pUBP2 (which contain pHV2 and pHH1 replicons, respectively), although it was readily transformed by plasmids containing a pHK2 replicon, indicating a role for radA in the maintenance or replication of some halobacterial plasmids . Despite its slower growth rate, Hf . volcanii DS52 was still easy to culture and transform, and should be suitable for use in studies where a recombination-deficient background is desired.

Naunyn Schmiedebergs Arch Pharmacol, 1997 Feb, 355(2), 150 - 60
Expression of beta 2-adrenoceptors in halobacteria; Sohlemann P et al.; Halobacteria are halophilic representatives of the recently defined domain, the Archaea . Halobacterium salinarium belongs to this group of microorganisms and contains large amounts of bacteriorhodopsin in its membrane . Bacteriorhodopsin is a seven-transmembrane protein that consists of bacterio-opsin (BO), and the chromophore retinal, which is covalently attached to BO . We have investigated whether the expression machinery for BO can be utilized for synthesis of the human beta 2-adrenoceptor (beta 2-AR), a protein with a similar seven-transmembrane-helix topology . An expression vector for BO synthesis was modified to express beta 2-ARs under the control of BO regulatory clements in H . salinarium . Homologous recombination into the genome was verified by polymerase chain reactions . Northern blots revealed transcripts of the calculated size and significant amounts of epitope-tagged beta 2-ARs were detected in Western blots . However, binding of the beta-AR antagonist 125I-cyanopindolol revealed low levels of functional receptors, and the ligand binding properties of these receptors were altered when compared to native receptors . Expression of chimeras containing larger amino terminal portions of BO did not result in higher receptor levels . Expression of beta 2-AR in Haloferax volcanii, another member of halobacteria, was achieved with a vector carrying the ferredoxin promoter . The levels of functional receptor as determined by 125I-cyanopindolol binding were 180 fmol/mg protein . The beta-AR ligands isoprenaline and propranolol showed affinities expected for functional beta 2-ARs . Thus, functional human beta 2-ARs were expressed in halobacteria, constituting a first approach for expression of a eukaryotic protein in the domain of Archaea.

FEMS Microbiol Lett, 1997 Feb 1, 147(1), 81 - 8
Haloanaerobium congolense sp . nov., an anaerobic, moderately halophilic, thiosulfate- and sulfur-reducing bacterium from an African oil field; Ravot G et al.; A strictly anaerobic, moderately halophilic, Gram-negative, non-motile rod-shaped bacterium was isolated from an oil-well head sample of an offshore Congolese oil field . The strain, designated SEBR 4224T (T = type strain), grew optimally at 42 degrees C and pH 7.0 in a complex medium containing 10% NaCl with a generation time of 2.5 h . Strain SEBR 4224T grew on a range of carbohydrates including fructose, galactose, D-glucose, maltose, D-mannose, D-ribose, sucrose, and trehalose . Yeast extract and/or bio-Trypcase was required for growth on carbohydrates and could not be replaced with amino acids and/or vitamins . The end-products from glucose fermentation were acetate, H2, and CO2 . Thiosulfate and elemental sulfur were used as electron acceptors . Thiosulfate improved carbohydrate utilization and biomass yields . The G + C content of the isolate was 34 mol% . Ribosomal 16S rRNA sequence analysis showed that strain SEBR 4224T is a new member of the genus Haloanaerobium . The lack of DNA homology with H . acetoethylicum, its closest relative, as determined by DNA-DNA hybridization supports the designation of strain SEBR 4224T as a new species, Haloanaerobium congolense sp . nov . The type strain is SEBR 4224T (= DSM 11287).

J Bacteriol, 1997 Feb, 179(4), 1180 - 5
Biochemical and serological evidence for an RNase E-like activity in halophilic Archaea; Franzetti B et al.; Endoribonuclease RNase E appears to control the rate-limiting step that mediates the degradation of many mRNA species in bacteria . In this work, an RNase E-like activity in Archaea is described . An endoribonucleolytic activity from the extreme halophile Haloarcula marismortui showed the same RNA substrate specificity as the Escherichia coli RNase E and cross-reacted with a monoclonal antibody raised against E . coli RNase E . The archaeal RNase E activity was partially purified from the extreme halophilic cells and shown, contrary to the E . coli enzyme, to require a high salt concentration for cleavage specificity and stability . These data indicate that a halophilic RNA processing enzyme can specifically recognize and cleave mRNA from E . coli in an extremely salty environment (3 M KCI) . Having recently been shown in mammalian cells (A . Wennborg, B . Sohlberg, D . Angerer, G . Klein, and A . von Gabain, Proc . Natl . Acad . Sci . USA 92:7322-7326, 1995), RNase E-like activity has now been identified in all three evolutionary domains: Archaea, Bacteria, and Eukarya . This strongly suggests that mRNA decay mechanisms are highly conserved despite quite different environmental conditions.

Curr Microbiol, 1997 Feb, 34(2), 85 - 90
Salt-Sensitive and Auxotrophic Mutants of Halomonas elongata and H . meridiana by Use of Hydroxylamine Mutagenesis
Cánovas D, Vargas C, Ventosa A, Nieto JJ.
The killing action and induced mutagenicity inhydroxylamine (HA)-treated cells of two moderately halophilic species of thegenus Halomonas, H . elongata and H . meridiana, wereinvestigated . A high sensitivity of H . elongata and especially ofH . meridiana to HA was found . The efficiency of the mutagenicityobtained with the HA treatment was tested at different salinities . Optimalexperimental conditions for HA mutagenesis of these two moderate halophileswere determined . A clear, efficient mutagenicity of both H . elongataand H . meridiana after HA mutagenesis was achieved . The optimalprocedures yielded a number of useful auxotrophic mutants of H.elongata as well as different salt-sensitive mutants of H.elongata and H . meridiana for further studies . Some of theselatter mutants appeared to be affected in the synthesis of compatiblesolutes.

Arch Microbiol, 1997 Jan 29, 167(1), 11 - 8
Chromatium glycolicum sp . nov., a moderately halophilic purple sulfur bacterium that uses glycolate as substrate
Caumette P, Imhoff JF, Suling J, Matheron R.
From the microbial mats that develop in Solar Lake, a new purple sulfur bacterium was isolated . This strain (Chromatium strain SL 3201) was morphologically similar to Chromatium gracile and Chromatium minutissimum . Chromatium SL 3201 was found to be a moderate halophile with a growth range between 2 and 20% NaCl (optimum 4-5% NaCl) and was able to grow photo-organotrophically using glycolate and glycerol . It is the first described phototrophic sulfur bacterium able to use glycolate . According to NaCl requirements and utilization of organic compounds, the strain is not related to any known species of the genus Chromatium . On the basis of its 16S rRNA gene sequence, it clusters with other Chromatium species and is most similar to Chromatium salexigens and Chr . gracile, but it is sufficiently separated to be considered as a new species of the genus . It is, therefore, described as Chromatium glycolicum sp . nov.

Eur J Biochem, 1997 Jan 15, 243(1-2), 141 - 50
Seryl-tRNA synthetase from the extreme halophile Haloarcula marismortui--isolation, characterization and sequencing of the gene and its expression in Escherichia coli; Taupin CM et al.; The seryl-tRNA synthetase from the extreme halophilic archaebacterium Haloarcula marismortui, belonging to the group Euryarchaeota, has been purified and its hyperhalophilic behavior demonstrated by activity and stability tests in KCl, NaCl and MgCl2 solutions . Although the natural external environment of this archaebacterium is rich in sodium ions and poor in potassium ions, the converse being the case in the bacterial cytosol . there is no large significant difference in activity and stability in vitro of the enzyme between solutions of NaCl and KCl . Low, but not high, concentrations of MgCl2 stabilize the enzyme . The enzyme aminoacylates tRNA from Escherichia coli even under the high salt conditions of the assay . A fluorescence study indicated that low salt denaturation of the hyperhalophilic enzyme is a biphasic process . The hyperhalophilic enzyme demonstrated immunological reactivity with antisera against the catalytic domain of the homologous E . coli enzyme . The gene coding for the H . marismortui enzyme has been isolated and sequenced . The derived amino acid sequence is the first of a hyperhalophilic aminoacyl-tRNA synthetase . The wild-type gene and a mutant gene with a deletion of the halophile-specific insertion were expressed in E . coli using the T7 RNA polymerase and the Thiofusion expression systems . None of the expressed proteins were enzymically active . A structural model has been produced by comparison with other seryl-tRNA synthetases which illustrates the high negative-charge density of the surface of the hyperhalophilic enzyme.

Protein Sci, 1997 Jan, 6(1), 156 - 61
3-Hydroxy-3-methylglutaryl-coenzyme A reductase of Haloferax volcanii: role of histidine 398 and attenuation of activity by introduction of negative charge at position 404; Bischoff KM et al.; Mutant 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases of the halophilic archaeon Haloferax volcanii were constructed to test the proposed mechanism that phosphorylation downregulates the activity of higher eukarya HMG-CoA reductases via charge-charge interaction with the active site histidine . To first verify the sequence-based inference that His 398 is the catalytic histidine of the H . volcanii enzyme, enzyme H398Q was constructed, purified, and assayed for catalysis of three reactions: {1} reductive deacylation of HMG-CoA, {2} reduction of mevaldehyde, and {3} oxidative acylation of mevaldehyde . Enzyme H398Q had low activity for catalysis of reaction {1} or {3}, but readily catalyzed mevaldehyde reduction . By analogy to hamster HMG-CoA reductase, we conclude that His 398 is the active site histidine . Mutant forms of the 403-residue H . volcanii enzyme were constructed to model phosphorylation and infer whether attenuated activity involved interaction with His 398 . Chimeric H . volcanii-hamster enzymes constructed in an effort to create an active, phosphorylatable chimeric enzyme were inactive or not phosphorylated . We therefore added Asp at position 404 to mimic the introduction of negative charge that would accompany phosphorylation . Enzyme 404D/H398Q was inactive for reaction {1} or {3}, but catalyzed reaction {2} at 35% the wild-type rate . These observations are consistent with the model that attenuation of catalytic activity results from an ionic interaction between the imidazolium cation of His 398 and the carboxylate anion of Asp 404.

Int J Syst Bacteriol, 1997 Jan, 47(1), 122 - 6
Thermonema rossianum sp . nov., a new thermophilic and slightly halophilic species from saline hot springs in Naples, Italy; Tenreiro S et al.; Six slightly halophilic, thermophilic bacterial strains were isolated from saline hot springs along the Bay of Naples, Italy . These strains produce bright yellow colonies and have a filamentous morphology and an optimum growth temperature of about 60 degrees C . Lipid composition and 16S ribosomal DNA sequence analyses showed that these strains belong to the genus Thermonema, a member of the cytophaga-flavobacter-bacteroides phylum . Growth was observed in medium containing 1 to 3% NaCl . The DNA G + C content was 50.9 mol% . DNA-DNA hybridization studies showed that these strains represent a new species of the genus Thermonema . We propose that strain NR-27T (T = type strain) and the other strains represent a new species, Thermonema rossianum . Strain NR-27 (= DSM 10300) is the type strain of this species.

Int J Syst Bacteriol, 1997 Jan, 47(1), 73 - 7
Haloarcula argentinensis sp . nov . and Haloarcula mukohataei sp . nov., two new extremely halophilic archaea collected in Argentina; Ihara K et al.; Strains arg-1T (T = type strain) and arg-2T, two new strains of extremely halophilic archaea, were isolated from the soils of the Argentine salt flats . The taxonomic features of arg-1T were similar to, but distinct from, those of the type strain of Haloarcula vallismortis and other Haloarcula species . On the 16S rRNA phylogenetic tree, strain arg-1T formed a cluster together with Haloarcula species . Strain arg-2T differed in its glycolipid composition but still was more closely related to the genus Haloarcula than to other established genera . We propose that strain arg-1T be classified as a member of a new species, Haloarcula argentinensis, and that strain arg-2T be classified as a member of Haloarcula mukohataei sp . nov., although arg-2T may belong to a new genus or a subgenus of the genus Haloarcula . The type strain of H . argentinensis is strain arg-1 (= JCM 9737), and the type strain of H . mukohataei is strain arg-2 (= JCM 9738).

J Bacteriol, 1997 Jan, 179(2), 548 - 51
Isolation, sequence, and expression of the gene encoding halocin H4, a bacteriocin from the halophilic archaeon Haloferax mediterranei R4; Cheung J et al.; The first gene to encode a haloarchaeal bacteriocin (halocin H4) has been cloned and sequenced from Haloferax mediterranei R4 . Both the signal sequence in the halocin H4 preprotein and the monocistronic halH4 gene have some unusual features . The physiology of halH4 expression reveals that although halH4 transcripts are present at low basal levels during exponential growth, halocin H4 activity first appears as the culture enters stationary phase . As halocin activity levels increase, so do transcript levels, but then activity levels decrease precipitously while transcript levels remain elevated.

J Inorg Biochem, 1997 Jan, 65(1), 35 - 43
Mn-superoxide dismutase from the halophilic halotolerant bacterium Ba1--isolation and active site spectroscopic studies; Yorkovsky Y et al.; The superoxide-dismutase (SOD) enzyme, isolated from the halophilic halotolerant bacterium Ba1, was found to be a dimer with a molecular weight of 40 kD and a subunit weight of 23.5 kD . The partial N-terminal sequence showed significant homology to SODs isolated from various sources . Metal analysis showed that SOD from Ba1 contains manganese and iron with the following stoichiometries: 0.9 +/- 0.4 Mn/dimer and 0.6 +/- 0.2 Fe/dimer . Two bands were obtained by isoelectric-focusing, at pI of 4.45 and at 4.40 . Native SOD from Ba1 at room temperature was ESR silent . An ESR spectrum of hydrated Mn(II) was obtained from denaturated enzyme . Native enzyme cooled to 97 K showed an ESR spectrum identified as being due to Fe(III) . The spectrum was pH-independent . SOD from Ba1 was not inactivated by H2O2 . On the basis of these observations, SOD from Ba1 was characterized as MnSOD . The excitation fluorescence spectrum of SOD from Ba1 showed four main peaks in the visible region . The effects on the spectra of KSCN, NaN3, NaF, and ascorbate were examined . Measurements of H2(17)O-nmr relaxation times T1 and T2, for solutions containing E . coli MnSOD and FeSOD, showed no paramagnetic contribution . These results support the assumption that the water molecule at the active site is strongly bound.

Carbohydr Res, 1996 Dec 13, 295, 147 - 56
The structure of the exopolysaccharide produced by the halophilic Archaeon Haloferax mediterranei strain R4 (ATCC 33500); Parolis H et al.; The halophilic Archaeon Haloferax mediterranei exudes into the growth medium a high molecular weight sulfated polysaccharide . The structure of the repeating unit of this polymer was determined by a combination of glycose, methylation, and sulfate analysis, periodate oxidation, and 1D and 2D NMR spectroscopic analysis of the native and periodate-oxidised/reduced polysaccharides . The location of the sulfate group was established from the 1H and 13C NMR data . The structure of the repeating unit of the polysaccharide may be written as {formula: see text}

J Mol Biol, 1996 Dec 6, 264(3), 567 - 84
Ectothiorhodospira halophila ferrocytochrome c551: solution structure and comparison with bacterial cytochromes c; Bersch B et al.; The solution structure of the Ectothiorhodospira halophila ferrocytochrome c551 has been determined . This molecule belongs to a separate class of small bacterial cytochromes c for which no 3D structure has been reported so far . It is characterized by a very low redox potential (58 mV) and is isolated from the periplasm of halophilic purple phototrophic bacteria . For the 78 residue protein, 1445 NOE derived distance constraints were used in a combined simulated annealing/restrained molecular dynamics calculation . The final ensemble of 37 structures presents a backbone r.m.s.d . of less than 0.5 A compared to the mean structure . The physical viability of these structures was investigated by subjecting eight of them to a constraint free molecular dynamics simulation . No systematic conformational change was observed and the average backbone r.m.s.d . compared to the initial structures was less than 1.5 A . The structure of the E . halophila cytochrome c551 shows a striking resemblance to Azotobacter vinelandii cytochrome c5 . Significant differences in backbone conformations occur in three small regions which are implicated in solvent protection of the heme propionates and thiomethyl-8(1) . Comparison with Pseudomonas aeruginosa cytochrome c551 reveals that only the common cytochrome c core, i.e . three helices, is conserved . The folding of the protein chain around the heme propionates is very different and results in more efficient solvent protection in Ps . aeruginosa . The electrostatic surface of E . halophila cytochrome c551 was found to be significantly different from mitochondrial cytochromes c and bacterial cytochromes c2 but similar to that of Ps . aeruginosa cytochrome c551.

Arch Microbiol, 1996 Dec, 166(6), 394 - 8
Taxonomic relationships of Thiobacillus halophilus, T . aquaesulis, and other species of Thiobacillus, as determined using 16S rDNA sequencing; McDonald IR et al.; Total base sequences of the 16S rRNA genes of Thiobacillus halophilus and Thiobacillus aquaesulis show that these bacteria fall into the gamma- and beta-subdivisions, respectively of the Proteobacteria . The closest relative of T . halophilus is Thiobacillus hydrothermalis (with 98.7% similarity), and the closest relative of T . aquaesulis is Thiobacillus thioparus (93.2% similarity) . Physiological properties and mol% G+C content of their DNA serve to confirm that these four organisms are all distinct species . It is reiterated that the species currently assigned to the genus Thiobacillus are clearly so diverse that they need reclassification into several genera . The type species, T . thioparus, is unequivocally placed in the beta-subdivision of the Proteobacteria, thus requiring that the use of the genus name Thiobacillus be restricted to the chemolithoautotrophic species falling into that group . T . aquaesulis and T . thioparus may thus be regarded as true species of Thiobacillus . The relatively large number of obligately chemolithoautotrophic Thiobacillus species falling in the gamma-subdivision of the Proteobacteria need further study in order to assess the case for reclassification into one or more new or different genera.

Microbiologia, 1996 Dec, 12(4), 571 - 84
Free-living spirochetes from Cape Cod microbial mats detected by electron microscopy; Teal TH et al.; Spirochetes from microbial mats and anaerobic mud samples collected in salt marshes were studied by light microscopy, whole mount and thin section transmission electron microscopy . Enriched in cellobiose-rifampin medium, selective for Spirochaeta bajacaliforniensis, seven distinguishable spirochete morphotypes were observed . Their diameters ranged from 0.17 micron to > 0.45 micron . Six of these morphotypes came from southwest Cape Cod, Massachusetts: five from Microcoleus-dominated mat samples collected at Sippewissett salt marsh and one from anoxic mud collected at School Street salt marsh (on the east side of Eel Pond) . The seventh morphotype was enriched from anoxic mud sampled from the north central Cape Cod, at the Sandy Neck salt marsh . Five of these morphotypes are similar or identical to previously described spirochetes (Leptospira, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirosymplokos deltaeiberi and Treponema), whereas the other two have unique features that suggest they have not been previously described . One of the morphotypes resembles Spirosymplokos deltaeiberi (the largest free-living spirochete described), in its large variable diameter (0.4-3.0 microns), cytoplasmic granules, and spherical (round) bodies with composite structure . This resemblance permits its tentative identification as a Sippewissett strain of Spirosymplokos deltaeiberi . Microbial mats samples collected in sterile Petri dishes and stored dry for more than four years yielded many organisms upon rewetting, including small unidentified spirochetes in at least 4 out of 100 enrichments.

Plant Physiol, 1996 Dec, 112(4), 1693 - 702
Primary structure and effect of pH on the expression of the plasma membrane H(+)-ATPase from Dunaliella acidophila and Dunaliella salina; Weiss M et al.; The plasma membrane H(+)-ATPase gene was cloned and sequenced from the extremely acidophilic green alga Dunaliella acidophila and from the extremely halotolerant Dunaliella salina . A special feature of the Dunaliella H(+)-ATPase is an extended C-terminal domain . The deduced amino acid sequences of the two proteins are 75% identical but differ in their C terminus . A hydrophilic loop within this domain in D . salina, which presumably faces the cell exterior, has a high ratio of acidic over basic amino acids, typical of halophilic proteins . The amount of the ATPase protein in plasma membranes and the level of its mRNA transcript in D . acidophila are far higher than in D . salina, suggesting that D . acidophila overexpresses the enzyme . A pH shift from 9.0 to 7.0 induces in D . salina a large increase in the level of the H(+)-ATPase mRNA and in the amount of the H(+)-ATPase protein . This suggests that the expression of the H(+)-ATPase in D . salina is pH-regulated at the transcriptional level . The implications of these findings are discussed with respect to the adaptive pressures imposed on these algal species by their exceptional environmental conditions.

J Bacteriol, 1996 Dec, 178(24), 7221 - 6
Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline-betaine pathway; Canovas D et al.; The osmoregulatory pathways of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been investigated . This strain grew optimally at 1.5 to 2 M NaCl in M63 glucose-defined medium . It required at least 0.5 M NaCl for growth, which is a higher concentration than that exhibited by the H . elongata type strain ATCC 33173 . Externally provided betaine, choline, or choline-O-sulfate (but not proline, ectoine, or proline betaine) enhanced the growth of H . elongata on 3 M NaCl-glucose-M63 plates, demonstrating the utilization of these compounds as osmoprotectants . Moreover, betaine and choline stimulated the growth of H . elongata DSM 3043 over the entire range of salinity, although betaine was more effective than choline at salinities below and above the optimum . We found that H . elongata DSM 3043 has at least one high-affinity transport system for betaine (K(m) = 3.06 microM and Vmax = 9.96 nmol of betaine min(-1) mg of protein(-1)) . Competition assays demonstrated that proline betaine and ectoine, but not proline, choline, or choline-O-sulfate, are also transported by the betaine permease . Finally, thin-layer chromatography and 13C-nuclear magnetic resonance analysis showed that exogenous choline was taken up and transformed to betaine by H . elongata, demonstrating the existence of a choline-glycine betaine pathway in this moderately halophilic bacterium.

Biochemistry, 1996 Nov 12, 35(45), 14047 - 53
Photoreaction cycle of photoactive yellow protein from Ectothiorhodospira halophila studied by low-temperature spectroscopy; Imamoto Y et al.; The photocycle of photoactive yellow protein (PYP) from Ectothiorhodospira halophila was studied by low-temperature spectroscopy . Irradiation of PYP at -190 degrees C produced a photo-steady-state mixture composed of bathochromic and hypsochromic photoproducts (PYPB and PYPH) . Upon warming, PYPH was thermally converted to a slightly blue-shifted intermediate (PYPHL) above -150 degrees C and then to a red-shifted one (PYPL) above -80 degrees C . PYPB was thermally converted to the blue-shifted intermediate (PYPBL) above -180 degrees C and then to PYPL above -90 degrees C . PYPL thermally reverted to PYP above -50 degrees C, completing the photocycle . The spectral properties of PYPL formed at low temperature suggest that it corresponds to the red-shifted photoproduct detected in the nano- to microsecond time scale at room temperature (A465) . The absolute absorption spectra of PYPH, PYPB, and PYPL were estimated, and their absorption maxima were determined to be 442 and 489 nm at -190 degrees C and 456 nm at -80 degrees C, respectively . Although a near-UV intermediate (A355) is observed in the recovery process of PYP from A465 at room temperature, it was not detected at low temperatures, probably due to the effects of temperature and the presence of glycerol . A scheme of the photocycle of PYP is presented.

Int J Food Microbiol, 1996 Nov, 33(1), 121 - 37
Microbiological spoilage of fish and fish products; Gram L et al.; Spoilage of fresh and lightly preserved fish products is caused by microbial action . This paper reviews the current knowledge in terms of the microbiology of fish and fish products with particular emphasis on identification of specific spoilage bacteria and the qualitative and quantitative biochemical indicators of spoilage . Shewanella putrefaciens and Pseudomonas spp . are the specific spoilage bacteria of iced fresh fish regardless of the origin of the fish . Modified atmosphere stored marine fish from temperate waters are spoiled by the CO2 resistant Photobacterium phosphoreum whereas Gram-positive bacteria are likely spoilers of CO2 packed fish from fresh or tropical waters . Fish products with high salt contents may spoil due to growth of halophilic bacteria (salted fish) or growth of anaerobic bacteria and yeasts (barrel salted fish) . Whilst the spoilage of fresh and highly salted fish is well understood, much less is known about spoilage of lightly preserved fish products . It is concluded that the spoilage is probably caused by lactic acid bacteria, certain psychotrophic Enterobacteriaceae and/or Photobacterium phosphoreum . However, more work is needed in this area.

Gene, 1996 Oct 17, 176(1-2), 27 - 33
Protein-encoding genes in the sulfothermophilic archaea Sulfolobus and Pyrococcus; De Vendittis E et al.; A number of unrelated protein-encoding genes from sulfothermophilic archaea, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Pyrococcus furiosus and Pyrococcus woesei, has been analyzed . In the Sulfolobus genus, the content of A + T is significantly higher than that of C + G and the base usage follows the order, A > T > G > C . In Pyrococcus, the A + T content is also higher than that of C + G, but with lower values; in the order of base usage, G precedes T . The codon usage of these sulfothermophiles has been determined; alternative start codons are frequently used in both genera; codon preferences reflect the rich A + T composition of the corresponding genomes; for both genera the codon bias is particularly evident within the different arginine triplets, where AGA and AGG are predominant . From the similarities in the codon usage, close taxonomic relationships become evident within the Sulfolobus or the Pyrococcus genus; a lower, but significant similarity is also clear between these genera . The synonymous codon usage of these sulfothermophiles shows similarities with that of Saccharomyces cerevisiae and bovine mitochondria, whereas clear divergences are observed with the halophilic archaeal genus, Halobacterium, or the eubacterium, Escherichia coli . The unrelated proteins of the considered sulfothermophiles have been analyzed for the content of hydrophobic residues; the comparison with mesophiles reveals a significant increase in the average hydrophobicity of amino acid residues . This finding could indicate a mechanism of adaptation of proteins in organisms living under extreme environments . It is noteworthy that an opposite trend, i.e . a decreased average hydrophobicity, occurs in unrelated halophilic proteins.

Eur J Biochem, 1996 Oct 15, 241(2), 440 - 52
The solution structure refinement of the paramagnetic reduced high-potential iron-sulfur protein I from Ectothiorhodospira halophila by using stable isotope labeling and nuclear relaxation; Bertini I et al.; The reduced high-potential iron sulfur protein I from Ectothiorhodospira halophila which contains the {4Fe-4S}2+ polymetallic center has been fully labeled with 15N and 13C . The protein is paramagnetic, the nuclear relaxation times of nuclei close to the paramagnetic ion are drastically shortened and some strategic dipolar connectivities are lost . Notwithstanding, the solution structure has been reported {Banci, L., Bertini, I., Eltis, L . D., Felli, I . C., Kastrau, D . H . W., Luchinat, C., Piccioli, M., Pierattelli, R . & Smith, M . (1994) Eur . J . Biochem . 225, 715-725} . We have performed classical HNHA, HNCA soft-COSY, soft-HCCH E . COSY and 15N-1H correlated NOESY experiments in order to obtain a set of 3J scalar coupling constants . Some experiments have been optimized to counterbalance the effect of paramagnetism . From heteronuclear single-quantum experiments preceded by a 180 degrees pulse and variable delay times, the non-selective magnetization recovery has been followed from which the contribution to dipolar relaxation of nuclei due to the interaction with the paramagnetic metal ions (rho para) has been estimated . Finally, the intensities of NOEs have been corrected for the presence of paramagnetic metal ions and these corrected values together with 3J values and rho para data have been used to obtain a well defined solution structure . The aim is that of obtaining a structure with enough constraints to be well resolved all over the protein, including the vicinity of the paramagnetic metal cluster, which is anchored to the protein through the rho para constraints . In total, 1226 corrected NOESY crosspeaks (of which 945 were found to be meaningful), 37 one-dimensional NOEs, 39 3JHNH alpha and 37 3JHNC' (providing 45 phi dihedral angle constraints) 54 3JH alpha H beta and 31 3JNH beta (providing 26 chi 1 dihedral angle constraints), 4 chi 2 dihedral angle constraints of the coordinated cysteines, obtained from the hyperfine shifts of the beta CH protons, and 58 rho para constraints, have been used for structure calculation . Restrained molecular dynamics simulations have also been performed to provide the final family of structures . This research demonstrates that stable isotope labeling provides specific advantages for the NMR investigation of paramagnetic molecules, as the small magnetic moment of heteronuclei minimizes the paramagnetic influence of unpaired electrons.

Biochim Biophys Acta, 1996 Oct 2, 1284(1), 79 - 85
Voltage-dependent absorbance change of carotenoids in halophilic archaebacteria; Seki SI et al.; Membrane vesicles of wild-type Halobacterium sp . mex strain show a wavy absorbance change which has not been so far reported in halophilic archaebacteria . A white mutant strain lacking carotenoids did not show the wavy absorbance change . The wavy absorbance change in the range of 440-590 nm was induced by a red flash (600-640 nm), which photoexcited electrogenic ion pumps, mex bacteriorhodopsin and mex halorhodopsin but not carotenoids . The wavy change was also caused by K+ diffusion potentials without light . These results suggest that the wavy absorbance change in the membrane vesicles is the voltage-dependent absorbance change of the carotenoids.

Appl Environ Microbiol, 1996 Oct, 62(10), 3779 - 86
Phylogenetic analyses of some extremely halophilic archaea isolated from Dead Sea water, determined on the basis of their 16S rRNA sequences; Arahal DR et al.; Twenty-two extremely halophilic aerobic archaeal strains were isolated from enrichments prepared from Dead Sea water samples collected 57 years ago . The isolates were phenotypically clustered into five different groups, and a representative from each group was chosen for further study . Almost the entire sequences of the 16S rRNA genes of these representatives, and of Haloarcula hispanica ATCC 33960, were determined to establish their phylogenetic positions . The sequences of these strains were compared to previously published sequences of 27 reference halophilic archaea (members of the family Halobacteriaceae) and two other archaea, Methanobacterium formicicum DSM 1312 and Methanospirillum hungatei DSM 864 . Phylogenetic analysis using approximately 1,400 base comparisons of 16S rRNA-encoding gene sequences demonstrated that the five isolates clustered closely to species belonging to three different genera--Haloferax, Halobacterium, and Haloarcula . Strains E1 and E8 were closely related and identified as members of the species Haloferax volcanii, and strain E12 was closely related and identified as a member of the species Halobacterium salinarum . However, strains E2 and E11 clustered in the Haloarcula branch with Haloarcula hispanica as the closest relative at 98.9 and 98.8% similarity, respectively . Strains E2 and E11 could represent two new species of the genus Haloarcula . However, because strains of these two new species were isolated from a single source, they will not be named until additional strains are isolated from other sources and fully characterized.

Mutat Res, 1996 Sep 2, 364(1), 25 - 32
The repair of ultraviolet light-induced DNA damage in the halophilic archaebacteria, Halobacterium cutirubrum, Halobacterium halobium and Haloferax volcanii; McCready S; Extremely halophilic archaebacteria have been reported to have no capacity for dark repair (excision repair) of ultraviolet damage and to rely on very efficient photoreactivation for recovery after UVC irradiation . Post-UV incubation in the light restores 100% survival in these organisms . This has been taken to indicate that cyclobutane dimers are the only significant UV-induced lesions and that they are completely repaired by photoreactivation . However, in all organisms studied to date, pyrimidine (6-4) pyrimidone photoproducts are a significant cytotoxic and mutagenic lesion and constitute 10-30% of UV photoproducts . The question arises, therefore--are 6-4 photoproducts induced in the halophilic archaebacteria and, if they are, how are they repaired? This paper shows that both cyclobutane dimers and 6-4 photoproducts are induced in the extremely halophilic archaebacteria, Halobacterium cutirubrum, Halobacterium halobium and Haloferax volcanii, at similar levels as in other organisms . Furthermore, contrary to previous reports, there is dark repair of both lesions . As in other organisms, 6-4 photoproducts are removed more efficiently than cyclobutane dimers in the dark . In the light, cyclobutane dimers are repaired very rapidly and there is also photoenhanced repair of 6-4 photoproducts . This work confirms that organisms such as Halobacterium and Haloferax which live in conditions of high exposure to sunlight have very efficient rates of repair of UV lesions in the light.

Arch Microbiol, 1996 Sep, 166(3), 169 - 75
The porins from the halophilic species Ectothiorhodospira shaposhnikovii and Ectothiorhodospira vacuolata; Wolf E et al.; Major outer membrane proteins with porin activity were isolated from cell envelopes of the halophilic strains Ectothiorhodospira shaposhnikovii N1 and Ectothiorhodospira vacuolata beta1 . The porins were obtained as oligomers . They dissociated into monomers by heat or EDTA treatment . The molecular masses of the monomers were determined by mass spectrometry to be 39,285 and 37,160 Da for E . shaposhnikovii N1 and E . vacuolata beta1, respectively . Both were shown by analytical ultracentrifugation to be trimers of about 112, 000 Da . Circular dichroism spectra indicated predominantly beta-sheet structure . The 18 N-terminal amino acid sequences of the two porins were identical except for the amino acids in positions 12 and 14 . No sequence similarity with the primary structure of known porins was found . In reconstitution experiments with lipid bilayers, the porins of E . shaposhnikovii N1 and E . vacuolata beta1 formed channels with a single-channel conductance of 1.5 and 0.7 nS, respectively, in 1 M KCl . The single-channel conductance saturated with increasing salt concentration, indicating a putative binding-site for anions in the channel since both porins exhibited anion-selectivity . For the porin of E . vacuolata beta1, but not for that of E . shaposhnikovii N1, an influence of detergent concentration on the single-channel conductance was observed.

J Biol Chem, 1996 Aug 16, 271(33), 19724 - 31
Isolation and chromosomal distribution of natural Z-DNA-forming sequences in Halobacterium halobium; Kim J et al.; Conditions favoring left-handed Z-DNA such as high salinity (> 4 ), high negative DNA supercoiling, and GC-rich DNA {statistically favoring d(CG)n repeat sequences}, are all found in the extremely halophilic archaeum (archaebacterium) Halobacterium halobium . In order to identify and study Z-DNA regions of the H . halobium genome, an affinity chromatography method with high Z-DNA selection efficiency was developed . Supercoiled plasmids were incubated with a Z-DNA-specific antibody (Z22) and passed over a protein A-agarose column, and the bound plasmids were eluted using an ethidium bromide gradient . In control experiments using mixtures of pUC12 (Z-negative) and a d(CG)5-containing (Z-positive) pUC12 derivative, up to 4,000-fold enrichment of the Z-DNA-containing plasmid was demonstrated per cycle of the Z-DNA selection procedure . The selection efficiency was determined by transformation of Escherichia coli DH5alpha with eluted plasmids and blue-white screening on X-gal plates . Twenty recombinant plasmids containing Z-DNA-forming sequences of H . halobium were isolated from a genomic library using affinity chromatography . Z-DNA-forming sequences in selected plasmids were identified by bandshift and antibody footprinting assays using Z22 monoclonal antibody . Alternating purine-pyrimidine sequences ranging from 8 base pairs (bp) to 13 bp with at least a 6-bp alternating d(GC) stretch were found in the Z22 antibody binding regions of isolated plasmids . The distribution of Z-DNA-forming sequences in the Halobacterium salinarum GRB chromosome was analyzed by dot-blot hybridization of an ordered cosmid library using the cloned H . halobium Z-DNA segments as probe . Among the 11 Z-DNA segments tested, five were found to be clustered in a 100-kilobase pair region of the genome, whereas six others were distributed throughout the rest of the genome.

Biodegradation, 1996 Aug, 7(4), 297 - 302
Bacterial influence on partitioning rate during the biodegradation of styrene in a biphasic aqueous-organic system; Osswald P et al.; The degradation by a consortium of slightly-halophile marine bacteria of styrene initially dissolved in silicone oil was monitored in batch reactors stirred at 75, 125 and 500 rpm, respectively . In the 75 and 125 rpm cases, the styrene biodegradation rate was higher than the rate of spontaneous partitioning of styrene from the oil to the water, determined under abiotic conditions . Abiotic transfer tests carried out after biodegradation runs revealed that bacterial activity had resulted in a significant increase in the rate of styrene partitioning between the two liquid phases . Even though bacterial adsorption was noticeable at the oil-water interface, this effect appeared to be due to the release by the bacteria of chemicals in the aqueous phase . Similarity with observations made with Triton X-100 suggested that the chemicals released may have been biosurfactants or solubilizing agents.

Mol Microbiol, 1996 Aug, 21(4), 683 - 93
Photobiology of microorganisms: how photosensors catch a photon to initialize signalling; Hellingwerf KJ et al.; Photobiological processes are relevant for microorganisms for energy generation, protection against excess and/or damaging radiation, and for signalling . In this review we give an overview of the knowledge on the functioning of photosensors in microorganisms, with special emphasis on the conformational changes that lead to signal generation and transduction . Light is absorbed by specific chromophores, which are tuned, by their proteinaceous environment, to function optimally . These chromophores belong to three classes: tetrapyrroles, polyenes and aromatics . The chemical structure of photosensing pigment/protein complexes has been resolved for many of the photobiological processes that have a characteristic sensitivity in the visible and infrared part of the spectrum of (solar) radiation . However, knowledge about the structure of photoreceptors responsible for several physiologically well-characterized photoresponses to UV- and blue light is still lacking . For a limited number of phototransduction processes, the details of light-induced signal transfer are beginning to be understood in atomic detail . This applies particularly to two photosensors involved in phototactic responses in bacteria: sensory rhodopsin I (SR-I) from Halobacterium salinarium and photoactive yellow protein (PYP) from Ectothiorhodospira halophila . The SR-1 system is of special interest because the transducer accepting the signal from SR-1 was recently identified as Htr-1, a homologue of the methyl-accepting chemotaxis proteins which have been characterized in Escherichia coli . PYP, on the other hand, may be the first photosensor to actually reveal all relevant details of the kinetics, thermodynamics, and molecular motion of light-induced signal generation, through an understanding of how the photo-isomerization of the chromophore forces the sensor protein into the signalling state . Here we compare these photosensors and discuss common themes in the initiation of photosensory signal transduction in microorganisms in terms of the molecular properties of photosensors and their signalling state.

J Bacteriol, 1996 Aug, 178(15), 4737 - 41
Conserved sequence elements involved in regulation of ribosomal protein gene expression in halophilic archaea; Shimmin LC et al.; A region of the Haloferax volcanii genome encoding ribosomal proteins L11e, L1e, L10e, and L12e was cloned and sequenced, and the transcripts derived from the cluster were characterized . Flanking and noncoding regions of the sequence were analyzed phylogenetically by comparison with the homologous sequences from two other halophilic archaea, i.e., Halobacterium cutirubrum and Haloarcula marismortui . Motifs, identified by high-level sequence conservation, include both transcriptional and translational regulatory elements and other elements of unknown function.

J Biol Chem, 1996 Jul 26, 271(30), 17718 - 23
A salt-resistant plasma membrane carbonic anhydrase is induced by salt in Dunaliella salina; Fisher M et al.; The mechanisms allowing proliferation of the unicellular green alga Dunaliella salina in up to saturating NaCl concentrations are only partially understood at present . Previously, the level of a plasma membrane Mr 60,000 protein, p60, was found to increase with rising external salinities . Based on cDNA cloning and enzymatic assays, it is now shown that p60 is an internally duplicated carbonic anhydrase, with each repeat homologous to animal and Chlamydomonas reinhardtii carbonic anhydrases, but exceptional in the excess of acidic over basic residues . Increasing salinities, alkaline shift, or removal of bicarbonate induced in D . salina parallel increases in the levels of p60, its mRNA, and external carbonic anhydrase activity . Moreover, purified p60 exhibited carbonic anhydrase activity comparable to other carbonic anhydrases . A p60-enriched soluble preparation showed maximal carbonic anhydrase activity at approximately 1.0 M NaCl and retained considerable activity at higher salt concentrations . In contrast, a similar preparation from C . reinhardtii was approximately 90% inhibited in 0.6 M NaCl . These results identified p60 as a structurally novel carbonic anhydrase transcriptionally regulated by CO2 availability and exhibiting halophilic-like characteristics . This enzyme is potentially suited to optimize CO2 uptake by cells growing in hypersaline media.

FEMS Microbiol Lett, 1996 Jul 15, 141(1), 59 - 63
NADP-glutamate dehydrogenase from the halophilic archaeon Haloferax mediterranei: enzyme purification, N-terminal sequence and stability; Ferrer J et al.; An NADP(H)-specific glutamate dehydrogenase of Haloferax mediterranei has been purified to apparent homogeneity and characterised . The purified enzyme was stabilized by glycerol in absence of salt . Glutamate dehydrogenase from Hf . mediterranei is a hexameric enzyme with a native molecular mass of 320 kDa composed of monomers each with a molecular mass of 55 kDa . At pH 8.5 the enzyme has Kms of 0.018, 0.34 and 4.2 mM for NADP+, 2-oxoglutarate and ammonium, respectively . Amino acid composition and sequence of the first 16 residues of the N-terminus have been determined.

Microb Drug Resist, 1996 Summer, 2(2), 269 - 72
beta-Lactamases are absent from Archaea (archaebacteria); Martin HH et al.; beta-Lactamases, enzymes that hydrolyze and inactive beta-lactam antibiotics, are of widespread occurrence in Bacteria and are related to the metabolism of bacterial cell wall murein . So far, no information exists on beta-lactamases in Archaea, a separate domain of prokaryotes with diverse types of unique cell wall polymers . Different mesophilic methanogenic and extremely halophilic Archaea containing methanochondroitin, pseudomurein, or S-layer protein or glycoprotein cell walls, were tested for beta-lactamase activity with the chromogenic beta-lactam nitrocefin as substrate . Also tested were representative microbial Eucarya from algae, yeasts, and protozoa . No beta-lactamase activity was detected in any of the archaeal and eukaryotic organisms . This supports the view that beta-lactamases are restricted to the domain of Bacteria.

Biophys J, 1996 Jul, 71(1), 365 - 80
Protein folding thermodynamics applied to the photocycle of the photoactive yellow protein; Van Brederode ME et al.; Two complementary aspects of the thermodynamics of the photoactive yellow protein (PYP), a new type of photoreceptor that has been isolated from Ectothiorhodospira halophila, have been investigated . First, the thermal denaturation of PYP at pH 3.4 has been examined by global analysis of the temperature-induced changes in the UV-VIS absorbance spectrum of this chromophoric protein . Subsequently, a thermodynamic model for protein (un)folding processes, incorporating heat capacity changes, has been applied to these data . The second aspect of PYP that has been studied is the temperature dependence of its photocycle kinetics, which have been reported to display an unexplained deviation from normal Arrhenius behavior . We have extended these measurements in two solvents with different hydrophobicities and have analyzed the number of rate constants needed to describe these data . Here we show that the resulting temperature dependence of the rate constants can be quantitatively explained by the application of a thermodynamic model which assumes that heat capacity changes are associated with the two transitions in the photocycle of PYP . This result is the first example of an enzyme catalytic cycle being described by a thermodynamic model including heat capacity changes . It is proposed that a strong link exists between the processes occurring during the photocycle of PYP and protein (un)folding processes . This permits a thermodynamic analysis of the light-induced, physiologically relevant, conformational changes occurring in this photoreceptor protein.

Int J Syst Bacteriol, 1996 Jul, 46(3), 817 - 21
Analysis of 16S rRNA gene sequences of Vibrio costicola strains: description of Salinivibrio costicola gen . nov., comb . nov; Mellado E et al.; The phylogenetic positions of six Vibrio costicola strains were determined by direct sequencing and analysis of their PCR-amplified 16S ribosomal DNAs . A comparative analysis of the sequence data revealed that the moderate halophile V . costicola forms a monophyletic branch that is distinct from other Vibrio species and from moderately halophilic species of other genera . These results complement phenotypic and genotypic data determined previously . The molecular evidence, together with several phenotypic differences, distinguishes V . costicola from species of the genus Vibrio and other species belonging to the gamma subclass of the Proteobacteria and indicates that V . costicola should be placed in a new and separate genus . The name Salinivibrio costicola gen . nov., comb . nov . is proposed for this bacterium . The guanine-plus-cytosine content of the DNA is 49.4 to 50.5 mol% . The type strain of S . costicola is strain NCIMB 701 (= ATCC 33508).

Int J Syst Bacteriol, 1996 Jul, 46(3), 710 - 5
Desulfovibrio gabonensis sp . nov., a new moderately halophilic sulfate-reducing bacterium isolated from an oil pipeline; Tardy-Jacquenod C et al.; Two moderately halophilic sulfate-reducing bacteria were isolated from an African oil pipeline and designated strains SEBR 3640 and SEBR 2840T (T = type strain) . Both of these strains possess traits that define the genus Desulfovibrio . The cells of both isolates were motile curved rods that had a single polar flagellum and contained desulfoviridin, and both isolates utilized lactate, pyruvate, malate, fumarate, succinate, and ethanol in the presence of sulfate . Sulfite, thiosulfate, and elemental sulfur were also used as an electron acceptors in the presence of lactate . However, both strains tolerated higher concentrations of NaCl (up to 17%) than all other Desulfovibrio species except Desulfovibrio halophilus, which tolerated a similar level of NaCl . The results of a 16S rRNA gene sequence analysis also placed the designated type strain, strain SEBR 2840, in the genus Desulfovibrio but revealed that this organism was significantly different from D . halophilus and all other validly described Desulfovibrio species . On the basis of our results, we propose that strain SEBR 2840T is a member of a new species of the genus Desulfovibrio, Desulfovibrio gabonensis . The type strain of D . gabonensis is strain SEBR 2840 (= DSM 10636).

Appl Environ Microbiol, 1996 Jul, 62(7), 2673 - 5
Identification of Vibrio proteolyticus with a differential medium and a specific probe; Muniesa-Perez M et al.; A differential medium (VP8) and a specific probe, based on the variable region V3 of the 16S rRNA gene, for the detection of Vibrio proteolyticus are defined . The medium contains 8% NaCl, which allows selective growth of moderately halophilic Vibrio strains . D-Sorbitol, as the main carbon source, differentiates the species that can ferment it by the pH indicators cresol red and bromothymol blue . V . proteolyticus and 8 of 418 strains studied grew on the medium and used the D-sorbitol, forming bright yellow colonies . An oligonucleotide, based on the variable region V3 of the 16S rRNA gene (5'CGCTAACGTCAAATAATGCATCTA3'), was used as the specific probe (V3VPR) . Only three strains of Vibrio sp . and one strain identified as V . natriegens cross-hybridized with the probe . However, unlike V . proteolyticus, none of the strains grew on VP8 . The combined use of VP8 medium and the probe allowed an unequivocal identification of V . proteolyticus.

Microbiology, 1996 Jul, 142 ( Pt 7), 1715 - 23
Influence of salt on the transcription of the gas-vesicle genes of Haloferax mediterranei and identification of the endogenous transcriptional activator gene; Roder R et al.; The transcription of the 14 gvp genes of the gas-vesicle-encoding mc-vac region was investigated, using RNA from 25% and 15% (w/v) salt cultures of the moderately halophilic archaeon Haloferax mediterranei . Transcription occurred only from two promoters, located in front of the mc-gvpA and mc-gvpD genes . In both cultures transcripts spanning the entire mc-gvpDEFGHIJKLM transcription unit were formed only during the exponential growth phase . Amounts of these transcripts were larger in the 25% salt culture, in which the 2.0 kb mc-gvpD transcripts were also synthesized during the stationary phase . The levels of the mc-gvpD transcripts and of the 324 nt mc-gvpA mRNA increased in parallel during the stationary phase of the 25% salt culture . Only under these conditions were mRNAs spanning the entire mc-gvpACNO transcription unit observed, and gas-vesicles were formed . Investigation of the influence of the mc-gvpDE genes on both mc-vac promoters in transformants revealed that by themselves they were nearly inactive . The addition of mc-gvpE, however, resulted in a high level of constitutively produced mc-gvpA and mc-gvpD mRNA, indicating a transcriptional activator function for the mc-gvpE product.

J Appl Bacteriol, 1996 Jul, 81(1), 109 - 12
Phylogenetic characterization of a novel salt-tolerant Bacillus species: description of Bacillus dipsosauri sp . nov; Lawson PA et al.; The taxonomic position of a novel halophilic endospore-forming bacterium previously isolated from a desert iguana was investigated by 16S rRNA gene sequencing . Comparative sequence analyses showed the unidentified bacterium to be phylogenetically loosely associated with some other spore-forming (Bacillus pantothenticus, Sporosarcina halophila) and non-spore-forming (Marinococcus albus) halotolerant bacteria . Based on the phenotypic and phylogenetic distinctiveness of the unidentified bacterium, it is proposed that it is classified in the genus Bacillus as a new species, Bacillus dipsosauri.

EMBO J, 1996 Jul 1, 15(13), 3209 - 18
The xanthopsins: a new family of eubacterial blue-light photoreceptors; Kort R et al.; Photoactive yellow protein (PYP) is a photoreceptor that has been isolated from three halophilic phototrophic purple bacteria . The PYP from Ectothiorhodospira halophila BN9626 is the only member for which the sequence has been reported at the DNA level . Here we describe the cloning and sequencing of the genes encoding the PYPs from E.halophila SL-1 (type strain) and Rhodospirillum salexigens . The latter protein contains, like the E.halophila PYP, the chromophore trans p-coumaric acid, as we show here with high performance capillary zone electrophoresis . Additionally, we present evidence for the presence of a gene encoding a PYP homolog in Rhodobacter sphaeroides, the first genetically well-characterized bacterium in which this photoreceptor has been identified . An ORF downstream of the pyp gene from E.halophila encodes an enzyme, which is proposed to be involved in the biosynthesis of the chromophore of PYP . The pyp gene from E.halophila was used for heterologous overexpression in both Escherichia coli and R.sphaeroides, aimed at the development of a holoPYP overexpression system (an intact PYP, containing the p-coumaric acid chromophore and displaying the 446 nm absorbance band) . In both organisms the protein could be detected immunologically, but its yellow color was not observed . Molecular genetic construction of a histidine-tagged version of PYP led to its 2500-fold overproduction in E.coli and simplified purification of the heterologously produced apoprotein . HoloPYP could be reconstituted by the addition of p-coumaric anhydride to the histidine-tagged apoPYP (PYP lacking its chromophore) . We propose to call the family of photoactive yellow proteins the xanthopsins, in analogy with the rhodopsins.

Appl Environ Microbiol, 1996 Jun, 62(6), 1897 - 902
Gram-positive, catalase-positive cocci from dry cured Iberian ham and their enterotoxigenic potential; Rodriguez M et al.; Iberian ham is an uncooked, cured meat product ripened under natural uncontrolled conditions for 18 to 24 months . Gram-positive, catalase-positive cocci are the main microbial population in Iberian ham for most of the ripening time . Since some of these organisms are able to produce enterotoxins, adequate characterization and toxicological study are needed . For this, 1,327 gram-positive, catalase-positive cocci, isolated from Iberian hams at different stages and locations, were characterized by physiological and biochemical tests . Selected isolates were further characterized by guanine-cytosine (G+C) content and restriction enzyme analysis of genes coding for 16S rRNA . The toxigenic potential of these organisms was tested with specific DNA gene probes for staphylococcal enterotoxins A, B, C, and D and confirmed by semiquantitative sandwich enzyme immunoassay . The majority of the isolates were identified as Staphylococcus spp . and Micrococcus spp . Non-identified gram-positive, catalase-positive cocci which were moderately halophilic and showed a 42 to 52% G+C content were detected . A great variety of staphylococcal strains were found within the different species at any sampling time . Two strains of Staphylococcus xylosus, one Staphylococcus cohnii strain, and four of the non-identified organisms with 42 to 52% G+C contents hybridized with some of the DNA probes for C and D staphylococcal enterotoxin genes . S . xylosus hybridizing with C-enterotoxin probe reacted with both C and D enterotoxins in the immunological test . In addition, enterotoxin D was confirmed in the nonidentified strains . Some toxigenic organisms were isolated from the final product, posing a health hazard for the consumer.

Nucleic Acids Res, 1996 Jun 1, 24(11), 2125 - 32
recA-like genes from three archaean species with putative protein products similar to Rad51 and Dmc1 proteins of the yeast Saccharomyces cerevisiae; Sandler SJ et al.; The process of homologous recombination has been documented in bacterial and eucaryotic organisms . The Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins are the archetypal members of two related families of proteins that play a central role in this process . Using the PCR process primed by degenerate oligonucleotides designed to encode regions of the proteins showing the greatest degree of identity, we examined DNA from three organisms of a third phylogenetically divergent group, Archaea, for sequences encoding proteins similar to RecA and Rad51 . The archaeans examined were a hyperthermophilic acidophile, Sulfolobus sofataricus (Sso); a halophile, Haloferax volcanii (Hvo); and a hyperthermophilic piezophilic methanogen, Methanococcus jannaschii (Mja) . The PCR generated DNA was used to clone a larger genomic DNA fragment containing an open reading frame (orf), that we refer to as the radA gene, for each of the three archaeans . As shown by amino acid sequence alignments, percent amino acid identities and phylogenetic analysis, the putative proteins encoded by all three are related to each other and to both the RecA and Rad51 families of proteins . The putative RadA proteins are more similar to the Rad51 family (approximately 40% identity at the amino acid level) than to the RecA family (approximately 20%) . Conserved sequence motifs, putative tertiary structures and phylogenetic analysis implied by the alignment are discussed . The 5' ends of mRNA transcripts to the Sso radA were mapped . The levels of radA mRNA do not increase after treatment with UV irradiation as do recA and RAD51 transcripts in E.coli and S.cerevisiae . Hence it is likely that radA in this organism is a constitutively expressed gene and we discuss possible implications of the lack of UV-inducibility.

Curr Microbiol, 1996 Jun, 32(6), 308 - 13
Process of Carbonate Precipitation by Deleya halophila
Rivadeneyra MA, Ramos-Cormenzana A, Delgado G, Delgado R.
Scanning electron microscopy and X-ray dispersive energy microanalysis were used to investigate the formation of carbonate crystals by Deleya halophila . The formation of calcium carbonate crystals (polymorphous aragonite) by D . halophila is a sequential process that commences with a nucleus formed by the aggregation of a few calcified bacterial cells and the subsequent accumulation of more calcified cells and carbonate, which acts to weld the bacteria together . The process leads to the formation of spherical bioliths measuring approximately 50 &mgr;m in diameter . The mechanism of carbonate precipitation by D . halophila under our working conditions represents a process of induced biomineralization.

J Bacteriol, 1996 Jun, 178(11), 3044 - 8
Dihydrolipoamide dehydrogenase from the halophilic archaeon Haloferax volcanii: homologous overexpression of the cloned gene; Jolley KA et al.; The gene encoding dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii, has been subcloned and overexpressed in the parent organism by using the halophilic archaeal rRNA promoter . The recombinant protein has been purified to homogeneity and characterized with respect to its kinetic, molecular, and salt-dependent properties . A dihydrolipoamide dehydrogenase-minus mutant of H . volcanii has been created by homologous recombination with the subcloned gene after insertion of the mevinolin resistance determinant into the protein-coding region . To explore the physiological function of the dihydrolipoamide dehydrogenase, the growth properties of the mutant halophile have been examined.

Biochim Biophys Acta, 1996 May 23, 1294(2), 159 - 67
Halolysin R4, a serine proteinase from the halophilic archaeon Haloferax mediterranei; gene cloning, expression and structural studies; Kamekura M et al.; A gene encoding a halophilic serine proteinase, halolysin R4, from a halophilic archaeon Haloferax mediterranei strain R4 was cloned, its nucleotide sequence determined, and expressed in Haloferax volcanii WFD11 . The deduced amino-acid sequence (403 aa in length) showed the highest similarity to halolysin 172P1, produced by another halophilic archaeon, strain 172P1 (now designated as Natrialba asiatica) . Both halolysins belong to the thermitase branch of class I subtilases, but show long C-terminal extensions of 117 and 123 amino acids, respectively . Removal of this "tail' region from halolysin R4 abolished proteinase activity, indicating it provides an essential (but as yet unknown) function . Substitution of the two cysteine residues in the C-terminal extension with serine decreased enzyme stability in hypotonic solutions, possibly owing to disruption of potential disulfide bonds or perturbation of calcium binding site(s).

Microbiology, 1996 May, 142 ( Pt 5), 1115 - 22
Degradation of 2,4-dichlorophenoxyacetic acid by haloalkaliphilic bacteria; Maltseva O et al.; Three 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial isolates were obtained from the highly saline and alkaline Alkali Lake site in southwestern Oregon contaminated with 2,4-D production wastes . While similar in most respects, the three isolates differed significantly in 2,4-D degradation rates, with the most active strain, I-18, demonstrating an ability to degrade up to 3000 mg 2,4-D I-1 in 3 d . This strain was well adapted to the extreme environment from which it was isolated, growing optimally on 2,4-D at pH 8.4-9.4 and at sodium ion concentrations of 0.6-1.0 M . According to its optimum salt concentration and pH for growth, this isolate was a moderately halophilic, alkaliphilic bacterium . The 16S RNA gene sequence (303 nt) was identical for all three isolates and most closely resembled those of the moderately halophilic eubacteria of the family Halomonadaceae (91% identity) . Biochemical and genetic examination revealed strain I-18 utilizes the same 2,4-D degradation pathway as most of the 2,4-D-degrading bacteria from non-extreme environments . Hybridization data and comparison of the partial sequences of the tfdA gene from the Alkali Lake isolates with those of bacteria from non-extreme environments suggested a common genetic origin of the 2,4-D degradation pathway in the two groups of micro-organisms.

Nat Struct Biol, 1996 May, 3(5), 452 - 8
Insights into protein adaptation to a saturated salt environment from the crystal structure of a halophilic 2Fe-2S ferredoxin; Frolow F et al.; Haloarcula marismortui is an archaebacterium that flourishes in the world's saltiest body of water, the Dead Sea . The cytosol of this organism is a supersaturated salt solution in which proteins are soluble and active . The crystal structure of a 2Fe-2S ferredoxin from H . marismortui determined at 1.9 A is similar to those of plant-type 2Fe-2S ferredoxins of known structure, with two important distinctions . The entire surface of the protein is coated with acidic residues except for the vicinity of the iron-sulphur cluster, and there is an insertion of two amphipathic helices near the N-terminus . These form a separate hyperacidic domain whose postulated function to provide extra surface carboxylates for solvation . These data and the fact that bound surface water molecules have on the average 40% more hydrogen bonds than in a typical non-halophilic protein crystal structure support the notion that haloadaptation involves better water binding capacity.

J Biol Chem, 1996 Apr 19, 271(16), 9340 - 6
Analysis of left-handed Z-DNA formation in short d(CG)n sequences in Escherichia coli and Halobacterium halobium plasmids . Stabilization by increasing repeat length and DNA supercoiling but not salinity; Kim J et al.; To evaluate the relative importance of alternating d(CG) sequence length, DNA supercoiling, and salt in left-handed Z-DNA formation, plasmids containing short d(CG)n sequences (n = 3-17) with the capability of replicating in either Escherichia coli or the halophilic archaeum Halobacterium halobium were constructed . Z-DNA conformation in the d(CG)n sequences was assayed by (i) a band shift assay using the Z-DNA-specific Z22 monoclonal antibody (ZIBS assay); (ii) an S1 nuclease cleavage-primer extension assay to map B-Z junctions; and (iii) a BssHII restriction inhibition assay . Using the ZIBS assay on plasmids purified from E . coli, the transition from B-DNA to Z-DNA occurred from d(CG)4, to d(CG)5, with 20% of d(CG)4, and 90% of d(CG)5 in Z-DNA conformation . These findings were consistent with the results of S1 nuclease cleavage observed at B-Z junctions flanking d(CG)4 and d(CG)5 sequences . Resistance to BssHII restriction endonuclease digestion was observed only in supercoiled plasmids containing d(CG)8 or longer sequences, indicating that shorter d(CG)n sequences are in dynamic equilibrium between B- and Z-DNA conformations . When a plasmid containing d(CG)4, was isolated from a topA mutant of E . coli, it contained 25% greater linking deficiency and 40% greater Z-DNA conformation in the alternating d(CG) region . In plasmids purified from H . halobium, which showed 30% greater linking deficiency than from E . coli, 20-40% greater Z-DNA formation was found in d(CG)4-6 sequences . Surprisingly, no significant difference in Z-DNA formation could be detected in d(CG)3-17 sequences in plasmids from either E . coli or H . halobium in the NaCl concentration range of 0.1-4 M.

Gene, 1996 Apr 17, 170(1), 77 - 80
Vectors using the phospho-alpha-(1,1)-glucosidase-encoding gene treA of Bacillus subtilis as a reporter; Schock F et al.; The intracellular phospho-alpha-(1,1)-glucosidase, TreA, from Bacillus subtilis (Bs) hydrolyses trehalose 6-phosphate into glucose and glucose 6-phosphate . The enzyme is also able to cleave p-nitrophenyl alpha-D-glucopyranoside (PNPG) . This enzymatic reaction can be easily monitored in a beta-galactosidase analogous enzyme assay . The vectors we have constructed can be used to study promoter activity in transcriptional treA fusions and may prove especially useful under high-salt conditions due to the halophilic character of TreA . The treA gene is useful as a reporter in either Bs or Escherichia coli (Ec) . Such fusions can be integrated in the Bs amyE locus and selected on either kanamycin or chloramphenicol, or used as plasmids in Ec . As an example of the general utility, we demonstrate treA expression under xylA-operator-promoter control.

FEBS Lett, 1996 Apr 1, 383(3), 227 - 9
Glucose dehydrogenase from the halophilic Archaeon Haloferax mediterranei: enzyme purification, characterisation and N-terminal sequence; Bonete MJ et al.; An NAD(P)-glucose dehydrogenase from the extremely halophilic Archaeon, Haloferax mediterranei, has been purified to electrophoretic homogeneity . The purified enzyme has been characterised with respect to its cofactor specificity, subunit composition and its salt and thermal stability . The N-terminal amino acid sequence has been determined and N-terminus alignment with sequences of other glucose dehydrogenases shows that the halophilic enzyme most closely resembles the NAD(P)-linked glucose dehydrogenase from the thermophilic Archaeon Thermoplasma acidophilum . However, the halophilic glucose dehydrogenase appears to be a dimeric protein, in contrast to the tetrameric enzyme from the thermophile.

Microbiologia, 1996 Mar, 12(1), 75 - 84
Optimization of the production of a bacteriocin from Haloferax mediterranei Xia3; Platas G et al.; The optimal conditions for the production of the halocin H1, a 31 kDa bacteriocin-like molecule produced by the extreme halophilic Archaea Haloferax mediterranei Xia3 active against Gram-negative haloarchaea, was characterized . The physico-chemical conditions required for the optimal production of halocin H1 are similar to those found in the habitat in which the microorganism was isolated: 20% salt concentration and temperature range between 37 and 42 degrees C . Optimal antimicrobial activity was obtained using 0.5% of N-Z amine E as nutrient.

Microbiologia, 1996 Mar, 12(1), 55 - 60
{Effect of nutritional conditions on the viscosity and emulsifying capacity of V2-7 biopolymer from Volcaniella eurihalina}; Martinez-Checa F et al.; Volcaniella eurihalina is a moderately halophilic bacterium able to produce an exopolysaccharide (EPS) under different culture conditions . Rheological behavior of 1% EPS solutions varied depending on the conditions under which EPS were produced . The maximum viscosity was reached when maltose was used as carbon source . Limitations of phosphorus and sulfur also increased its viscosity power . On the other hand, the addition of residual oil products to the culture medium enhanced the production of this biosurfactant polymer.

Appl Environ Microbiol, 1996 Mar, 62(3), 766 - 71
Diversity of uncultured microorganisms associated with the seagrass Halophila stipulacea estimated by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes; Weidner S et al.; The diversity of microorganisms associated with the leaves of the seagrass Halophila stipulacea in the northern Gulf of Elat was examined by culture-independent analysis . Microorganisms were harvested by a sonication treatment for total-community genomic DNA isolation . Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used for PCR amplification . The 16S rDNA PCR products were subcloned and further characterized by a restriction fragment length analysis termed ARDRA (amplified rDNA restriction analysis) . These analyses were carried out after reamplifying the cloned fragments with two primers binding symmetrically to the plasmid immediately on both sides of the cloned insert . Computer-aided clustering was performed after separate restriction analysis with enzymes HinfI and HpaII . By this method, 103 cloned 16S rDNA fragments were clustered into a total of 58 different groups . Sequence analysis of clones with an identical ARDRA pattern confirmed that members of an ARDRA group were closely related to each other . The sequenced clones were found to be affiliated with a marine snow-associated plastid-like rRNA clone and with a marine Hyphomonas strain, respectively . The method applied in this study could be useful for the routine study of other microbial communities of interest.

J Bacteriol, 1996 Mar, 178(5), 1320 - 7
Isolation of an ftsZ homolog from the archaebacterium Halobacterium salinarium: implications for the evolution of FtsZ and tubulin; Margolin W et al.; We have isolated a homolog of the cell division gene ftsZ from the extremely halophilic archaebacterium Halobacterium salinarium . The predicted protein of 39 kDa is divergent relative to eubacterial homologs, with 32% identity to Escherichia coli FtsZ . No other eubacterial cell division gene homologs were found adjacent to H . salinarium ftsZ . Expression of the ftsZ gene region in H . salinarium induced significant morphological changes leading to the loss of rod shape . Phylogenetic analysis demonstrated that the H . salinarium FtsZ protein is more related to tubulins than are the FtsZ proteins of eubacteria, supporting the hypothesis that FtsZ may have evolved into eukaryotic tubulin.

Biochemistry, 1996 Feb 27, 35(8), 2526 - 34
Sequence evidence for strong conservation of the photoactive yellow proteins from the halophilic phototrophic bacteria Chromatium salexigens and Rhodospirillum salexigens; Koh M et al.; The photoactive yellow proteins (PYP) have been found to date only in three species of halophilic purple phototrophic bacteria . They have photochemical activity remarkably similar to that of the bacteria rhodopsins . In contrast to rhodopsins, however, the PYPs are small water-soluble proteins . We now report the complete amino acid sequences of Rhodospirillum salexigens and Chromatium salexigens PYP which allow comparison with the known sequence and three-dimensional structure of the prototypic protein from Ectothiorhodospira halophila . Although isolated from three different families of bacteria, the PYP sequences are 70-76% identical . All three contain 125 amino acid residues, and no insertions or deletions are necessary for alignment . This is a remarkable result when it is considered that electron transfer proteins from these purple bacterial species are only 25-40% identical and that insertions and deletions are needed for their proper alignment . It thus appears that PYP has the same important function in each of the purple bacteria and that most of the amino acid residues are necessary to maintain structure and function . By most standards, PYP would be called a "slowly evolving protein" . R . salexigens PYP is uniquely degraded by proteolysis at low ionic strength, probably as a consequence of unfolding due to electrostatic repulsion of the excess negative charge . Therefore it may also be classified as a "halophilic protein".

Biochim Biophys Acta, 1996 Feb 9, 1289(1), 14 - 24
NAD-glutamate dehydrogenase from Halobacterium halobium: inhibition and activation by TCA intermediates and amino acids; Bonete MJ et al.; A variety of metabolites have been found to elicit a form of inhibition or activation on an NAD-specific glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from Halobacterium halobium . The purified halophilic enzyme was tested with several compounds known to be allosteric modifiers of mammalian glutamate dehydrogenases to determine their effects on enzyme activity . GTP, ATP, ADP and AMP did not affect the enzyme, so these effectors of bovine glutamate dehydrogenase do not play a role in the regulation of the halophilic enzyme . However, the halophilic enzyme was subject to strong inhibition by TCA intermediates . When measuring the initial rate of the reaction, the oxidative deamination of L-glutamate was inhibited by TCA metabolites such as: fumarate, oxalacetate, succinate and malate; by substrate analogues such as: NADP+, D-glutamate and glutarate; and by dicarboxylic compounds such as adipate . On the other hand, all the amino acids tested were activators of this enzyme, except the D-isomer of the substrate L-glutamate that acted as an inhibitor . The relative effectiveness of each inhibitor or activator (Ki or Ka values) was correlated with the dipole moment (mu), HOMO and LUMO molecular orbital energies, optimal distance between two carboxyl groups, and hydrophobicity . Compounds with high dipole moment acted as good activators while compounds with low dipole moment were inhibitors . We have also found that the best activators were amino acids with no polar lateral chain.

Proteins, 1996 Feb, 24(2), 158 - 64
A complete relaxation matrix refinement of the solution structure of a paramagnetic metalloprotein: reduced HiPIP I from Ectothiorhodospira halophila; Bertini I et al.; We have accounted for the effect of paramagnetism on the intensities of NOEs in a 73-residue paramagnetic metalloprotein, the reduced high-potential iron sulfur protein ISO I from Ectothiorhodospira halophila, whose solution structure had been recently solved by us . The paramagnetic effects were dealt with through a suitably modified complete relaxation matrix approach . We have then recalculated the structure through a distance geometry program by minimizing the difference between the sixth roots of the calculated and experimental NOEs . The average RMSD, calculated on residues 4-71, within the structures constituting the two families decreased from 0.67 to 0.46 angstrom for backbone atoms and from 1.23 to 1.06 angstroms for all heavy atoms . The structures in the new family are for the most part within the indetermination of the previous, less resolved, family . A few specific differences are detected and related to the presence of non-negligible paramagnetic effects, which are now properly evaluated.

Arch Microbiol, 1996 Feb, 165(2), 97 - 105
Primary structure and properties of the formyltransferase from the mesophilic Methanosarcina barkeri: comparison with the enzymes from thermophilic and hyperthermophilic methanogens; Kunow J et al.; The ftr gene encoding formylmethanofuran: tetrahydromethanopterin formyltransferase (Ftr) from Methanosarcina barkeri was cloned, sequenced, and functionally expressed in Escherichia coli . The overproduced enzyme was purified eightfold to apparent homogeneity, and its catalytic properties were determined . The primary structure and the hydropathic character of the formyltransferase from Methanosarcina barkeri were compared with those of the enzymes from Methanobacterium thermoautotrophicum, Methanothermus fervidus, and Methanopyrus kandleri . The amino acid sequence of the enzyme from Methanosarcina barkeri was 64%, 61%, and 59% identical to that of the enzyme from Methanobacterium thermoautotrophicum, Methanothermus fervidus, and Methanopyrus kandleri, respectively . A negative correlation between the hydrophobicity of the enzymes and both the growth temperature optimum and the intracellular salt concentration of the four organisms was observed . The hydrophobicity of amino acid composition was +21.6 for the enzyme from Methanosarcina barkeri (growth temperature optimum 37 degrees C, intracellular salt concentrationapproximately 0.3 M), +9.9 for the enzyme from Methanobacterium thermoautotrophicum (65 degrees C,approximately 0.7 M), -20.8 for the enzyme from Methanothermus fervidus (83 degrees C,approximately 1.0 M) and -31.4 for the enzyme from Methanopyrus kandleri (98 degrees C, > 1.1 M) . Generally, a positive correlation between hydrophobicity and thermophilicity of enzymes and a negative correlation between hydrophobicity and halophilicity of enzymes are observed . The findings therefore indicate that the hydropathic character of the formyltransferases compared is mainly determined by the intracellular salt concentration rather than by temperature . Sequence similarities between the formyltransferases from methanogens and an open reading frame from Methylobacterium extorquens AM1 are discussed.

Arch Microbiol, 1996 Feb, 165(2), 106 - 13
The phylogenetic relationship among Ectothiorhodospiraceae: a reevaluation of their taxonomy on the basis of 16S rDNA analyses; Imhoff JF et al.; Sequences of the 16S rRNA gene were determined from all type strains of the recognized Ectothiorhodospira species and from a number of additional strains . For the first time, these data resolve the phylogenetic relationships of the Ectothiorhodospiraceae in detail, confirm the established species, and improve the classification of strains of uncertain affiliation . Two major groups that are recognized as separate genera were clearly established . The extremely halophilic species were removed from the genus Ectothiorhodospira and reassigned to the new genus Halorhodospira gen . nov., to recognize that the most halophilic eubacteria are species of this genus . These species are Halorhodospira halophila comb . nov., Halorhodospira halochloris comb . nov., and Halorhodospira abdelmalekii comb . nov . Among the slightly halophilic Ectothiorhodospira species, the classification of strains belonging to Ectothiorhodospira mobilis and Ectothiorhodospira shaposhnikovii was improved . Several strains that were tentatively identified as Ectothiorhodospira mobilis form a separate cluster on the basis of their 16S rDNA sequences and are recognized as two new species: Ectothiorhodospira haloalkaliphila sp . nov., which includes the most alkaliphilic strains originating from strongly alkaline soda lakes, and Ectothiorhodospira marina, describing isolates from the marine environment.

Biochemistry, 1996 Jan 30, 35(4), 1274 - 81
Chemical reactivity and spectroscopy of the thiol ester-linked p-coumaric acid chromophore in the photoactive yellow protein from Ectothiorhodospira halophila; Hoff WD et al.; We have recently identified p-coumaric acid as the chromophore of the photoactive yellow protein (PYP) from the purple sulfur bacterium Ectothiorhodospira halophila, a blue-light photoreceptor with rhodopsin-like photochemistry {Hoff, W . D., Dux, P., Hard, K., Nugteren-Roodzant, I . M., Crielaard, W., Boelens, R., Kaptein, R., Van Beeumen, J., & Hellingwerf, K . J . (1994) Biochemistry 33, 13959-13962} . Here we report on the chemistry of the linkage of this new photoactive cofactor to apoPYP: (i) Analysis of chromophore-peptide conjugates of PYP by high-resolution mass spectrometry unambiguously shows that the p-coumaric acid molecule is bound to Cys 69 via a thiol ester bond . The PYP chromophore is the first cofactor known to be stably thiol ester-linked to its apoprotein . (ii) The chemical reactivity of this thiol ester bond with respect to dithiothreitol, performic acid, and high pH is similar to that of disulfide bridges . These treatments result in the cleavage of the thiol ester bond, concomitant with strong shifts in the UV/vis absorbance band of the chromophore . (iii) The spectral properties of the PYP chromophore under different conditions are related to the structural integrity of the protein, the presence of the thiol ester bond, and the ionization state of the phenolic proton of the chromophore . These results are important for the general problem of spectral tuning in photoreceptor proteins.

FEBS Lett, 1996 Jan 22, 379(1), 43 - 6
Evidence for farnesol-mediated isoprenoid synthesis regulation in a halophilic archaeon, Haloferax volcanii; Tachibana A et al.; Farnesol strongly inhibited growth of a halophilic archaeon, Haloferax volcanii, with an IC50 value of only 2 microM (0.4 microgram/ml) in rich medium and 50 nM (0.01 microgram/ml) in minimal medium without lysis . Other isoprenoid alcohols such as isopentenol, dimethylallyl alcohol, geraniol, and geranylgeraniol at 500 microM did not affect its growth . Mevalonate, which is the precursor of all isoprenoid membrane lipids in archaea, led to recovery of the growth inhibition of H . volcanii, but acetate had no such effect . Farnesol inhibited incorporation of acetate, but not mevalonate, into the lipid fraction . These results suggest that farnesol inhibited the biosynthetic pathway from acetate (acetyl-CoA) to mevalonate . Farnesol is known to be derived from the important intermediate of isoprenoids, farnesyl diphosphate (FPP), and found in neutral lipid fraction from this archaeon . Moreover, the cell-free extracts from H . volcanii could phosphorylate farnesol with ATP to generate farnesyl monophosphate and FPP . We conclude that farnesol-mediated isoprenoid synthesis regulation system by controlling farnesol concentration is present in H . volcanii.

Biochim Biophys Acta, 1996 Jan 4, 1292(1), 140 - 4
Total reconstitution of active small ribosomal subunits of the extreme halophilic archaeon Haloferax mediterranei; Sanchez ME et al.; The small ribosomal subunit of the halophilic archaeon Haloferax mediterranei has been reconstituted from its dissociated rRNA and protein components . Efficient reconstitution of particles, fully active in poly(U)-dependent polyphenylalanine synthesis, occurs after 2 h of incubation at 36 degrees C in the presence of 1.5 M of (NH4)2SO4, 100 mM of MgAc2, 20 mM Tris-HCl (pH 8.2) and 6 mM 2-mercaptoethanol . Important differences in the optimal ionic conditions for the reconstitution of the 30S and the 50S ribosomal subunits from Haloferax mediterranei have been found . K+ and NH4+ ions have differing abilities to promote the reconstitution of the particles . The assembly of 30S ribosomal subunits of H . mediterranei has a higher tolerance to ionic strength than the assembly of the 50S subunits and it is independent of the Mg2+ concentration present in the system.

Biochimie, 1996, 78(6), 488 - 501
Enzymatic conversion of adenosine to inosine and to N1-methylinosine in transfer RNAs: a review; Grosjean H et al.; Inosine (6-deaminated adenosine) is a characteristic modified nucleoside that is found at the first anticodon position (position 34) of several tRNAs of eukaryotic and eubacterial origins, while N1-methylinosine is found exclusively at position 37 (3' adjacent to the anticodon) of eukaryotic tRNA(Ala) and at position 57 (in the middle of the psi loop) of several tRNAs from halophilic and thermophilic archaebacteria . Inosine has also been recently found in double-stranded RNA, mRNA and viral RNAs . As for all other modified nucleosides in RNAs, formation of inosine and inosine derivative in these RNA is catalysed by specific enzymes acting after transcription of the RNA genes . Using recombinant tRNAs and T7-runoff transcripts of several tRNA genes as substrates, we have studied the mechanism and specificity of tRNA-inosine-forming enzymes . The results show that inosine-34 and inosine-37 in tRNAs are both synthesised by a hydrolytic deamination-type reaction, catalysed by distinct tRNA:adenosine deaminases . Recognition of tRNA substrates by the deaminases does not strictly depend on a particular "identity' nucleotide . However, the efficiency of adenosine to inosine conversion depends on the nucleotides composition of the anticodon loop and the proximal stem as well as on 3D-architecture of the tRNA . In eukaryotic tRNA(Ala), N1-methylinosine-37 is formed from inosine-37 by a specific SAM-dependent methylase, while in the case of N1-methylinosine-57 in archaeal tRNAs, methylation of adenosine-57 into N1-methyladenosine-57 occurs before the deamination process . The T psi-branch of fragmented tRNA is the minimalist substrate for the N1-methylinosine-57 forming enzymes . Inosine-34 and N1-methylinosine-37 in human tRNA(Ala) are targets for specific autoantibodies which are present in the serum of patients with inflammatory muscle disease of the PL-12 polymyositis type . Here we discuss the mechanism, specificity and general properties of the recently discovered RNA:adenosine deaminases/editases acting on double-stranded RNA, intron-containing mRNA and viral RNA in relation to those of the deaminases acting on tRNAs.

J Clin Lab Anal, 1996, 10(2), 70 - 3
Purification, characterization, and pathogenicity of urease produced by Vibrio parahaemolyticus; Cai Y et al.; Vibrio parahaemolyticus is a halophilic bacterium associated with gastroenteritis and traveller's diarrhea . The urease-positive, Kanagawa-negative V . parahaemolyticus had been isolated from patients and the environment in the Pacific Northwest, first reported by Kelly et al . (5) . Recently, we purified the urease produced by a clinical isolated of V . parahaemolyticus, and its characterization and pathogenicity has been studied . The urease isolation procedure included a water extraction and anion exchange chromatography . The molecular weight for the native enzyme was 275 KDa, and the three subunits of 85 KDa, 59 KDa, and 33 KDa were determined . The isoelectric focusing of urease was 5.2 . The purified urease also can cause intestinal fluid accumulation and demonstrate a positive result in the suckling mouse test . These results suggested that the urease produced by V . parahaemolyticus may be another important indicator for the pathogenesis of the bacteria.

Z Naturforsch {C}, 1996 Jan-Feb, 51(1-2), 29 - 39
Isolation, characterization, and substrate specificity of the plasma membrane ATPase of the hlophilic achaeon Haloferax volcanii; Steinert K et al.; Isolated membranes of the moderate halophilic bacterium Haloferax volcanii are able to hydrolyze ATP via an ATPase, which needs the presence of Mg2+ or Mn2+, high concentrations of NaCl, a pH value of 9, and high temperatures with an optimum at 60 degrees C . We have not found any phosphatase activity in the preparations . We developed a purification method for the isolated enzyme with an enrichment factor of 90 . SDS-gel electrophoresis of the partially purified enzyme of Haloferax volcanii showed putative ATPase subunits of 63, 51, 37, and 12 kDa . N-ethylmaleimide (NEM) a specific inhibitor for V-ATPases, which alkylates cyteines, inhibited the enzyme slightly . Binding of tritiated NEM to the isolated ATPase fractions resulted in labelling of the 63 and 51 kDa peptides . Using PCR with degenerate oligonucleotides, we could clone and sequence a gene cluster encoding the A1 part of the halophilic ATPase . The described genes are organized in an operon in the order D, C, E, B, A, named alphabetically according to their decreasing size . The deduced products of 64.5, 52, 38.7, 22, and 11.6 kDa confirm the results of the partial purification of the ATPase . Biochemical characterization of the Haloferax volcanii ATPase gave the following results: In presence of Mn2+ higher rates of ATP hydrolysis could be observed than in presence of Mg2+, but free manganese ions inhibited the enzyme activity of the ATPase . Calculation of the true concentrations of the complex between ATP and the respective divalent metal ion led to determination of Michaelis-Menten constants for ATP in the hydrolysis direction of 1 mM in presence of MgCl2 and 0.24 mM in presence of MnCl2 . Sodium chloride concentrations in the molar range induce changes in KM by a factor of about 10 . The enzyme is specific for ATP; other nucleotides including GTP and ADP are competitive inhibitors of ATP hydrolysis.

Plant Mol Biol, 1996 Jan, 30(1), 65 - 75
Higher-plant chloroplast and cytosolic 3-phosphoglycerate kinases: a case of endosymbiotic gene replacement; Brinkmann H et al.; Previous studies indicated that plant nuclear genes for chloroplast and cytosolic isoenzymes of 3-phosphoglycerate kinase (PGK) arose through recombination between a preexisting gene of the eukaryotic host nucleus for the cytosolic enzyme and an endosymbiont-derived gene for the chloroplast enzyme . We readdressed the evolution of eukaryotic pgk genes through isolation and characterisation of a pgk gene from the extreme halophilic, photosynthetic archaebacterium Haloarcula vallismortis and analysis of PGK sequences from the three urkingdoms . A very high calculated net negative charge of 63 for PGK from H . vallismortis was found which is suggested to result from selection for enzyme solubility in this extremely halophilic cytosol . We refute the recombination hypothesis proposed for the origin of plant PGK isoenzymes . The data indicate that the ancestral gene from which contemporary homologues for the Calvin cycle/glycolytic isoenzymes in higher plants derive was acquired by the nucleus from (endosymbiotic) eubacteria . Gene duplication subsequent to separation of Chlamydomonas and land plant lineages gave rise to the contemporary genes for chloroplast and cytosolic PGK isoenzymes in higher plants, and resulted in replacement of the preexisting gene for PGK of the eukaryotic cytosol . Evidence suggesting a eubacterial origin of plant genes for PGK via endosymbiotic gene replacement indicates that plant nuclear genomes are more highly chimaeric, i.e . contain more genes of eubacterial origin, than is generally assumed.

Int J Syst Bacteriol, 1996 Jan, 46(1), 305 - 12
Spirochaeta alkalica sp . nov., Spirochaeta africana sp . nov., and Spirochaeta asiatica sp . nov., alkaliphilic anaerobes from the Continental Soda Lakes in Central Asia and the East African Rift; Zhilina TN et al.; During a study of microbial communities in athalassic bodies of water, three new species within the genus Spirochaeta were described . These are alkaliphilic Spirochaeta alkalica sp . nov . Z-7491 (DSM 8900) and halophilic S . africana sp . nov . Z-7692 (DSM 8902) from the soda-depositing Lake Magadi in Central Africa and haloalkaliphilic S . asiatica sp . nov . Z-7591 (DSM 8901) from Lake Khatyn, Central Asia . These mesophilic spirochetes develop at pHs of > 9 as anaerobic saccharolytic dissipotrophs . The DNA base compositions (moles percent G+C) of the strains were as follows: S . alkalica Z-7491, 57.1; S . africana Z-7692, 56.1; and S . asiatica Z-7591, 49.2 . The optimum growth parameters (temperature, pH, and NaCl concentration {percent, wt/vol}, respectively) were as follows: for S . alkalica Z-7491, 35 degrees C, 9.2, and 5 to 7%; for S . africana Z-7692, 35 degrees C, 9.3, and 5 to 7%; and for S . asiatica Z-7591, 35 degrees C, 8.9, and 3 to 6% . The products of glucose fermentation were acetate, hydrogen, ethanol, and lactate, in different proportions, for S . alkalica and S . africana; for S . asiatica, they were acetate, ethanol, and lactate . S . asiatica is strictly anaerobic, while S . alkalica and S . africana are rather aerotolerant . All three species group within the radiation of the majority of the species of the genus Spirochaeta . Studies of the genes encoding 16S rRNA indicate a possible fanning out of the phylogenetic tree of spirochetes.

J Bacteriol, 1996 Jan, 178(1), 309 - 13
Differentially transcribed regions of Haloferax volcanii genome depending on the medium salinity; Ferrer C et al.; To identify genomic regions involved in osmoregulation in the extremely halophilic archaeon Haloferax volcanii, we used a technique which involves hybridization of cDNAs obtained at different salinities against a cosmid library of the organism . Both low and high salt concentrations trigger differential expression; however, adaptation to low salinities seems to elicit a wider response . The presence of a large domain within the largest of the megaplasmids with a strong response to low salt concentrations is noteworthy.

Biochim Biophys Acta, 1995 Dec 6, 1253(2), 181 - 8
Occurrence and purification of the photoactive yellow protein of Ectothiorhodospira halophila (PYP) and of immunologically related proteins of Rhodospirillum salexigens and Chromatium salexigens and intracellular localization of PYP; Thiemann B et al.; The photoactive yellow protein of Ectothiohodospira halophila (PYP) was purified to homogeneity by an advanced method and applied as an affinity ligand for the isolation of an anti-PYP IgG fraction which was used for immunoscreening . The distribution of proteins immunologically related to PYP was investigated in protein fractions of 51 strains from 38 species of non-halophilic and halophilic phototrophic and chemotrophic eubacteria and archaeobacteria . Strong immunoreactive bands indicating the presence of authentic PYP on Western blots (apparent mass 17.8 kDa) was only found in the strains of E . halophila . Additionally, two soluble proteins of Chromatium salexigens and Rhodospirillum salexigens (apparent molecular masses 16.4 and 19 kDa, respectively) cross-reacted to approx . 6% and 4% . Analyses of cell fractions of E . halophila revealed that PYP is a cytoplasmic protein.

J Nat Prod, 1995 Dec, 58(12), 1950 - 4
Chemical constituents of halophilic facultatively anaerobic bacteria, 1; Fu X et al.; Two new nitrotyramine derivatives, 1 and 2, along with five known aromatic compounds, were isolated from the culture broth of a facultatively anaerobic, halophilic bacterium isolated from a sediment from the Great Salt Plains, Alfalfa County, Oklahoma . The structures of the new compounds were determined from spectral data and were confirmed by synthesis from tyramine hydrochloride . Compound 1 showed cytotoxicity against the murine leukemia P-388 cell line (IC50 3 micrograms/ml).

Plant Mol Biol, 1995 Dec, 29(6), 1127 - 42
Atypical phytochrome gene structure in the green alga Mesotaenium caldariorum; Lagarias DM et al.; The phytochrome photoreceptor in the green alga Mesotaenium caldariorum is encoded by a small family of highly related genes . DNA sequence analysis of two of the algal phytochrome genes indicates an atypical gene structure with numerous long introns . The two genes, termed mesphy1a and mesphy1b, encode polypeptides which differ by one amino acid in the region of overlap that was sequenced . RT-PCR studies have established the intron-exon junctions of both genes and show that both are expressed . RNA blot analysis indicates a single transcript of ca . 4.1 kb in length . The deduced amino acid sequence of the mesphy1b gene reveals that the photoreceptor consists of 1142 amino acids, with an overall structure similar to other phytochromes . Phylogenetic analyses indicate that the algal phytochrome falls into a distinct subfamily with other lower plant phytochromes . Profile analysis of an internal repeat found within the central hinge region of the phytochrome polypeptide indicates an evolutionary relatedness to the photoactive yellow protein from the purple bacterium Ectothiorhodospira halophila, to several bacterial sensor kinase family members, and to a family of eukaryotic regulatory proteins which includes the period clock (per) and single-minded (sim) gene products of Drosophila . Since mutations which alter phytochrome activity cluster within the region delimited by these direct repeats (P.H . Quail et al., Science 268 (1995): 675-680), this conserved motif may play an important role in the signal transducing function of these disparate protein families.

Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 432 - 8
Gene cloning, purification, and characterization of thermostable and halophilic leucine dehydrogenase from a halophilic thermophile, Bacillus licheniformis TSN9; Nagata S et al.; A halophilic and thermophilic isolate from the sand of Tottori Dune was found to produce a thermostable and halophilic leucine dehydrogenase (EC 1.4.1.9) . It was identified to be a new strain of Bacillus licheniformis . The enzyme gene was cloned into Escherichia coli JM109 with a vector plasmid pUC18 . The enzyme was purified to homogeneity from the clone cell extract by ion-exchange column chromatography with a yield of 31% . The enzyme was found to be composed of eight subunits identical in relative molecular mass (43,000) . The amino acid sequence of the enzyme, deduced from the nucleotide sequence of the gene, showed an identity of 84.6% with that of the B . stearothermophilus enzyme {Nagata S, Tanizawa K, Esaki N, Sakamoto Y, Oshima T, Tanaka H, Soda K (1988) Biochemistry 27:9056-9062}, although both enzymes were similar to each other in various enzymological properties such as thermostability, substrate and coenzyme specificities, and stereospecificity for hydrogen transfer from the C-4 of NADH . However, they were markedly distinct from each other in halophilicity; the B . licheniformis enzyme was much more stable than the other in the presence of high concentrations of salts.

FEMS Microbiol Lett, 1995 Dec 1, 134(1), 85 - 90
Isocitrate dehydrogenases from Haloferax volcanii and Sulfolobus solfataricus: enzyme purification, characterisation and N-terminal sequence; Camacho ML et al.; The isocitrate dehydrogenases from the extremely halophilic Archaeon, Haloferax volcanii, and from the hyperthermophilic Archaeon, Sulfolobus solfataricus, have been purified to electrophoretic homogeneity . The purified enzymes have been characterised with respect to their cofactor specificities, subunit compositions and their salt and thermal stabilities . N-terminal amino acid sequences have been determined for both enzymes, and multiple alignments with sequences of bacterial and eukaryotic isocitrate dehydrogenases show that the archaeal enzymes most closely resemble the NADP-linked dimeric isocitrate dehydrogenases from the Bacteria.

FEMS Microbiol Lett, 1995 Dec 1, 134(1), 115 - 9
Reevaluating the classification of Halobacteroides and Haloanaerobacter species based on sequence comparisons of the 16S ribosomal RNA gene; Patel BK et al.; The 16S rRNA gene (rDNA) sequence analysis of four halophilic anaerobes: Halobacteroides halobius, H . lacunaris . Haloanaerobacter (Hb.) chitinovorans and H . acetoethylicus confirmed that they were all members of the family Haloanaerobiaceae . H . lacunaris and H . halobius were found to be more closely related to each other and were distantly related to Sporohalobacter lortetti and the members of the genera Haloanaerobium and Halothermothrix . These data are in agreement with their assignment to the genus Halobacteroides . Further analysis indicated that Hb . chitinovorans was closely affiliated to members of the genus Halobacteroides, and therefore we propose to transfer it to the genus Halobacteroides as H . chitinovorans comb . nov . This transfer would invalidate the genus Haloanaerobacter, as Hb . chitinovorans is the only member of this genus . The 16S rDNA sequence analysis of H . acetoethylicum indicated that it was very closely related to members of the genus Haloanaerobium, viz . Haloanaerobium (Ha.) praevalens, Ha . salsugo, and Ha . alcaliphilum, and hence we propose to transfer it to the genus Haloanaerobium as Ha . acetoethylicus comb . nov.

Protein Sci, 1995 Dec, 4(12), 2562 - 72
The role of a conserved tyrosine residue in high-potential iron sulfur proteins; Iwagami SG et al.; Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A) . Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification . Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-11.0 . Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 O delta 1 and Lys-59 NH, respectively . The effect of the loss of the latter interaction is propagated through the Lys-59/Val-58 peptide bond, thereby perturbing Gly-46 . The delta delta GDapp of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter . CD measurements show that Tyr-12 influences several electronic transitions within the cluster . The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), respectively, higher than that of rcWT HiPIP . The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.

Curr Microbiol, 1995 Dec, 31(6), 365 - 71
Influence of salt concentration on the susceptibility of moderately halophilic bacteria to antimicrobials and its potential use for genetic transfer studies; Coronado MJ et al.; The influence of salinity on the susceptibility of 13 moderately halophilic collection strains belonging to the genera Chromohalobacter, Deleya, Halomonas, Vibrio, and Volcaniella to 10 common antimicrobials has been studied . Three different patterns of tolerance were found when salinity was varied from 10 to 1% (wt/vol) total salts in the testing media . The first one included the responses to ampicillin and rifampicin, where only minimal effects on the susceptibility were found . All moderate halophiles showed a high sensitivity to rifampicin regardless of the salt concentration . In the second group, including the responses to the aminoglycosides gentamycin, kanamycin, neomycin, and streptomycin, a remarkable and gradual increase of the toxicity was detected at lower salinities . Thirdly, the highest heterogeneity was found for the rest of antimicrobials assayed (trimethoprim, nalidixic acid, spectinomycin, and tetracycline), where the effect of salinity was moderate and dependent on both the individual strain and the antimicrobial tested . The data presented here should facilitate genetic studies on moderate halophiles . Thus, they simplify the design of selection media for genetic exchange experiments . Besides, by using low-salinity media, genes encoding resistance to a number of antimicrobials, especially to aminoglycosides, can be used as genetic markers for plasmids or transposons to be transferred to these extremophiles.

Eur J Biochem, 1995 Nov 15, 234(1), 24 - 31
Cartography of ribosomal proteins of the 30S subunit from the halophilic Haloarcula marismortui and complete sequence analysis of protein HS26; Engemann S et al.; By two-dimensional polyacrylamide gel electrophoresis of 30S ribosomal subunit proteins (S proteins) from Haloarcula marismortui we identified 27 distinct spots and analyzed all of them by protein sequence analysis . We demonstrated that protein HmaS2 (HS2) is encoded by the open reading frame orfMSG and has sequence similarities to the S2 ribosomal protein family . The proteins HmaS5 and HmaS14 were identified as spots HS7 and HS21/HS22, respectively . Protein HS4 was characterized by amino-terminal sequence analysis . The spot HS25 was recognized as an individual protein and also characterized by sequence analysis . Furthermore, the complete primary sequence of HS26 is reported, showing similarity only to eukaryotic ribosomal proteins . The sequence data of a further basic protein shows a high degree of similarity to ribosomal protein S12, therefore, it was designated HmaS12 . Slightly different results compared to published sequence data were obtained for the protein HS12 and HmaS19 . The putative 'ribosomal' protein HSH could not be localized in the two-dimensional pattern of the total 30S ribosomal subunit proteins of H . marismortui . Therefore, it seems to be unlikely that this protein is a real constituent of the H . marismortui ribosome.

Nucleic Acids Res, 1995 Nov 11, 23(21), 4312 - 9
A novel enzymatic pathway leading to 1-methylinosine modification in Haloferax volcanii tRNA; Grosjean H et al.; Transfer RNAs of the extreme halophile Haloferax volcanii contain several modified nucleosides, among them 1-methylpseudouridine (m1 psi), pseudouridine (psi), 2'-0-methylcytosine (Cm) and 1-methylinosine (m1l), present in positions 54, 55, 56 and 57 of the psi-loop, respectively . At the same positions in tRNAs from eubacteria and eukaryotes, ribothymidine (T-54), pseudouridine (psi-55), non-modified cytosine (C-56) and non-modified adenosine or guanosine (A-57 or G-57) are found in the so-called T psi-loop . Using as substrate a T7 transcript of Haloferax volcanii tRNA(Ile) devoid of modified nucleosides, the enzymatic activities of several tRNA modification enzymes, including those for m1 psi-54, psi-55, Cm-56 and m1l-57, were detected in cell extracts of H.volcanii . Here, we demonstrate that modification of A-57 into m1l-57 in H.volcanii tRNA(Ile) occurs via a two-step enzymatic process . The first step corresponds to the formation of m1A-57 catalyzed by a S-adenosylmethionine-dependent tRNA methyltransferase, followed by the deamination of the 6-amino group of the adenine moiety by a 1-methyladenosine-57 deaminase . This enzymatic pathway differs from that leading to the formation of m1l-37 in the anticodon loop of eukaryotic tRNA(Ala) . In the latter case, inosine-37 formation preceeds the S-adenosylmethionine-dependent methylation of l-37 into m1l-37 . Thus, enzymatic strategies for catalyzing the formation of 1-methylinosine in tRNAs differ in organisms from distinct evolutionary kingdoms.

Gene, 1995 Nov 7, 165(1), 17 - 24
Genetic organization of a small cryptic plasmid of Helicobacter pylori; Heuermann D et al.; A 2.9-kb cryptic plasmid of Helicobacter pylori (Hp), pHel1, was isolated and the complete nucleotide (nt) sequence was determined . An open reading frame (ORF1) was identified encoding a putative polypeptide of 63,709 Da, the existence and correct size of which was confirmed by T7 promoter expression analysis . The ORF1 sequence showed strong amino-acid sequence identity to a recently identified putative ORF1 protein of a cryptic Hp plasmid, pHPM180, and significant homologies to putative Rep proteins of Campylobacter coli (RepB) and Pediococcus halophilus (RepA), and was therefore designated RepA . A functional role of RepA in replication of pHel1 was demonstrated by the fact that only pHel1 plasmid derivatives with an intact repA gene were able to autonomously replicate in Hp . Upstream of repA, a 22-bp sequence was recognized which was tandemly repeated four and a half times, a feature typical for many replication origins (ori) and commonly termed a DNA iteron . Analysis of the repA upstream region by primer extension identified a transcription start point for the repA mRNA, but did not correspond to known consensus promoter sequences . Southern hybridizations using pHel1 as a probe under stringent conditions revealed that homologous sequences to pHel1 were present in nearly all plasmid-carrying Hp strains, but not in a plasmid-carrying Helicobacter felis strain, suggesting that this type of replicon is predominantly found in the Hp species.

Plasmid, 1995 Nov, 34(3), 157 - 64
Construction of novel shuttle vectors for use between moderately halophilic bacteria and Escherichia coli; Mellado E et al.; Shuttle vectors useful for the genetic manipulation of several moderately halophilic bacteria have been constructed . These vectors are based on the minimal replicon of pCM1, a cryptic plasmid from Chromohalobacter marismortui, combined with the useful properties of pUC18 plasmid (i.e., small size, high copy number, multiple cloning sites, lacZ fragment), as well as with the trimethoprim resistance gene as a selection marker for moderate halophiles . These vectors can be efficiently transferred by RP4-mediated conjugation from Escherichia coli to the moderate halophiles Chromohalobacter marismortui, Deleya halophila, Halomonas elongata, Halomonas subglaciescola, and Volcaniella eurihalina.

Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 739 - 49
A milestone in ribosomal crystallography: the construction of preliminary electron density maps at intermediate resolution; Schlunzen F et al.; Preliminary electron density maps of the large and the small ribosomal particles from halophilic and thermophilic sources, phased by the isomorphous replacement method, have been constructed at intermediate resolution . These maps contain features comparable in size with what is expected for the corresponding particles, and their packing arrangements are in accord with the schemes obtained by ab-initio procedures as well as with the motifs observed in thin sections of the crystals by electron microscopy . To phase higher resolution data, procedures are being developed for derivatization by specific labeling of the ribosomal particles at selected locations with rather small and dense clusters . Potential binding sites are being inserted either by site directed mutagenesis or by chemical modifications to facilitate cluster binding on the surface of the halophilic large and the thermophilic small ribosomal particles, which yield the crystals diffracting to highest resolution (2.9 and 7.3 A (1 A = 0.1 nm), respectively) . For this purpose, the surface of these ribosomal particles is being characterized and procedures are being developed for quantitative detachment of selected ribosomal proteins and for their incorporation into core particles . The genes of these proteins are being cloned, sequenced, mutated to introduce reactive side groups, mainly cysteines, and overexpressed . In parallel, two in situ small and stable complexes were isolated from the halophilic ribosome . Procedures for their crystal production in large quantities are currently being developed . Models, reconstructed at low resolution from crystalline arrays of ribosomes and their large subunits, are being used for initial low-resolution phasing of the X-ray amplitudes . The interpretation of these models stimulated the design and the crystallization of complexes mimicking defined functional states of a higher quality than those obtained for isolated ribosomes . These models also inspired modelling experiments according to results of functional studies, performed elsewhere, focusing on the progression of nascent proteins.

FEMS Microbiol Lett, 1995 Nov 1, 133(1-2), 127 - 30
Identification of the replication region of Lactobacillus acidophilus plasmid pLA103; Kanatani K et al.; The structure of the region necessary for replication of the plasmid pLA103 from Lactobacillus acidophilus TK8912 has been characterized . Sequence analysis revealed that the replication region contained an open reading frame (OrfA) encoding a 282-amino acid peptide preceded by a 22-bp tandem repeat sequence region . The predicted OrfA protein showed homology to the replication protein of a plasmid from Pediococcus halophilus . The plasmid containing the repeat sequence region preceding OrfA was able to replicate in the Lactobacillus host when provided with OrfA in trans, suggesting that the repeat sequence region contains the origin sequence essential for the pLA103 replication.

J Laryngol Otol, 1995 Nov, 109(11), 1082 - 4
Complicated suppurative otitis media in a Greek diver due to a marine halophilic Vibrio sp; Tsakris A et al.; Halophilic vibrios are distinct from non-cholera vibrios and have been recognized increasingly as potentially pathogenic bacteria in extraintestinal infections . A case of suppurative chronic otitis media in a Greek diver with Vibrio alginolyticus recovered from an ear drainage culture, is reported . The patient received appropriate antimicrobial therapy and her hearing improved significantly after a tympanoplasty type I was performed . The association of halophilic Vibrio spp . infections with prolonged seawater contact, particularly in subtropical climates, is discussed . In swimmers with extensive exposure to salt water, individual preventive measures and aetiological treatment of ear infections seems to be required in order to reduce the severity of possible Vibrio spp . ear infections.

Appl Environ Microbiol, 1995 Nov, 61(11), 3821 - 5
Development of a gene reporter system in moderately halophilic bacteria by employing the ice nucleation gene of Pseudomonas syringae; Arvanitis N et al.; The expression of the ice nucleation gene inaZ of Pseudomonas syringae in several moderate halophiles was investigated to establish its utility as a reporter for promoter activity and gene expression studies in these biotechnologically and environmentally important bacteria . A promoterless version of inaZ was introduced in two different restriction sites and at both orientations in a recombinant plasmid able to replicate in moderate halophiles and, in particular, within the sequence of its pHE1 part, a native plasmid of Halomonas elongata . One orientation of both recombinant constructs expressed high levels of ice nucleation activity in H . elongata and Volcaniella eurihalina cells, indicating that inaZ was probably introduced in the correct orientation downstream of putative native promoters . A recombinant construct carrying a tandem duplication of inaZ at the same orientation gave significantly higher ice nucleation activity, showing that inaZ is appropriate for gene dosage studies . The ice nucleation gene was also expressed in H . elongata and V . eurihalina under the control of Pbla (the promoter of the beta-lactamase gene of Escherichia coli) and Ppdc (the promoter of the pyruvate decarboxylase gene of Zymomonas mobilis) . One of the inaZ reporter plasmids expressing high levels of ice nucleation activity under the control of a native putative promoter was also transferred in Halomonas subglaciescola, Halomonas meridiana, Halomonas halodurans, and Deleya halophila . In all cases, Ice+ transconjugants were successfully isolated, demonstrating that inaZ is expressed in a wide spectrum of moderately halophilic species.

Eur J Biochem, 1995 Nov 1, 233(3), 809 - 14
Absolute requirement of ammonium sulfate for reconstitution of active 70S ribosomes from the extreme halophilic archaeon Haloferax mediterranei; Sanchez E et al.; Active 70S ribosomes from the halophilic archaeon Haloferax mediterranei have been reconstituted from their isolated rRNAs and proteins . The reconstitution procedure consists of a two-step incubation; first with 1 M ammonium sulfate and 100 mM magnesium acetate for 1 h at 42 degrees C, followed by a 90-min incubation at 50 degrees C after increasing the ammonium sulfate to 2 M final concentration . The total reconstitution of halophilic 70S ribosomes is a process with its own identity, which does not correspond to the conditions required for the reconstitution of the isolated subunits . Ammonium sulfate is the only salt capable of promoting the assembly of active ribosomes . The increase of ammonium sulfate salts in the second incubation step is obligatory for the isolation of functional particles.

Biochemistry, 1995 Oct 3, 34(39), 12669 - 72
Resonance Raman evidence that the thioester-linked 4-hydroxycinnamyl chromophore of photoactive yellow protein is deprotonated; Kim M et al.; Resonance Raman spectra of the ground state of photoactive yellow protein (PYP), a photoactive pigment found in Ectothiorhodospira halophila, have been obtained with excitation at 413.1 nm using a microspinning sample cell . The resonance Raman spectra of the thioester-linked 4-hydroxycinnamyl chromophore in the protein are compared with the preresonance Raman spectra of the 4-hydroxycinnamyl phenyl thioester and 4-hydroxycinnamic acid model compounds at various pH values . Bands at 1568, 1542, 1500, 1434, and 1166 cm-1 in the Raman spectrum of the anionic form of the 4-hydroxycinnamyl phenyl thioester are shown to be characteristic for the deprotonation of the chromophore . The observation of bands in PYP exhibiting very similar frequency and intensity patterns provides strong evidence that the chromophore in PYP is stabilized as a phenolate anion at pH 7.4, in support of conclusions from crystallographic studies . Furthermore, the insensitivity of the PYP Raman spectrum to placement of the protein in D2O buffer is consistent with the absence of the exchangeable phenolic proton on the cinnamyl chromophore . These results establish the feasibility of elucidating the molecular mechanism of light-to-information transduction by this new photosensory pigment with resonance Raman spectroscopy.

Int J Syst Bacteriol, 1995 Oct, 45(4), 790 - 7
Haloanaerobium lacusroseus sp . nov., an extremely halophilic fermentative bacterium from the sediments of a hypersaline lake; Cayol JL et al.; A new extremely halophilic chemoorganotrophic bacterium (strain H200T {T = type strain}) was isolated from the hypersaline sediments of Retba Lake in Senegal . This organism was a sluggishly motile, rod-shaped, non-spore-forming, gram-negative, obligate anaerobe that grew optimally at 40 degrees C in the presence of 180 to 200 g of NaCl per liter . The DNA base composition was 32 mol% guanine plus cytosine . The fermentation products from glucose were ethanol, acetate, H2, and CO2 . Yeast extract was required for growth . The fermentable substrates included D-fructose, galactose, D-xylose, cellobiose, lactose, maltose, sucrose, starch, D-mannitol, glycerol, and Casamino Acids . On the basis of the results of a 16S rRNA sequence analysis, strain H200T was found to be related to Haloanaerobium species . The 16S rRNA sequence of strain H200T differed from the sequences of the three previously described Haloanaerobium species, and strain H200T also differed from these organisms in its NaCl range for growth (60 to 340 g/liter); strain H200T grew in the presence of the highest NaCl concentration recorded for any halophilic anaerobic organism, including the three previously described Haloanaerobium species . We propose that strain H200T (= DSM 10165) belongs to a new Haloanaerobium species, Haloanaerobium lacusroseus.

Int J Syst Bacteriol, 1995 Oct, 45(4), 762 - 6
Natronococcus amylolyticus sp . nov., a haloalkaliphilic archaeon; Kanal H et al.; The alpha-amylase-producing haloalkaliphilic archaeon Natronococcus sp . strain Ah-36T (T = type strain) was isolated previously from a Kenyan soda lake, Lake Magadi . Most cells of strain Ah-36T occurred in irregular clusters, and the colonies were orange-red . The polar lipids of this organism were composed of C20, C20 and C20, C25 derivatives of phosphatidylglycerol and phosphatidylglycerophosphate . Phosphatidylglycero-(cyclo-) phosphate, which is characteristic of Natronococcus occultus, was not detected . The complete nucleotide sequence of the 16S rRNA gene revealed that the closest relative of strain Ah-36T is N . occultus ATCC43101T (level of similarity, 96.4%), an extremely halophilic archaeon . However, strain Ah-36T did not exhibit a significant level of DNA homology to N . occultus ATCC43101T, which represents the only previously described species in the genus Natronococcus . We describe a new species for strain Ah-36T, for which we propose the name Natronococcus amylolyticus.

Int J Syst Bacteriol, 1995 Oct, 45(4), 747 - 54
Halobaculum gomorrense gen . nov., sp . nov., a novel extremely halophilic archaeon from the Dead Sea; Oren A et al.; A novel extremely halophilic archaeon was isolated from the Dead Sea . This isolate is rod shaped and, like Halobacterium sodomense, requires a relatively low level of sodium ions for growth and a very high level of magnesium; optimal growth occurs in the presence of 0.6 to 1.0 M Mg2+ . The new strain resembles members of the Halobacterium saccharovorum-Halobacterium sodomense-Halobacterium trapanicum group in many physiological properties . However, the polar lipid composition of this organism is characteristic of representatives of the genus Haloferax; a sulfated diglycosyl diether is present, and the glycerol diether analog of phosphatidylglycerosulfate is absent . The G+C content of the DNA is 70 mol% . We found that on the basis of 16S rRNA sequence data our new isolate occupies a position intermediate between the position of the Halobacterium saccharovorum group and the position of the genus Haloferax and is sufficiently different from the previously described members of the Halobacteriaceae to justify classification in a new species and a new genus . We propose the name Halobaculum gomorrense gen . nov., sp . nov . for this organism; the type strain is strain DSM 9297.

Int J Syst Bacteriol, 1995 Oct, 45(4), 712 - 6
Phylogenetic inferences and taxonomic consequences of 16S ribosomal DNA sequence comparison of Chromohalobacter marismortui, Volcaniella eurihalina, and Deleya salina and reclassification of V . eurihalina as Halomonas eurihalina comb . nov; Mellado E et al.; The phylogenetic positions of the moderately halophilic bacteria Chromohalobacter marismortui, Volcaniella eurihalina, and Deleya salina were determined by PCR amplification of rRNA genes and direct sequencing . The resulting data were compared with data for other bacteria obtained from 16S rRNA sequence databases . C . marismortui, V . eurihalina, and D . salina clustered phylogenetically within the gamma subclass of the Proteobacteria and are closely related to other species on the Halomonas-Deleya branch . C . marismortui belongs in the family Halomonadaceae and has the characteristic 16S rRNA signatures defined for this family, including the distinctive cytosine residue at position 486 found in all members of the Halomonadaceae . V . eurihalina is closely related to the type species of the genus Halomonas, Halomonas elongata, and we formally propose that V . eurihalina should be transferred to the genus Halomonas as Halomonas eurihalina comb . nov . The type strain of this species is strain F9-6 (= ATCC 49336) . D . salina is not as closely related to other species belonging to the Halomonas-Deleya complex, but is more closely related to Halomonas elongata than to Deleya aquamarina, the type species of the genus Deleya . A polyphasic approach will be necessary to determine the natural taxonomic positions of the species belonging to the genera Halomonas and Deleya, as well as C . marismortui, V . eurihalina, Halovibrio variabilis, and Paracoccus halodenitrificans.

Anal Biochem, 1995 Sep 20, 230(2), 290 - 4
Halophilic protein stabilization by the mild solubilizing agents nondetergent sulfobetaines; Vuillard L et al.; In this work, experiments performed on pig heart and halophilic malate dehydrogenase as well as halophilic elongation factor Tu demonstrate a protein stabilization property from the recently described mild solubilizing agents nondetergent sulfobetaines . A practical application is given by the separation of halophilic bacteria elongation factor Tu and halophilic malate dehydrogenase by high-performance ion-exchange chromatography achieved at reduced salt levels without significant loss of activity.

Biochem Mol Biol Int, 1995 Sep, 37(1), 107 - 15
Organization and nucleotide sequence of a gene cluster comprising the translation elongation factor 1 alpha, ribosomal protein S10 and tRNA(Ala) from Halobacterium halobium; Fujita T et al.; Lambda EMBL clone containing a gene cluster coding for the translation elongation factor 1alpha, ribosomal protein S10 and tRNA(ala) was identified in a genomic library for the halophilic archaebacterium Halobacterium halobium using a PCR probe amplified by two oligonucleotide primers for conserved amino acid sequences of the elongation factor 1 alpha family . The gene coding for elongation factor EF-2 was also found 4.3kb upstream from the 5'end of the elongation factor 1 alpha by hybridization analysis using a DNA fragment specific for EF-2 from Halobacterium halobium {1} . Halobacterial and eukaryotic elongation factor 1 alpha homologues are very similar in sequence and in length and appear to be more closely related to each other than to the eubacterial protein.

Res Microbiol, 1995 Sep, 146(7), 543 - 50
The genus Methylophaga, a new line of descent within phylogenetic branch gamma of Proteobacteria; Janvier M et al.; The genus Methylophaga with two species, M . marina and M . thalassica, comprises halophilic methylotrophic bacteria . These organisms utilise C1 compounds through the ribulose monophosphate pathway and are unable to grow on methane . Nearly complete 16S rRNA sequences were obtained for both Methylophaga species by directly sequencing the amplified 16S rRNA gene . These sequences were compared with published 16S rRNA sequences of methylotrophic strains and a large number of marine bacterial strains including several members of the alpha, beta and gamma subclasses of Proteobacteria . Phylogenetic trees were inferred using both parsimony and distance matrix methods . Each topology was analysed by bootstrap . The genus Methylophaga was found to be clearly separated from other methylotrophic bacteria and formed a distinct branch within the gamma subclass of Proteobacteria.

Biochem Mol Biol Int, 1995 Aug, 36(6), 1225 - 34
Effects of divalent cations and nucleotides on the 14CO2-oxaloacetate exchange catalyzed by the phosphoenol pyruvate carboxykinase from the moderate halophile, Vibrio costicola; Salvarrey MS et al.; The phosphoenolpyruvate carboxykinase (PEPCK) from Vibrio costicola catalyzed a 14CO2-oxaloacetate exchange reaction with an unusual nucleotide specificity . ATP gave the higher apparent catalytic efficiency (Vmax/Km, 6.78), followed by GTP (1.30), CTP (0.87) and ITP (0.66) . Maximal activity required a divalent cation; CdCl2 and MgCl2 synergistically activated the enzyme, when added in the presence of MnCl2 . The sigmoidal saturation curve for MnCl2 (apparent n 2.11) was converted into a hyperbola by 0.01 mM CdCl2 (apparent n 1) . The results suggest a double role of the divalent cation in the reaction mechanism, namely as part of the MeATP2- substrate and as free Me2+ . Mn2+ would be the best for the first, and Cd2+ for the second role . Preincubation with 0.01 mM CdCl2 increased the activity of the enzyme assayed with MgATP2- through an increase in Vmax; addition of CdCl2 to the reaction mixture elicited further activation, through a 17-fold decrease in the apparent Km for MgATP2- . These results, together with the biphasic curve of activation by CdCl2 when used alone, suggest the existence of two different sites for free Cd2+ on the enzyme.

Biokhimiia, 1995 Aug, 60(8), 1261 - 7
{Halophilic archaea flagella: biochemical and genetic analysis}; Serganova IS et al.; The protein compositions of archaebacteria (Halobacterium salinarium, Halobacterium volcanii, Halobacterium saccharovorum and Natronobacterium pharaonis 12) flagella have been studied . It was found that flagella of these archaebacterial species are made up of flagellins . The flagellins of H . salinarium, H . volcanii and H . saccharovorum are glycosylated . Based on the known primary sequences of Halobacterium halobium R1M1 flagellin genes, oligonucleotides to the 5'- and 3'-ends of locus A containing two out of five such genes have been synthesized . The amplified by primers fragment of chromosomal DNA coding for H . halobium flagellins A1 and A2 was used as a probe for detecting homologous sites in archaebacterial DNA . Southern blotting hybridization revealed that the DNA of all archaebacterial species tested in this study contains sequences that are homologous to genes flg A1 and flg A2 of H . halobium.

Infect Immun, 1995 Jul, 63(7), 2418 - 23
Purification and characterization of a Chinese hamster ovary cell elongation factor of Vibrio hollisae; Kothary MH et al.; The halophilic bacterium Vibrio hollisae, isolated from patients with diarrhea, produces an extracellular toxin which elongates Chinese hamster ovary (CHO) cells . We purified this toxin to homogeneity by sequential ammonium sulfate precipitation, gel filtration with Sephacryl S-200, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, ion-exchange chromatography with DEAE-Sephadex A-50, and affinity chromatography . The toxin is heat labile and sensitive to proteases, with an isoelectric point of about 6.5 and molecular weights of about 83,000 and 80,000, as estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively . The toxin did not react with immunoaffinity-purified antibodies to cholera toxin in a plate enzyme-linked immunosorbent assay and in a Western blot, and its activity could not be neutralized by anti-cholrea toxin serum . Mixed gangliosides and gangliosides GM1, GD1a, GD1b, Gq1b, GT1b, GD2, GD3, GM2, and GM3 failed to block its activity . Elongation of CHO cells induced by the toxin was not accompanied by an increase in the levels of cyclic AMP . The toxin induced intestinal fluid accumulation in suckling mice . These results and the lack of homology between V . hollisae DNA and DNA coding for cholera toxin or the heat-labile toxin of Escherichia coli suggest that the V . hollisae toxin is structurally and functionally different from other CHO cell-elongating toxins.

Mol Microbiol, 1995 Jul, 17(1), 85 - 93
Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning; Mojica FJ et al.; We report the presence of long stretches of tandem repeats in the genome of the halophilic Archaea Haloferax mediterranei and Haloferax volcanii . A 30 bp sequence with dyad symmetry (including 5 bp inverted repeats) was repeated in tandem, interspersed with 33-39 bp unique sequences . This structure extends for long stretches--1.4 kb at one location in H . mediterranei chromosome and about 3 kb in the H . volcanii chromosome . The tandem repeats (designated TREPs) show a similar distribution in both organisms, appearing once or twice in the H . volcanii and H . mediterranei chromosomes, and once in the largest, probably essential megaplasmid of each organism but not in the smaller replicons . Sequencing of the structures in both H . volcanii replicons revealed an extremely high sequence conservation in both replicons within the species, as well as in the different organisms . Homologous sequences have also been found in other more distantly related halophilic members of the Archaea . Transformation of H . volcanii with a recombinant plasmid containing a 1.1 kb fragment of the TREPs produced significant alterations in the host cells, particularly in terms of cell viability . The introduction of extra copies of TREPs within the vector significantly alters the distribution of the genome among the daughter cells, as observed by DAPI staining . Although the precise biological role cannot be completely ascertained, all the data conform with the tandem repeats being involved in replicon partitioning in halobacteria.

Eur J Biochem, 1995 Jun 15, 230(3), 906 - 13
Formylmethanofuran:tetrahydromethanopterin formyltransferase (Ftr) from the hyperthermophilic Methanopyrus kandleri . Cloning, sequencing and functional expression of the ftr gene and one-step purification of the enzyme overproduced in Escherichia coli; Shima S et al.; Methanopyrus kandleri is a methanogenic Archaeon that grows on H2 and CO2 at a temperature optimum of 98 degrees C . The gene ftr encoding the formylmethanofuran:tetrahydromethanopterin formyltransferase, an enzyme involved in CO2 reduction to methane, has been cloned, sequenced, and overexpressed in Escherichia coli . The overproduced enzyme could be purified in yields above 90% by simply heating the cell extract to 90 degrees C in 1.5 M K2HPO4 pH 8.0 for 30 min . From 1 g wet cells (70 mg protein) approximately 14 mg formyltransferase was obtained . The purified enzyme showed essentially the same catalytic properties as that purified from M . kandleri cells . The primary structure and properties of the formyltransferase are compared with those of the enzyme from Methanobacterium thermoautotrophicum (growth temperature optimum 65 degrees C) and Methanothermus fervidus (83 degrees C) . Of the three enzymes that from M . kandleri had the lowest isoelectric point (4.2) and the lowest hydrophobicity of the amino acid composition . The enzyme from M . kandleri had the relatively highest content in alanine, glutamate and glutamine and the relatively lowest content in isoleucine, leucine and lysine . These properties, some of which are unusual for enzymes from other hyperthermophilic organisms, may reflect that the formyltransferase from M . kandleri is adapted to both hyperthermophilic and halophilic conditions.

Eur J Biochem, 1995 Jun 15, 230(3), 1088 - 95
Mutation at a single acidic amino acid enhances the halophilic behaviour of malate dehydrogenase from Haloarcula marismortui in physiological salts; Madern D et al.; In a statistical analysis of the amino acid compositions of 26 halophilic proteins, 24 showed an increase in acidic amino acids and a decrease in basic ones when compared to their non-halophilic homologues . The role of acidic residues in halophilic adaptation was investigated by site-directed mutagenesis of malate dehydrogenase (MalDH) from Haloarcula marismortui . In all of 40 non-halophilic homologous proteins, the position aligned with E243 in halophilic MalDH is occupied by a non-acidic amino acid, most frequently by arginine . The E243R mutant of halophilic MalDH was constructed, over-expressed in Escherichia coli, renatured and purified . Its salt-dependent catalytic activity was not affected compared to the wild-type enzyme and both proteins have the same Km values for their substrates . The resistance to denaturation of the mutant was compared to that of the wild-type protein in different physiological salt (NaCl or KCl) and temperature conditions and interpreted in terms of classical quasi-thermodynamic parameters . The mutant is more halophilic than the wild-type protein; it is more sensitive to temperature and requires significantly higher concentrations of NaCl or KCl for equivalent stability . These results highlight the role of acidic amino acids in halophilic behaviour and are in agreement with a model in which these amino acids act cooperatively to organise hydrated ion binding to the protein.

Biochim Biophys Acta, 1995 Jun 12, 1249(2), 137 - 44
Nucleotide sequence of the ATPase A- and B-subunits of the halophilic archaebacterium Haloferax volcanii and characterization of the enzyme; Steinert K et al.; Using PCR with degenerate oligonucleotides, we amplified two conserved regions of the plasma membrane ATPase A (alpha)- and B (beta)-subunit-encoding genes from Haloferax volcanii, a halophilic archaeon . The amplified fragments were cloned and sequenced, and then used as homologous probes to clone genomic restriction fragments, one of which contained the nearly complete atpA- and atpB-gene . To complete the latter one, we sequenced a region of an overlapping fragment using synthetic oligonucleotides as primers . The deduced amino-acid sequence showed a high degree of similarity with the A and B sequences of Halobacterium halobium-ATPase, and a relatively high degree with Daucus carota V-ATPase, but less similarity to F-ATPase alpha- and beta-subunits . Like in V-ATPases, the A-subunit is more related to the catalytic F-ATPase beta-subunit, whereas B corresponds to alpha . Cross-reaction of antibodies against CF1-alpha (synthetic peptide) with B and CF1-beta with A of Haloferax volcanii confirmed this finding . The ATPase of Haloferax volcanii is a halophilic enzyme, the amino-acid sequences contain about 20% negatively charged residues . This is discussed in terms of adaption to hypersaline conditions . The enzyme is specific for ATP, we determined KM values for ATP in presence of Mn2+ and Mg2+, respectively . Other nucleotides, including GTP and ADP are competitive inhibitors of ATP hydrolysis . Despite of the high degree of similarity to the Halobacterium enzyme, the ATPase showed no sensitivity to most of the tested inhibitors of ATP hydrolysis . NEM, a specific inhibitor for V-ATPases inhibited the enzyme only slightly . Bafilomycin, NBD-Cl and nitrate showed no effect . These results will be discussed in context with some specific differences in the primary structure of the Haloferax A-subunit.

J Bacteriol, 1995 Jun, 177(12), 3443 - 50
Characterization of the basic replicon of pCM1, a narrow-host-range plasmid from the moderate halophile Chromohalobacter marismortui; Mellado E et al.; The moderately halophilic bacterium Chromohalobacter marismortui contains a 17.5-kb narrow-host-range plasmid, pCM1, which shows interesting properties for the development of cloning vectors for the genetic manipulation of this important group of extremophiles . Plasmid pCM1 can stably replicate and is maintained in most gram-negative moderate halophiles tested . The replication origin has been identified and sequenced, and the minimal pCM1 replicon has been localized to a 1,600-bp region which includes two functionally discrete regions, the oriV region and the repA gene . oriV, located on a 700-bp fragment, contains four iterons 20 bp in length adjacent to a DnaA box that is dispensable but required for efficient replication of pCM1, and it requires trans-acting functions . The repA gene, which encodes a replication protein of 289 residues, is similar to the replication proteins of other gram-negative bacteria.

Biophys Chem, 1995 Jun-Jul, 55(1-2), 31 - 41
Crystallography of ribosomes: attempts at decorating the ribosomal surface; Sagi I et al.; Crystals of various ribosomal particles, diffracting best to 2.9 A resolution were grown . Crystallographic data were collected from shock frozen crystals with intense synchrotron radiation at cryo temperature . For obtaining phase information, monofunctional reagents were prepared from an undecagold and a tetrairidium cluster, by attaching to them chemically reactive handles, specific for sulfhydryl moieties . Heavy-atom derivatives were prepared by a specific and quantitative binding of the undecagold cluster to an exposed sulfhydryl prior to the crystallization . To create potential binding sites on the halophilic and thermophilic ribosomal particles, which yield our best and most interesting crystals, exposed reactive moieties were inserted, using genetic and chemical procedures . In order to choose the appropriate locations for these insertions, the surfaces of the ribosomal particles were mapped by direct chemical determination of exposed amino and sulfhydryl groups.

Biophys Chem, 1995 Jun-Jul, 55(1-2), 137 - 51
Bacteriorhodopsin: the mechanism of 2D-array formation and the structure of retinal in the protein; Watts A; Bacteriorhodopsin, the light driven proton pump of the extreme halophilic bacterium H . salinarium, is an integral membrane protein (M(r) ca . 26000) which forms 2D arrays in the purple membrane of the bacterium . It is this feature which has permitted the use of electron diffraction methods to resolve the protein structure to some degree of atomic detail, although the prosthetic group has not been fully resolved . However, the features which induce the protein to form these arrays have not been previously clarified . We have now shown that the protein array formation is driven by specific interaction of the protein with the charged phospholipid, phosphatidyl glycerol phosphate (or the sulphate derivative), a major (ca . 60%) lipid of the bacterial host membrane . In addition, in an effort to provide further structural information about the chromophore, retinal, of this protein, the orientation of the individual methyl groups of retinal have been determined from solid state deuterium NMR studies of the deuterated chromophore when in the protein binding site . This approach to structural resolution of the prosthetic group is ab initio, agrees with other studies on the chromophore and resolves new features of the bound retinal to a high degree (+/- 2 degrees) of precision . Here, these two studies on this integral membrane protein will be reviewed.

FEMS Microbiol Lett, 1995 May 15, 128(3), 293 - 9
Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion of transposon Tn5 and Tn1732 in Halomonas elongata; Kunte HJ et al.; Molecular genetic studies of halophilic eubacteria have been limited by the lack of a suitable method for mutagenesis . To overcome this, we established a transposon mutagenesis procedure for the ectoine-producing, halophilic bacterium Halomonas elongata . We used suicide plasmids pSUP101 and pSUP102-Gm to introduce the transposons Tn5 and Tn1732 respectively into H . elongata via Escherichia coli SM10 mediated conjugation . Our finding that H . elongata is sensitive to aminoglycoside antibiotics at low salinity enabled us to apply transposons that mediate kanamycin resistance . The insertions of transposon Tn1732 occurred at different sites in the chromosome of H . elongata, as proved by Southern hybridization analysis . Phenotypic analysis revealed that different auxotrophic and salt sensitive mutants were generated by mutagenesis with transposon Tn1732 . To our knowledge this is the first report of a successful application of a transposon for direct generalized mutagenesis in a halophilic eubacterium.

FEMS Microbiol Lett, 1995 May 1, 128(2), 167 - 75
pUCL287 plasmid from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315 represents a new theta-type replicon family of lactic acid bacteria; Benachour A et al.; A cryptic plasmid, pUCL287, was isolated from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315 . It had a theta-type mechanism of replication in its natural host . Its minimal replicon, Rep287, was isolated on a 1.6-kb EcoRI fragment . The Rep287 host range included the genera Pediococcus, Enterococcus, Lactobacillus and Leuconostoc but not genus Lactococcus . Plasmids hybridizing to pUCL287 are rare among lactic acid bacteria . As assessed by hybridization, Rep287 is dissimilar to pAM beta 1, pIP501 and pUCL22, representatives of the most common theta-type replicon groups in Gram-positive bacteria . Therefore, pUCL287 appears to represent a new theta-type replicon family from lactic acid bacteria.

J Protein Chem, 1995 May, 14(4), 189 - 95
Amino acid sequence of the ribosomal protein HS23 from the halophilic Haloarcula marismortui and homology studies to other ribosomal proteins; Engemann S et al.; The ribosomal protein HS23 from the 30S subunit of the extreme halophilic Haloarcula marismortui, belonging to the group of archaea, was isolated either by RP-HLPLC or two-dimensional polyacrylamide gel electrophoresis . The complete amino acid sequence was determined by automated N-terminal microsequencing . The protein consists of 123 residues with a corresponding molecular mass of 12,552 Da as determined by electrospray mass spectroscopy; the pI is 11.04 . Homology studies reveal similarities to the eukaryotic ribosomal protein S8 from Homo sapiens, Rattus norvegicus, Leishmania major, and Saccharomyces cerevisiae.

Biochemistry, 1995 Apr 11, 34(14), 4828 - 38
Effects of substitution of tyrosine 57 with asparagine and phenylalanine on the properties of bacteriorhodopsin; Govindjec R et al.; Tyrosine 57 is one of the residues present in the retinal binding pocket and is conserved in all the halophilic retinal proteins . We have studied mutants of bacteriorhodopsin, expressed in Halobacterium salinarium, in which tyrosine 57 is replaced by an asparagine (Y57N) or phenylalanine (Y57F) . In Y57N the photocycle proceeds only up to the L intermediate; no M is formed at neutral pH . The lifetime of L intermediate is extremely long, ca . 500 ms . Proton release is severely affected in both the mutants which suggests that Y57 is associated with the proton release pathway . By comparing the pH-induced absorption changes in the UV in Y57N and Y57F with those in the wild-type (WT), we determined that the pKa of Y57 is 10.2 . In Y57F, which shows M formation, the rate constant of the L-->M transition is pH dependent (pKa 8.7) suggesting that Y57 is probably not the residue that normally controls the transition into the alkaline photocycle . Y57 is either part of the counterion complex or in close proximity to D85 since its mutation influences the pKa of Asp85 . In Y57F the pKa of D85 is approximately 4.9 (compared to approximately 2.9 in the WT) . The Y57N mutant shows two pKa's in the purple to blue transition, approximately 3.8 and < 1 . In the presence of hydroxylamine, at neutral pH, Y57N is stable in the dark but bleaches very rapidly upon illumination compared to the WT . Since the lifetime of L intermediate is long in Y57N, we suggest that the Schiff base becomes accessible to hydroxylamine in this state.

J Virol, 1995 Apr, 69(4), 2322 - 7
Halophage HF2: genome organization and replication strategy; Nuttall SD et al.; Halophage HF2 is a lytic, broad-host-range bacteriophage of the extremely halophilic domain Archaea . It has a 79.7-kb double-stranded DNA genome which is linear, contains no modified nucleotides, and is not susceptible to cleavage by many type II restriction endonucleases . This insensitivity is attributed to selection against palindromic restriction sites, a commonly observed feature of broad-host-range phages . Interestingly, enzymes that did cut the genome recognized AT-rich sites, and five such enzymes, DraI, AseI, HpaI, HindIII, and SspI, were used to construct a physical map of the genome . Southern hybridization experiments used to order fragments on the map indicated homologies between the phage termini, and subsequent sequence analysis showed that HF2 possessed 306-bp direct terminal repeats . The presence of such repeats suggested replication through concatameric intermediates, and this was confirmed by analysis of the state of the phage genome in infected cells . This is a replication strategy adopted by many well-studied bacterial phages, for example T3 and T7 . Other similarities between the terminal repeats of T3 or T7 and HF2 include a putative nick site at the repeat border and a series of short imperfect repeats . These observations suggest a long evolutionary history for concatamer-based strategies of phage replication, possibly predating the divergence of Archaea/Eucarya and Bacteria, or alternatively, indicate possible lateral transfer of phage genes or modules between the domains Archaea and Bacteria.

Arch Biochem Biophys, 1995 Apr 1, 318(1), 1 - 5
Life in unusual environments: progress in understanding the structure and function of enzymes from extreme halophilic bacteria; Eisenberg H; Extreme halophilic archaea are saturated with salt and the intracellular electrolyte concentration exceeds that of the extracellular environment . Enzymes and other proteins from extreme halophilic archaea have been purified for many years and studied by biochemical and biophysical solution methodologies . They are active and stable at multimolar salt concentrations and denature below 2 to 3 M NaCl or KCl . Adaptation to these high concentrations of salt, genetic and evolutionary aspects, and the possibility of biotechnological applications are problems of considerable interest . Since the status of this fascinating field of research was reviewed in 1992, malate dehydrogenase from Haloarcula marismortui, now known to be a tetramer, was sequenced, its gene was cloned and expressed in active form, and its physical properties were redefined . A single mutation of Arg100 (in the enzyme active site) to Gln switched the enzyme specificity from malate to lactate dehydrogenase . Recent determination of its molecular structure by X-ray crystallography (O . Dym et al., in press) provides an exciting basis for the understanding of the structure and function of extreme halophilic enzymes . A major problem which so far has not been tackled in the study of extreme halophilic archaea is the understanding of protein nucleic acid interactions which are essential for the performance of biological function . Whereas the stability and activity of enzymes and other proteins can be modified to perform at high salt concentrations by use of currently known structural concepts, the existence of meaningful protein nucleic acid interactions in physiological concentrations of 4 to 5 M KCl constitutes an unsolved enigma worth intensive investigation.

Int J Syst Bacteriol, 1995 Apr, 45(2), 319 - 26
Isolation and characterization of Rhodovulum strictum sp . nov . and some other purple nonsulfur bacteria from colored blooms in tidal and seawater pools; Hiraishi A et al.; Several strains of phototrophic purple nonsulfur bacteria were isolated from colored blooms occurring in tidal and seawater pools in Japan . All of these isolates had ovoid to rod-shaped cells that were motile by means of single polar flagella and contained vesicular intracytoplasmic membranes together with bacteriochlorophyll a and carotenoids of the spheroidene series . They produced ubiquinone 10 as the major quinone and contained straight-chain fatty acids, with C18:1 predominating . They were mesophilic, halophilic, and photoheterotrophic, utilized sulfide and thiosulfate as electron donors for phototrophic growth, and photoassimilated a wide variety of organic compounds as carbon sources . Our results suggested that all of these isolates are members of the recently described genus Rhodovulum . The isolates were classified into four groups (designated groups I through IV) on the basis of phenotypic and genotypic data . The group I isolates, which were the most abundant purple nonsulfur bacteria recovered from the blooms, grew in the presence of NaCl concentrations ranging from 0.5 to 3.0% (optimum NaCl concentration, 0.8%) and at pH values ranging from 7.5 to 9.0 (optimum pH, 8.0 to 8.5) . On the basis of these unique physiological traits, together with genotypic and phylogenetic data, we propose that the group I isolates should be classified as members of a new species, Rhodovulum strictum . The group II isolates were identified definitely as Rhodovulum sulfidophilum, and the group III and IV isolates were phenotypically most similar to R . sulfidophilum and Rhodovulum adriaticum, respectively, but could be differentiated from these species by DNA-DNA pairing data.

Int J Syst Bacteriol, 1995 Apr, 45(2), 301 - 7
Haloanaerobium alcaliphilum sp . nov., an anaerobic moderate halophile from the sediments of Great Salt Lake, Utah; Tsai CR et al.; A strictly anaerobic, moderately halophilic, gram-negative, rod-shaped bacterium was isolated from Great Salt Lake, Utah, sediments and designated GSLST (T = type strain) . Strain GSLST grew optimally at pH 6.7 to 7.0 but had a very broad pH range for growth (pH 5.8 to 10.0) . The optimum temperature for growth was 37 degrees C, and no growth occurred at 15 or 55 degrees C . The optimum salt concentration for growth was 10% . Strain GSLST required yeast extract and Trypticase peptone to ferment carbohydrates, pyruvate, and glycine betaine . Strain GSLST was resistant to penicillin, D-cycloserine, tetracycline, and streptomycin . The G + C content of this isolate was 31 mol% . The fermentation products from glucose utilization were acetate, butyrate, lactate, CO2, and H2, and in addition strain GSLST fermented glycine betaine to acetate and trimethylamine . All of these traits distinguish this organism from all previously described halophilic anaerobes . The 16S rRNA gene sequence of strain GSLST was found to be similar to, but also significantly different from, the 16S rRNA sequences of Haloanaerobium salsugo and Haloanaerobium praevalens . Therefore, strain GSLST (= DSM 8275T) is described as a new species, Haloanaerobium alcaliphilum.

Int J Syst Bacteriol, 1995 Apr, 45(2), 226 - 34
A new genus of marine budding phototrophic bacteria, Rhodobium gen . nov., which includes Rhodobium orientis sp . nov . and Rhodobium marinum comb . nov; Hiraishi A et al.; Strains of a previously undescribed species of purple nonsulfur phototrophic bacteria were isolated from coastal seawater in Japan . These new isolates were gram-negative, motile, budding rods that contained lamellar intracytoplasmic membranes and produced pink to red cultures . Cell extracts of photosynthetic cultures exhibited absorption maxima at 377, 468, 500, 530, 591, 802, and 870 nm, indicating that bacterio-chlorophyll a and carotenoids of the spirilloxanthin series were present . The new isolates were halophilic, facultatively aerobic photoheterotrophs that grew anaerobically in the light or aerobically in the dark . Maximum growth occurred in the presence of 4 to 5% NaCl . Anaerobic growth in the dark with nitrate as a terminal electron acceptor also occurred . Various organic compounds were used as photosynthetic electron donors and carbon sources . Sulfate was used as a sulfur source . Both menaquinone 10 and ubiquinone 10 were produced; these quinones were the major quinones . A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MB312T (T = type strain), a representative of the new phototrophs, was a member of a lineage that was distinct from members of the genus Rhodopseudomonas; Rhodopseudomonas marina was the closest relative . On the basis of the data described above, we propose the name Rhodobium orientis gen . nov., sp . nov . for the new isolates . We also propose that Rhodopseudomonas marina Imhoff 1983 should be transferred to the genus Rhodobium as Rhodobium marinum comb . nov.

Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 3036 - 40
The primary structure of sensory rhodopsin II: a member of an additional retinal protein subgroup is coexpressed with its transducer, the halobacterial transducer of rhodopsin II; Seidel R et al.; The blue-light receptor genes (sopII) of sensory rhodopsin (SR) II were cloned from two species, the halophilic bacteria Haloarcula vallismortis (vSR-II) and Natronobacterium pharaonis (pSR-II) . Upstream of both sopII gene loci, sequences corresponding to the halobacterial transducer of rhodopsin (Htr) II were recognized . In N . pharaonis, psopII and phtrII are transcribed as a single transcript . Comparison of the amino acid sequences of vHtr-II and pHtr-II with Htr-I and the chemotactic methyl-accepting proteins from Escherichia coli revealed considerable identities in the signal domain and methyl-accepting sites . Similarities with Htr-I in Halobacterium salinarium suggest a common principle in the phototaxis of extreme halophiles . Alignment of all known retinal protein sequences from Archaea identifies both SR-IIs as an additional subgroup of the family . Positions defining the retinal binding site are usually identical with the exception of Met-118 (numbering is according to the bacteriorhodopsin sequence), which might explain the typical blue color shift of SR-II to approximately 490 nm . In archaeal retinal proteins, the function can be deduced from amino acids in positions 85 and 96 . Proton pumps are characterized by Asp-85 and Asp-96; chloride pumps by Thr-85 and Ala-96; and sensors by Asp-85 and Tyr-96 or Phe-96.

Biochim Biophys Acta, 1995 Mar 22, 1234(2), 261 - 7
Isolation of photochemically active archaebacterial photoreceptor, pharaonis phoborhodopsin from Natronobacterium pharaonis; Tomioka H et al.; A photoreceptor, pharaonis phoborhodopsin for the negative phototaxis of extremely halophilic and alkalophilic archaebacterium, Natronobacterium pharaonis was isolated in a photochemically active state . A detailed examination of the chromatographic separation made it possible to separate contaminating proteins, such as cytochromes . The procedure resulted in a 2938-fold enrichment with a yield of 15.5% . The isolated pharaonis phoborhodopsin had an absorption maximum at 498 nm, an A280/A498 ratio of 1.27 and a single band near 24 kDa on the SDS-polyacrylamide gels . The isolated pharaonis phoborhodopsin underwent a photochemical reaction after flash excitation . The photocyclic reaction closely resembled that of the membrane-bound pharaonis phoborhodopsin.

Nucleic Acids Res, 1995 Mar 11, 23(5), 729 - 35
Singular over-representation of an octameric palindrome, HIP1, in DNA from many cyanobacteria; Robinson NJ et al.; An octameric palindrome (5'-GCGATCGC-3') is abundant in cyanobacterial sequences within databases (GenBank/EMBL) and was designated HIP1 (highly iterated palindrome) . The frequency of occurrence of all 256 octameric palindromes has now been determined in sub-databases revealing large and unique over-representation of HIP1 in cyanobacterial entries . DNA sequences from other bacteria were searched for any over-represented octameric palindromes analogous to HIP1 . Only two sequences were identified, in the genomes of a thermophile and halophilic archaebacteria, although these were less abundant than HIP1 in cyanobacteria and relate to codon usage . To test the proposed widespread distribution of HIP1 in DNA from the cyanobacterium Synechococcus PCC 6301, randomly selected genomic clones were partly sequenced . HIP1 constituted 2.5% of the novel sequences, equivalent to a site on average once every 320 nucleotides . An oligonucleotide including HIP1 was also tested in PCR . Multiple products were obtained using template DNA from cyanobacterial strains in which HIP1 is abundant in known sequences, and some strains generated characteristic HIP-PCR banding patterns . However, analysis of DNA from one strain (not previously represented in databases) by random sequencing, HIP-PCR and Pvul digestion, confirms that not all cyanobacterial genomes are rich in HIP1.

J Chromatogr B Biomed Appl, 1995 Mar 10, 665(1), 53 - 62
Isolation of individual amino acids from various microbiological sources using reversed-phase high-performance liquid chromatography; Egorova TA et al.; A new method for the preparative isolation of individual amino acids on a milligram scale based on reversed-phase high-performance liquid chromatography (RP-HPLC) after pre-column derivatization with carbobenzoxychloride (Z-Cl) has been developed . The chromatographic procedure was tested by the investigation of jack bean urease hydrolysate . The method has been applied to the preparative separation of Z-amino acids (from 10 up to 16) obtained from protein hydrolysates of various sources (green microalgae, blue-green algae, halophilic and methylotrophic microorganisms) and was proved to be reliable by the separation of deuterated amino acids (enrichment 97-99%) from Methylobacillus flagellatum (due to the bioconversion of CD3OD and D2O) . Independent of the biological source of the protein, the amino acids were isolated with high recovery (from 68% up to 89%) and chromatographic purity (from 96% up to 99%) . The method was also applied for the isolation of phenylalanine and leucine excreted by amino-acid overproducing microorganisms.

J Bacteriol, 1995 Mar, 177(5), 1129 - 36
Catabolic ornithine transcarbamylase of Halobacterium halobium (salinarium): purification, characterization, sequence determination, and evolution; Ruepp A et al.; Halobacterium halobium (salinarium) is able to grow fermentatively via the arginine deiminase pathway, which is mediated by three enzymes and one membrane-bound arginine-ornithine antiporter . One of the enzymes, catabolic ornithine transcarbamylase (cOTCase), was purified from fermentatively grown cultures by gel filtration and ammonium sulfate-mediated hydrophobic chromatography . It consists of a single type of subunit with an apparent molecular mass of 41 kDa . As is common for proteins of halophilic Archaea, the cOTCase is unstable below 1 M salt . In contrast to the cOTCase from Pseudomonas aeruginosa, the halophilic enzyme exhibits Michaelis-Menten kinetics with both carbamylphosphate and ornithine as substrates with Km values of 0.4 and 8 mM, respectively . The N-terminal sequences of the protein and four peptides were determined, comprising about 30% of the polypeptide . The sequence information was used to clone and sequence the corresponding gene, argB . It codes for a polypeptide of 295 amino acids with a calculated molecular mass of 32 kDa and an amino acid composition which is typical of halophilic proteins . The native molecular mass was determined to be 200 kDa, and therefore the cOTCase is a hexamer of identical subunits . The deduced protein sequence was compared to the cOTCase of P . aeruginosa and 14 anabolic OTCases, and a phylogenetic tree was constructed . The halobacterial cOTCase is more distantly related to the cOTCase than to the anabolic OTCase of P . aeruginosa . It is found in a group with the anabolic OTCases of Bacillus subtilis, P . aeruginosa, and Mycobacterium bovis.

Biophys J, 1995 Mar, 68(3), 1101 - 9
Photoinduced volume change and energy storage associated with the early transformations of the photoactive yellow protein from Ectothiorhodospira halophila; van Brederode ME et al.; The photocycle of the photoactive yellow protein (PYP) isolated from Ectothiorhodospira halophila was analyzed by flash photolysis with absorption detection at low excitation photon densities and by temperature-dependent laser-induced optoacoustic spectroscopy (LIOAS) . The quantum yield for the bleaching recovery of PYP, assumed to be identical to that for the phototransformation of PYP (pG), to the red-shifted intermediate, pR, was phi R = 0.35 +/- 0.05, much lower than the value of 0.64 reported in the literature . With this value and the LIOAS data, an energy content for pR of 120 kJ/mol was obtained, approximately 50% lower than for excited pG . Concomitant with the photochemical process, a volume contraction of 14 ml/photoconverted mol was observed, comparable with the contraction (11 ml/mol) determined for the bacteriorhodopsin monomer . The contraction in both cases is interpreted to arise from a protein reorganization around a phototransformed chromophore with a dipole moment different from that of the initial state . The deviations from linearity of the LIOAS data at photon densities > 0.3 photons per molecule are explained by absorption by pG and pR during the laser pulse duration (i.e., a four-level system, pG, pR, and their respective excited states) . The data can be fitted either by a simple saturation process or by a photochromic equilibrium between pG and pR, similar to that established between the parent chromoprotein and the first intermediate(s) in other biological photoreceptors . This nonlinearity has important consequences for the interpretation of the data obtained from in vitro studies with powerful lasers.

Can J Microbiol, 1995 Mar, 41(3), 302 - 7
Diversity of lactate metabolism in halophilic archaea; Oren A et al.; D-Lactate is readily used as a substrate for the growth of species of halophilic archaea belonging to the genera Haloferax and Haloarcula . L-Lactate was used by Haloferax species (Haloferax volcanii, Haloferax mediterranei) only when a substantial concentration of the D-isomer was also present in the medium . On the enzymatic level, considerable diversity was found in the lactate metabolism of the different representatives of the Halobacteriaceae . At least three types of lactate dehydrogenases were detected in halophilic archaea . A high level of activity of an NAD-linked enzyme was present constitutively in Haloarcula species, and a low level of activity was also detected in Haloferax mediterranei . NAD-independent lactate dehydrogenases, oxidizing L-lactate and D-lactate with 2,6-dichlorophenol-indophenol as electron acceptor, were detected in all nine species tested, but L-lactate dehydrogenase activity in Halobacterium species was very low, and Haloarcula species, which possess a high level of activity of NAD-linked lactate dehydrogenase, showed very low activities of both NAD-independent D- and L-lactate dehydrogenase . An inducible lactate racemase, displaying an unusually high pH optimum, was found in Haloferax volcanii . Lactate racemase activity was found constitutively in Haloarcula species, but no activity was detected in Halobacterium species and in Haloferax mediterranei.

Mikrobiologiia, 1995 Mar-Apr, 64(2), 252 - 8
{Interaction of halobacteria and cyanobacteria in a halophilic cyanobacterial community}; Zviagintseva IS et al.; From cyanobacterial mat, 36 strains of extremely halophilic archaebacteria were isolated . Basing on phenotypic and genotypic characteristics, two frequently occurring organisms among these strains were identified as Haloarcula japonica and Halobacterium distributum . Metabiotic interaction between halophilic Archaea and Microcoleus chthonoplastes, the primary organic matter producer, was demonstrated . It was shown that M . chthonoplastes secretes acids of tricarboxylic acid cycle into the medium and these are utilized by ecrysotrophic halobacteria . The obtained results suggest that extreme halophiles should be regarded as ecologically significant components of cyanobacterial communities.

Mol Gen Genet, 1995 Feb 20, 246(4), 411 - 8
Isolation of cryptic plasmids from moderately halophilic eubacteria of the genus Halomonas . Characterization of a small plasmid from H . elongata and its use for shuttle vector construction; Vargas C et al.; Three cryptic plasmids have been isolated from moderately halophilic eubacteria belonging to three species of the genus Halomonas . These three plasmids were designated pHE1 (4.2 kb, isolated from H . elongata ATCC 33174), pHI1 (48 kb, isolated from "H . israelensis" ATCC 43985), and pHS1 (ca . 70 kb, isolated from H . subglaciescola UQM 2927) . Because of its small size, the plasmid pHE1 was selected for further characterization and construction of a shuttle vector for Halomonas strains . pHE1 was cloned into pBluescript KS and a detailed restriction map was constructed . Hybridization experiments excluded the existence of sequences homologous to pHE1 in total DNA from other strains of the genus Halomonas . Moreover, no DNA homology with pMH1, the only plasmid described so far from moderate halophiles, was found . Since pHE1 appeared to be unable to replicate in Escherichia coli cells, a number of mobilizable pHE1-derived hybrid plasmids were constructed that could be selected and maintained both in E . coli and in H . elongata . Finally, an improved shuttle vector, pHS15, was generated . The vector pHS15 contains an origin of replication from E . coli as well as one from H . elongata, a streptomycin resistance gene for positive selection in moderate halophiles, a number of unique restriction sites commonly used for cloning, and the mobilization functions of the broad host range IncP plasmid RK2 . The vector pHS15 was readily mobilized by the RK2 derivative pRK2013 to all Halomonas strains tested so far . This is the first report on the development of a cloning vector useful for moderately halophilic eubacteria.

EMBO J, 1995 Feb 15, 14(4), 667 - 73
Chemotaxis and phototaxis require a CheA histidine kinase in the archaeon Halobacterium salinarium; Rudolph J et al.; Histidine kinases are part of the two-component signal transduction system responsible for eubacterial responses to diverse environmental signals . They have recently been detected in eukaryotes but their existence in the kingdom Archaea remains uncertain . Here we report the sequence and function of a histidine kinase (CheAH.s.) from Halobacterium salinarium, the first such transmitter in Archaea . The protein CheAH.s . (668 residues) has significant sequence identity with the CheA proteins known from eubacterial signal transduction (e.g . 34% identity with CheA from Bacillus subtilis) . Antibodies were raised against CheAH.s . as expressed in Escherichia coli and were used in Western blotting to demonstrate the expression of cheAH.s . in H . salinarium . As has been observed for other halophilic proteins, CheAH.s . has a deviant electrophoretic migration, with an apparent molecular weight of 103 kDa on SDS-PAGE compared with a calculated molecular weight of 72 kDa . Deletion of a part of the cheAH.s . gene leads to loss of both chemotactic and phototactic responses in H . salinarium as measured by swarm plate assays, motion analysis and tethering experiments . This indicates that CheAH.s . plays a crucial role in chemical and light signal integration, presumably interacting with at least two phototransducers and a number of chemoreceptors.

Gene, 1995 Feb 3, 153(1), 117 - 21
Analysis of the halobacterial plasmid pHK2 minimal replicon; Holmes ML et al.; The pMDS series of cloning vectors developed for use in halophilic archaea have utilized a 10.5-kb plasmid, pHK2, from Haloferax sp . Aa2.2 . The minimal replicon of pHK2 has now been determined (3359 bp) and completely sequenced . No significant sequence similarity was found between the pHK2 subfragment and plasmid pHV2 from the closely related H . volcanii . However, a long open reading frame (ORF), named rep, was identified which encodes a putative protein with approx . 30% sequence identity to ORFs within plasmids pGRB1, pHGN1 and pHSB1 from Halobacterium sp . All these putative Rep proteins contain sequence motifs conserved in bacterial plasmids and phage genomes known to replicate via a rolling-circle mechanism.

Biophys J, 1995 Feb, 68(2), 648 - 54
Photobleaching of the photoactive yellow protein from Ectothiorhodospira halophila promotes binding to lipid bilayers: evidence from surface plasmon resonance spectroscopy; Salamon Z et al.; The photoactive yellow protein (PYP) from the phototrophic bacterium Ectothiorhodospira halophila is a small, soluble protein that undergoes reversible photobleaching upon blue light irradiation and may function to mediate the negative phototactic response . Based on previous studies of the effects of solvent viscosity and of aliphatic alcohols on PYP photokinetics, we proposed that photobleaching is concomitant with a protein conformational change that exposes a hydrophobic region on the protein surface . In the present investigation, we have used surface plasmon resonance (SPR) spectroscopy to characterize the binding of PYP to lipid bilayers deposited on a thin silver film . SPR spectra demonstrate that the net negatively charged PYP molecule can bind in a saturable manner to electrically neutral, net positively, and net negatively charged bilayers . Illumination with either blue or white light of a PYP solution, which is in contact with the bilayer, at concentrations below saturation results in an increase in the extent of binding, consistent with exposure of a high affinity hydrophobic surface in the photobleached state, a property that may contribute to its biological function . A value for the thickness of the bound PYP layer (23 A), obtained from theoretical fits to the SPR spectra, is consistent with the structure of the protein determined by x-ray crystallography and indicates that the molecule binds with its long axis parallel to the membrane surface.

Res Microbiol, 1995 Feb, 146(2), 113 - 20
Convergent evolution of amino acid usage in archaebacterial and eubacterial lineages adapted to high salt; Gandbhir M et al.; Chemical composition and physical properties of the total protein of Haloferax mediterranei, a halophilic archaebacterium requiring high salt concentration for growth, of Halomonas elongata, a halotolerant eubacterium able to grow at any concentration of salt, and of Escherichia coli B, a eubacterium related to H . elongata, unable to grow at high salt concentration, were compared using robust standard biochemical methods . The distribution of amino acid abundancies in the bulk protein from H . elongata was found to be intermediate between that from H . mediterranei and that from E . coli . The two high-salt-adapted organisms displayed an enrichment in aspartic acid and glutamic acid together with an impoverishment in lysine as compared to E . coli . This signature in amino acid usage is reflected in the charge distribution of proteins, as revealed by anion exchange chromatography of crude cell extracts . Since H . elongata diverged from H . mediterranei more than three billion years ago, the resemblance of their amino acid usages can be interpreted as a convergent imprint of their common habitats onto the chemical constitution of their proteins.

Biochemistry, 1995 Jan 24, 34(3), 879 - 90
Optical studies of a bacterial photoreceptor protein, photoactive yellow protein, in single crystals; Ng K et al.; Photoactive yellow protein (PYP), isolated from Ectothiorhodospira halophila, is a water soluble, 14 kDa photoreceptor protein with a fully reversible photocycle resembling that of sensory rhodopsin II . We have established the presence of photoactivity in PYP crystals and defined the relaxation kinetics of spectroscopically distinguishable species in quantitative terms . The PYP crystal has a bright yellow color and displays pronounced anisotropic absorption properties . Linear dichroism measurements show that the transition moment of the PYP chromophore makes an angle of 73 degrees (or 107 degrees) with respect to the six-fold crystallographic symmetry axis . The crystal absorbance can be bleached reversibly as indicated by absorption changes . A bleached photostationary state in the crystal can be established via CW laser illumination, and the extent of crystal bleaching is found to be clearly dependent on excitation laser wavelength, intensity and illumination time . These results provide the information for designing time-resolved crystallography experiments in which a minimum perturbation is applied to the PYP crystals . Global exponential fitting shows that the relaxation from the photostationary state in the crystal is biphasic at -4 degrees C; a slower component of 1.4 +/- 0.2 s-1 accounts for 60% of the absorbance change and a faster component of 5.2 +/- 0.9 s-1 for the other 40% . As a control, we found that the kinetics for the same relaxation in solution are well described by one exponential and agree quantitatively with previous studies . The two rate constants observed in the crystal show similar temperature dependences, with activation energies for the slow and fast components of 11.7 +/- 1.2 and 5.5 +/- 2.3 kcal/mol, respectively . However, the amplitudes associated with the two exponents show different and opposite temperature dependence . Our results show that the solution kinetic model is not directly applicable to crystals . A kinetic model consistent with the optical data is important to extract the underlying structural intermediates from the time-resolved X-ray diffraction data obtained in parallel with the optical data described here . We propose an alternative model for the photocycle in the crystal which contains an additional bleached intermediate in parallel with the last long-lived intermediate in the solution model.

Biochim Biophys Acta, 1995 Jan 20, 1254(2), 155 - 60
Two new phospholipids, hydroxyarchaetidylglycerol and hydroxyarchaetidylethanolamine, from the Archaea Methanosarcina barkeri; Nishihara M et al.; The structures of two new ether phospholipids of the methanogenic Archaea, Methanosarcina barkeri, were determined as hydroxyarchaetidylglycerol and hydroxyarchaetidylethanolamine by means of chemical, chromatographic and enzymatic analyses, and fast atom bombardment-mass spectrometry . These lipids are hydroxy diether analogs of phosphatidylglycerol and phosphatidylethanolamine, respectively, with beta-hydroxyarachaeol (2-O-(3'-hydroxy)phytanyl-3-O-phytanyl-sn-glycerol) as a core lipid . In addition, two other ether phospholipids, usual archaetidylglycerol and archaetidylethanolamine, were also identified in the organism . The stereochemical structure of the unalkylated glycerophosphate of hydroxyarchaetidylglycerol and archaetidylglycerol was determined as sn-glycerol-3-phosphate by use of sn-glycerol-3-phosphate dehydrogenase . The stereochemical configuration of the glycerophosphoglycerol backbone of these lipids was a mirror image of that of diacylphosphatidylglycerol from the organisms of the domains Bacteria and Eucarya, and it was shared with extremely halophilic Archaea . These four phospholipids, in addition to five lipids that had already been reported, accounted for 88% of the total polar lipids of this organism.

Eur J Biochem, 1995 Jan 15, 227(1-2), 249 - 60
1H and 13C NMR assignments and structural aspects of a ferrocytochrome c-551 from the purple phototrophic bacterium Ectothiorhodospira halophila; Bersch B et al.; Two-dimensional nuclear magnetic resonance was used to assign the 1H and 13C resonances of ferrocytochrome c-551 from Ectothiorhodospira halophila, a halophilic phototrophic purple bacterium . This 78-residue protein belongs to a small subgroup of class I cytochromes c together with the analogous cytochromes c-551 from E . halochloris and E . abdelmalekii . A nearly complete assignment of 13C resonances was obtained at natural abundance using a gradient-enhanced 1H-detected heteronuclear single quantum coherence experiment (HSQC) . This was found to be extremely useful for the unambigous assignment of side chain protons . The secondary structure of the protein was determined from analyses of short- and medium-range nuclear Overhauser enhancements (NOE), amide proton exchange and 13C alpha chemical shifts . Three helices could be identified which are well conserved among the class I cytochromes c . There is some evidence for two other regions of less well defined helical structure . From a preliminary analysis of long-range NOE it is shown that in the E . halophila cytochrome c-551 the general cytochrome c fold is well conserved, including the three conserved helices (residues 2-8, 41-50, 63-76), the regions around the heme ligands (Cys14-Ser15-Ser16-Cys17-His18, Met55) and the omega loop (residues 18-28) . In addition, three variable segments of the protein are discussed in detail, one of those including a cis-proline, a feature so far unique in the cytochrome c family . Structural alignments of the E . halophila cytochrome c-551 with two other Pseudomonas cytochrome c5 homologs (Azotobacter vinelandii cytochrome c5 and Chlorobium limicola cytochrome c-555) are provided which are based on sequence similarities and secondary structure alignments.

Nucleic Acids Symp Ser, 1995, (34), 101 - 2
Structure and heterologous expression of the gene encoding the cell surface glycoprotein from Haloarcula japonica strain TR-1; Wakai H et al.; The gene encoding the cell surface glycoprotein (CSG) of Haloarcula japonica strain TR-1 was cloned and sequenced . The structural gene consisted from an open reading frame of 2,586 bp . A potential promoter sequence was found about 150 bp upstream of the ATG initiation codon . N-terminal amino acid sequence of the Ha . japonica CSG revealed that the mature CSG consisted of 828 amino acids . Five potential N-glycosylation sites were found in the mature sequence . The cloned CSG gene of Ha . japonica was expressed in closely-related halophilic archaea.

J Bacteriol, 1995 Jan, 177(2), 378 - 84
Purification and characterization of a mesohalic catalase from the halophilic bacterium Halobacterium halobium; Brown-Peterson NJ et al.; When subjected to the stress of growth in a relatively low-salt environment (1.25 M NaCl), the halophilic bacterium Halobacterium halobium induces a catalase . The protein has been purified to electrophoretic homogeneity and has an M(r) of 240,000 and a subunit size of approximately 62,000 . The enzyme is active over a broad pH range of 6.5 to 10.0, with a peak in activity at pH 7.0 . It has an isoelectric point of 4.0 . This catalse, which is not readily reduced by dithionite, shows a Soret peak at 406 nm . Cyanide and azide inhibit the enzyme at micromolar concentrations, whereas maleimide is without effect . The addition of 20 mM 3-amino-1,2,4-triazole results in a 33% inhibition in enzymatic activity . The tetrameric protein binds NADP in a 1:1 ratio but does not peroxidize NADPH, NADH, or ascorbate . Although the enzymatic activity is maximal when assayed in a 50 mM potassium phosphate buffer with no NaCl, prolonged incubation in a buffer lacking NaCl results in inactive enzyme . Moreover, purification must be performed in the presence of 2 M NaCl . Equally as effective in retaining enzymatic function are NaCl, LiCl, KCl, CsCl, and NH4Cl, whereas divalent salts such as MgCl2 and CaCl2 result in the immediate loss of activity . The catalase is stained by pararosaniline, which is indicative of a glycosidic linkage . The Km for H2O2 is 60 mM, with inhibition observed at concentrations in excess of 90 mM . Thus, the mesohalic catalase purified from H . halobium seems to be similar to other catalases, except for the salt requirements, but differs markedly from the constitutive halobacterial hydroperoxidase.

Zh Mikrobiol Epidemiol Immunobiol, 1995 Jan-Feb, (1), 15 - 8
{Phages of halophilic vibrios}; Libinzon AE et al.; The morphology and antigenic properties of 34 phages of Vibrio parahaemolyticus and V . alginolyticus strains isolated from Black Sea water, as well as their range of lytic activity and specificity of action, have been studied . Most of these phages have been found to lyze both V . parahaemolyticus and V . alginolyticus strains . In addition, highly specific phages capable of lyzing only V . parahaemolyticus serotype O5:K15, have been detected . The latter fact is a precedent indicating the possibility of search for other typing phages and the development of the scheme for the phage typing of V . parahaemolyticus in future.

Can J Microbiol, 1995 Jan, 41(1), 64 - 9
Taxonomy and protein fingerprinting of halophilic Vibrio isolates from bivalves of the Ebre delta; Montilla R et al.; A total of 380 isolates of halophilic Vibrio and related bacteria isolated from shellfish bred in the Ebro delta (in northeastern Spain) were studied by biochemical characterization; this allowed the use of numerical taxonomy programs . All but 25 isolates fell into 12 phenotypes . The analysis of whole-cell electrophoretic fingerprints of 100 isolates confirmed the numerical analysis of biochemical and morphological traits.

Mikrobiologiia, 1995 Jan-Feb, 64(1), 83 - 7
{Halophilic archaebacteria from the Kalamkass oilfield}; Zviagintseva IS et al.; Two strains of halophilic archebacteria, growing in the range from 10 to 25% NaCl, were obtained from the brines of the Kalamkass (Mangyshlak) oilfield . Both strains are extremely halophilic archaebacteria according to their total phenotypic properties . Strain M-11 was identified as Haloferax mediterranei on the basis of the composition of polar lipids and DNA-DNA homology . The composition of polar lipids and 16S rRNA sequences of M-18 strain permitted to include it in Haloferax genus . This strain differs from the affirmed species of Haloferax genus--H . volcanii and H . mediterranei . However, the additional investigations are necessary for its relation to new species.

Microbiol Immunol, 1995, 39(3), 177 - 83
Characterization of a cloned pR72H probe for Vibrio parahaemolyticus detection and development of a nonisotopic colony hybridization assay; Lee CY et al.; Vibrio parahaemolyticus is a halophilic bacterium often found in shellfish and is an important causative agent of food poisoning in Taiwan . A rapid and efficient detection method is required to identify this foodborne pathogen . A 0.76-Kb HindIII DNA fragment was cloned from the chromosomal DNA of V . parahaemolyticus strain no . 93, designated as pR72H fragment, was used as a polynucleotide probe . It was labeled with digoxigenin-11-dUTP (DIG) by the random primer-labeling method . The sensitivity and specificity of the digoxigenin-labeled 0.76-Kb DNA probe was determined by colony hybridization assay . Under stringent hybridization conditions, 122 of 124 isolates of V . parahaemolyticus showed positive hybridization reaction with DIG-0.76-Kb DNA probe; the negative strains were attributed to slow growth . The DIG-0.76-Kb probe did not hybridize with 86 isolates of other vibrios and a number of other enterics as well as nonenteric microorganisms . The sensitivity and specificity of this DIG probe are 98% and 100%, respectively . This nonisotopic colony hybridization assay can be very useful for routine monitoring of V . parahaemolyticus in the food industry, environmental analysis and clinical laboratories.

Biochem Biophys Res Commun, 1994 Dec 30, 205(3), 1736 - 40
Enhanced superoxide production by membrane vesicles from Halobacterium halobium in a hyposaline environment; Brown-Peterson NJ et al.; Membrane vesicles were prepared from the halophilic archaebacterium, Halobacterium halobium, which was grown either in medium containing 4 M NaCl or in a relatively hyposaline medium containing 1.25 M NaCl . Membrane vesicles prepared from bacteria grown in the lower salt environment consumed more oxygen, oxidized more NADH and generated more superoxide than vesicles prepared from cells grown in the normal 4 M NaCl containing medium . The enhanced respiratory activity of the membrane fragments obtained from the halophile which was grown and assayed in a hyposaline environment, along with the concomitant increased flux in superoxide, demonstrate a relation between an environmental perturbation and an altered electron transport activity.

FEMS Microbiol Lett, 1994 Dec 15, 124(3), 265 - 9
The use of arbitrarily primed PCR (AP-PCR) to develop taxa specific DNA probes of known sequence; Martinez-Murcia AJ et al.; A rapid polymerase chain reaction (PCR) method to obtain taxa-specific DNA probes has been developed . The oligonucleotide probes derived from the sequences of species-specific (or other taxa) random amplified polymorphic DNA (RAPD) fragments . The methodology was applied to design probes for the halophilic archaeal species Haloferax mediterranei . With this technique, DNA probes of known sequence can be generated easily and without any previous knowledge about the properties of the microorganisms.

J Mol Biol, 1994 Dec 9, 244(4), 436 - 47
Stability against denaturation mechanisms in halophilic malate dehydrogenase "adapt" to solvent conditions; Bonnete F et al.; Malate dehydrogenase from Haloarcula marisomortui (hMDH) is active, soluble and mildly unstable in an unusually wide range of salt conditions and temperatures, making it a particularly interesting model for the study of solvent effects on protein stability . Its denaturation (loss of activity due to concomitant dissociation and unfolding) kinetics was studied as a function of temperature and concentration of NaCl, potassium phosphate or ammonium sulphate in H2O or 2H2O . A transition-state-theory analysis was applied to the data . In all cases, stability (resistance to denaturation) increased with increasing salt concentration, and when 2H2O replaced H2O . Each salt condition was associated with a particular energy regime that dominated stability . In NaCl/H2O, a positive enthalpy term, delta H not equal to 0, always dominated the activation free energy of denaturation, delta G not equal to 0 . In potassium phosphate/H2O and ammonium sulphate/H2O, on the other hand, stability was dominated by a negative activation entropy, delta S not equal to 0 . and delta H not equal to 0 changed sign between 10 degrees C and 20 degrees C, consistent with a strong hydrophobic effect contribution, in these salting-out solvents . Decreasing stability at low temperatures, favouring cold denaturation, was observed . Replacing H2O by 2H2O strengthened the hydrophobic effect in all conditions . As a consequence, conditions were found in which hMDH was not halophilic; below 10 degrees C, it was stable in approximately 0.1 M NaCl/2H2O . The solution structure and preferential solvent interactions of hMDH in H2O or 2H2O solvents containing NaCl were studied by densimetry and neutron scattering . Despite the different stability of the protein in H2O or 2H2O, an experimentally identical invariant solution particle was formed in both solvents . It had a total volume of 1.165 cm3 g-1 and bound about 0.4 g of H2O (0.44 g of 2H2O) and about 0.08 g NaCl g protein . The impact of these results on a stabilisation model for hMDH, involving ion binding, is discussed.

Biochemistry, 1994 Dec 6, 33(48), 14369 - 77
Complete chemical structure of photoactive yellow protein: novel thioester-linked 4-hydroxycinnamyl chromophore and photocycle chemistry; Baca M et al.; The unique ability of photoactive proteins to capture and use energy from a photon of light depends on the chromophore, its linkage to the protein, and the surrounding protein environment . To understand the molecular mechanisms by which a chromophore and protein interact to undergo a light cycle, we are studying photoactive yellow protein (PYP), a 14-kDa water-soluble photoreceptor from Ectothiorhodospira halophila with a photocycle similar to that of sensory rhodopsin . Here, we report the cloning and sequencing of the pyp gene and the chemical identification of both the chromophore and its covalent linkage to the protein . Elemental composition data from high-resolution mass spectrometry of a proteolytically derived chromopeptide, pH titrations and UV-visible spectroscopy of the protein-bound and chemically released chromophore, and fragmentation mass spectrometry of the liberated chromophore amide were combined with results from the 1.4-A-resolution protein crystal structure to identify the chromophore in PYP as a 4-hydroxycinnamyl group covalently bound to the sole cysteine residue via a thioester linkage . While 4-hydroxycinnamate is a metabolic product of the phenylpropanoid pathway and a key molecule in plant stress response, this is the first report of covalent modification of a protein by this group . In the dark (yellow) state of PYP, the protein stabilizes the chromophore as the deprotonated phenolate anion . By combining our biochemical characterization of the chromophore with other published observations, we propose a chemical basis for the photocycle: following the initial absorption of a photon, the photocycle of PYP involves protonation of the chromophore to a neutral phenol form corresponding to the observed photobleached intermediate.

J Bacteriol, 1994 Dec, 176(23), 7274 - 9
Potassium extrusion by the moderately halophilic and alkaliphilic methanogen methanolobus taylorii GS-16 and homeostasis of cytosolic pH; Ni S et al.; Methanolobus taylorii GS-16, a moderately halophilic and alkaliphilic methanogen, grows over a wide pH range, from 6.8 to 9.0 . Cells suspended in medium with a pH above 8.2 reversed their transmembrane pH gradient (delta pH), making their cytosol more acidic than the medium . The decreased energy in the proton motive force due to the reversed delta pH was partly compensated by an increased electric membrane potential (delta psi) . The cytosolic acidification by M . taylorii at alkaline pH values was accompanied by K+ extrusion . The cytosolic K+ concentration was 110 mM in cells suspended at pH 8.7, but it was 320 mM in cells suspended at neutral pH values . High external K+ concentrations (210 mM or higher) inhibited the growth of M . taylorii at alkaline pH values, perhaps by preventing K+ extrusion . Cells suspended at pH 8.5 and 300 mM external K+ failed to acidify their cytosol . The key observation indicative of the involvement of K+ transport in cytosolic acidification was that valinomycin (0.8 microM), a K+ uniporter, inhibited the growth of M . taylorii only at alkaline pH values . Experiments with resting cells indicated that at alkaline pH values valinomycin uncoupled catabolic reactions from ATP synthesis . Thus, K+/H+ antiport activity was proposed to account for the K+ extrusion and the uncoupling effect of valinomycin at alkaline pH values . Such antiport activity was demonstrated by the sharp drop in pH of the bulk medium of the cell suspension upon the addition of 0.1 M KCl . The antiporter appeared to be active only at alkaline pH values, which was in accordance with a possible role in pH homeostasis by M . taylorii growing at alkaline pH values.

Biophys Chem, 1994 Dec, 53(1-2), 57 - 68
Protein and nucleic acid hydration and cosolvent interactions: establishment of reliable baseline values at high cosolvent concentrations; Eisenberg H; Hydration and cosolvent interactions of biological macromolecules can be derived, subject to excluded volume corrections, from studies of density increments at constant chemical potentials of diffusible solutes through a semipermeable membrane . In addition to precision density determinations of solutions dialyzed to equilibrium, the analytical ultracentrifuge, static and dynamic light and small angle X-ray and neutron scattering, and combined pairwise use of, for instance, ultracentrifugation and neutron scattering, considerably strengthen the experimental analysis and its interpretation . We have examined hydration of bovine serum albumin (BSA) in the native and denatured states, and binding of the denaturant guanidinium chloride (GdmCl) to the latter form; hydration of DNA and interaction with NaCl and CsCl; revised values of the halophilic malate dehydrogenase (hMDH) tetramer hydration and 'binding' of salts; probing of nucleosome core particle hydration as distinct from and additionally to the evaluation of volume exclusion (holes), by use of variously sized sugar related probes . Conclusions presented are compared to results from precision calorimetry and from X-ray crystallography structures, whenever applicable, and comparisons made with alternative interpretations and experimental approaches.

Trends Biotechnol, 1994 Dec, 12(12), 512 - 8
Heat-shock response in Archaea; Conway de Macario E et al.; The Archaea are one of the three phylogenetic domains into which all organisms have been classified, and include extreme halophiles, extreme thermophiles and methanogens . Some of these organisms inhabit inhospitable environments on Earth, and thus have evolved stress responses to cope with the extremes of heat, pH and salinity that they encounter . Although the archaeal stress or heat-shock response bears some similarity to the heat-shock responses of other organisms, it possesses some unique features . A better understanding of this response would facilitate its exploitation in the biotechnological industries; for example, in engineering cells that exhibit an improved ability to withstand, or recover from, stress.

Biochem Mol Biol Int, 1994 Dec, 34(6), 1109 - 20
Kinetic mechanism of Halobacterium halobium Mn(2+)-activated alkaline phosphatase; Bonet ML et al.; The extreme halophilic archaebacterium Halobacterium halobium contains an atypical alkaline phosphatase which is selectively activated by Mn2+ ions (Bonet et al., 1991: Int . J . Biochem . 23, 1445-1451) . Enzyme kinetic mechanism in the presence of Mn2+ with p-nitrophenylphosphate as substrate was analysed by initial rate and product and competitive inhibition studies . The results indicate that there is an ordered addition of activator and substrate, Mn2+ being first in binding to the phosphatase, and that inorganic phosphate is the last product in leaving the enzyme active site . Strong inhibition by vanadate suggests that a phosphoenzyme intermediate is formed during enzymatic phosphohydrolysis of substrate.

Biochim Biophys Acta, 1994 Nov 23, 1196(1), 38 - 44
Acylation of proteins of the archaebacteria Halobacterium cutirubrum and Methanobacterium thermoautotrophicum; Pugh EL et al.; Although the membrane lipids of extremely halophilic archaebacteria are exclusively derived from diphytanylglycerol diether, which is non-acylated, small amounts of fatty acids have been detected in these organisms . These fatty acids are formed by the action of a fatty acid synthase (FAS), shown to be present in the extreme halophile Halobacterium cutirubrum, despite the fact that only a fraction of the activity of FAS remains at the high salt concentration (> 4 M) present in the cytoplasm . It has now been demonstrated that fatty acids do not occur in lipid-bound form but largely in the form of acylated proteins in the red membrane of H . cutirubrum . In contrast, the bacteriorhodopsin of the purple membrane of this extreme halophile does not appear to be acylated . The thermophilic methanogen, Methanobacterium thermoautotrophicum had a much higher fatty acid synthase activity than the extreme halophile, and the synthase activity of the methanogen was optimal under its normal (anaerobic) growth conditions . The methanogen also utilized the resulting fatty acids to acylate its membrane proteins . The major fatty acids in both organisms were palmitic and stearic acids with small amounts of myristic and 18:1 acids, and these were bound to protein through both ester and amide linkages.

Biochim Biophys Acta, 1994 Nov 16, 1209(1), 1 - 9
Purification and properties of a halophilic catalase-peroxidase from Haloarcula marismortui; Cendrin F et al.; A heme protein, hCP, from the extreme halophile, Haloarcula marismortui, showing both peroxidatic and catalatic activity has been purified and characterized as a catalase-peroxidase . Catalatic activity is enhanced by molar concentrations of NaCl or (NH4)2SO4, while peroxidase activity decreases with increasing salt concentration . Optimal pH values are 6.0 for peroxidatic activity assayed in absence of NaCl and 7.5 for catalatic activity assayed in molar concentrations of NaCl . The two activities present saturation behaviour with increasing H2O2 concentration with apparent Km values of 0.5 and 2.5 mM for the peroxidatic and catalatic activities, respectively . A molecular mass of 81,292 +/- 9 Da was measured for the polypeptide by mass spectroscopy . One heme group (protoporphyrin IX with an iron atom in the ferric state) is associated with one molecule of hCP . Its amino-acid composition shows hCP to contain a high proportion of acidic residues . The EPR spectrum of the NO-compound of reduced (ferrous) hCP strongly suggests that the proximal ligand of the heme is the imidazole group of a histidine residue.

Arch Biochem Biophys, 1994 Nov 15, 315(1), 127 - 32
The novel ion pump rhodopsins from Haloarcula form a family independent from both the bacteriorhodopsin and archaerhodopsin families/tribes; Tateno M et al.; Extreme halophiles newly collected from Argentine salt flats were characterized, in one of which, Haloarcula (sp . arg-1), light-driven retinal protein ion pumps were found . The proton pump, cruxrhodopsin-1, shows amino acid sequence homologies of 52% to bacteriorhodopsin and 48% to archaerhodopsin-1 . The anion pump, cruxhalorhodopsin-1, identified partially as a 394bp polymerase chain reaction product, shows homologies of 70% to halorhodopsin, and 72% to pharaonis halorhodopsin . The ion pumps (and possibly sensors still to be found) in Haloarcula sp . arg-1, which constitute the cruxrhodopsin-1 family, are distinct from the bacteriorhodopsin and the archaerhodopsin families/tribes.

J Immunol Methods, 1994 Nov 10, 176(1), 1 - 7
Homogeneous immunoassay of antibody by use of liposomes made of a model lipid of archaebacteria; Tomioka K et al.; Liposomes made of 1,2-di(3RS,7R,11R-phytanyl)-sn-glycero-3-phosphocholine (DPhyPC), which was synthesized as one of the model lipids existing in archaebacterial halophiles, showed excellent stability . Because of this high stability, DPhyPC liposomes could be constituted high ratios (50%) of N-{4-(p-maleimidophenyl) butyryl} dipalmitoyl phosphatidylethanolamine (MPB-DPPE), and consequently could bind large amounts of antigen (alpha-chymotrypsinogen A) on the liposome surface in comparison with those made of ordinary lipids, such as dipalmitoylphosphatidylcholine (DPPC) . Though the characteristics of the DPhyPC liposomal membranes in lysis by the classical complement pathway were similar to those of DPPC liposomes, a high sensitivity and a low detection limit in the liposome immune lysis assay (LILA) of antibodies were attained by binding large amounts of the antigen . Further, by coupling sufficient amounts of antigen, almost all the DPhyPC liposome surface was covered with the antigen, and such liposomes showed higher resistance against non-specific lysis caused by complement activity in serum samples, which may be effective in reducing positive-false errors in LILA.

J Biochem (Tokyo), 1994 Nov, 116(5), 1030 - 8
Properties and sequence of the NhaA Na+/H+ antiporter of Vibrio parahaemolyticus; Kuroda T et al.; A gene encoding an Na+/H+ antiporter was cloned from chromosomal DNA of the slightly halophilic marine bacterium Vibrio parahaemolyticus . The host was an Escherichia coli mutant that lacked both of the two major Na+/H+ antiporters, NhaA and NhaB . Untransformed mutant cells were unable to grow in the presence of 0.6 M NaCl or 0.1 M LiCl, but Na+ and Li+ were non-toxic to cells transformed with a plasmid carrying the antiporter gene . Membrane vesicles prepared from the original E . coli mutant did not show any detectable Na+/H+ (and Li+/H+) antiport activity . However, we observed high Na+/H+ (and Li+/H+) antiport activity in membrane vesicles prepared from the transformed cells . The activity increased greatly when the pH of the assay medium was increased from 7.0 and 8.5 . This property is very similar to that of the NhaA Na+/H+ antiporter of E . coli . Drastic decreases in Km values for Li+ and Na+ were observed with membrane vesicles prepared from the transformed cells compared with those observed with V . parahaemolyticus vesicles . The amino acid sequence deduced from the nucleotide sequence of the cloned gene showed high homology (59% identity and 87% similarity) with the NhaA Na+/H+ antiporter of E . coli . Thus, we conclude that the gene we cloned and sequenced is the nhaA of V . parahaemolyticus . We also found that several regions of the NhaA protein showed sequence similarity with transport proteins from some other organisms . Such regions seem to be important for Na+ recognition, transport or amiloride binding.

Can J Microbiol, 1994 Nov, 40(11), 922 - 9
Construction of composite transposons for halophilic Archaea; Dyall-Smith ML et al.; Transposons with selectable marker genes (e.g., antibiotic resistance) have been extremely useful tools in bacterial genetics but have not been found naturally in Archaea . We constructed synthetic transposons consisting of halobacterial ISH elements (ISH2, ISH26, or ISH28) flanking a mevinolin resistance determinant . Introduction of these constructs into Haloferax volcanii cells can produce drug-resistant transformants through homologous recombination between the plasmid hmgA gene and the chromosomal hmgA locus . This problem was overcome by using another host, Haloarcula hispanica, the hmgA gene of which shares little homology with that from Haloferax volcanii . Introduction of an ISH28-based transposon (ThD28) into Haloarcula hispanica cells produced numerous transformants . Each of these was shown to contain an ISH-flanked mevinolin resistance determinant integrated into the cellular DNA . Integration was not obviously site specific . Transposon ThD26 (based on ISH26a), was less mobile, relative to ThD28, and the ISH2-based construct (ThD22) did not transpose at all in these cells . The further development of halobacterial transposons may provide useful genetic tools allowing rapid isolation and analysis of halobacterial genes, particularly those with no selectable phenotype.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 161 - 6
DNA intercalating drugs inhibit positive supercoiling induced by novobiocin in halophilic archaea; Gadelle D et al.; The two DNA intercalators, actinomycin D and 2-methyl-9-hydroxy-ellipticine, and the DNA minor groove ligant DAPI inhibited the growth of the haloarchaeon Halobacterium sp . GRB and bind to its plasmid pGRB-1 . In contrast to specific DNA topoisomerase II inhibitors, they produced neither double-stranded breaks nor relaxation of plasmidic DNA . The two DNA intercalators inhibited positive supercoiling induced by novobiocin, suggesting that positive supercoiling in haloarchaea is due to transcription, as in the domain Bacteria . Plasmids from haloarchaea could thus be used to prescreen for DNA intercalators and to discriminate between different drug families via their mode of action.

Eur J Biochem, 1994 Oct 15, 225(2), 715 - 25
The three-dimensional structure in solution of the paramagnetic high-potential iron-sulfur protein I from Ectothiorhodospira halophila through nuclear magnetic resonance; Banci L et al.; The three-dimensional structure in solution of reduced recombinant high-potential iron-sulfur protein iso-I from Ectothiorhodospira halophila was determined using 948 relevant interproton NOEs out of the 1246 observed NOEs . The determination was accomplished using the XEASY program for spectral analysis and the distance geometry (DG) program DIANA for generation of the structure as described by Wuthrich {Wuthrich, K . (1989) Acc . Chem . Res . 22, 36-44} . The FeS cluster was simulated using an amino acid residue constructed for the present work from a cysteinyl residue with an iron and a sulfur atom attached to the terminal thiol . The family of structures obtained from distance geometry were subjected to energy minimization and molecular dynamics simulations using previously defined force field parameters . The quality of these structures at each stage of the refinement process is discussed with respect to the dihedral angle order parameter and the root-mean-square deviation of the atomic coordinates . The latter values for the backbone atoms vary from 67 pm for the distance-geometry structures to 60 pm for the energy-minimized structures to 51 pm for the structures subjected to restrained molecular dynamics . Finally, the structure in best agreement with the NOE constraints has been further treated with extensive restrained molecular dynamics in water . The solution structure is well defined and is very similar to the available X-ray structure . We do not know of any previous determination of the structure of a paramagnetic protein in solution by NMR . The effect of paramagnetism on the quality of the structure determination is discussed.

Eur J Biochem, 1994 Oct 15, 225(2), 703 - 14
Sequence-specific assignment of the 1H and 15N nuclear magnetic resonance spectra of the reduced recombinant high-potential iron-sulfur protein I from Ectothiorhodospira halophila; Bertini I et al.; A 1H and 15N NMR investigation through two-dimensional and three-dimensional spectroscopy has been performed on the reduced form ({Fe4S4}2+) of the recombinant high-potential iron-sulfur protein (HiPIP) I from Ectothiorhodospira halophila expressed in Escherichia coli . {Fe4S4}2+ clusters in proteins are paramagnetic with a relatively low mu eff of about 0.8 mu B/iron ion, but the paramagnetic effects on nuclear relaxation are so strong as to yield T1 values of a few milliseconds and linewidths of hundreds of hertz for the nuclei closet to the paramagnetic center . Despite these features, 71 out of 73 residues were identified, most of which were assigned completely as far as proton resonances are concerned; as many as 68 residues could be assigned without any reference to the existing X-ray structure . A total of 88% of all protein protons and 58 out of 69 peptide HN nitrogen signals were assigned . To the best of our knowledge, this is the most extensive 1H assignment of a paramagnetic protein to date . Protons sensitive to the proximity of the cluster were assigned through suitable NOE spectroscopy experiments . Three out of the four coordinated cysteines were assigned, and two residues have been identified whose peptide HN protons give rise to H bonds with coordinated sulfur atoms . The inter-residue NOE cross peaks are in qualitative agreement with the secondary and tertiary structure as obtained from the available X-ray crystallographic analysis of the wild-type protein at 250-pm resolution . It is therefore shown that the expressed protein is properly folded and that it is a reliable model for the wild-type protein . These data are meaningful for the detection of structural differences among mutants in future studies.

Appl Environ Microbiol, 1994 Oct, 60(10), 3867 - 9
A transcriptional reporter for in vivo promoter analysis in the archaeon Haloferax volcanii; Palmer JR et al.; We have used a modified intron-containing tRNA(Pro(UGG) gene (tRNA(ProM), derived from the Saccharomyces cerevisiae tRNA(Pro(UGG) gene, as a reporter to measure in vivo transcription from a halophilic archaeon promoter . Coupling of the yeast tRNA(ProM) gene to the Haloferax volcanii tRNA(Lys) promoter on the H . volcanii plasmid pWL201 led to the production of a single stable transcript that was readily quantitated by Northern (RNA) blot analysis . Comparison of tRNA(ProM) RNA production from constructs containing the wild-type tRNA(Lys) promoter and those containing mutant tRNA(Lys) promoters demonstrated that this assay system can be used to measure expression from strong and weak promoters.

Int J Syst Bacteriol, 1994 Oct, 44(4), 694 - 703
Molecular characterization and application of random amplified polymorphic DNA analysis of Mrakia and Sterigmatomyces species; Messner R et al.; The qualitative and quantitative monosaccharide spectra of purified yeast cell walls revealed that there are three phylogenetically distinct lineages of sterigma-forming basidiomycetous yeasts: (i) Kurtzmanomyces and Sterigmatomyces species, which contain high levels of mannose; (ii) Tilletiopsis species, which contain glucose, galactose, and small amounts of mannose; and (iii) Fellomyces, Kockovaella, Sterigmatosporidium, and Tsuchiyaea species, which appear to be closely related on the basis of their high levels of glucose and the presence of xylose . The yeast cell wall neutral sugars of Sporobolomyces antarcticus and Sterigmatomyces aphidis were similar to those of members of the genus Tilletiopsis . However, the possibility that these taxa are conspecific was eliminated by the results of a random amplified polymorphic DNA (RAPD) analysis . The conspecificity of Mrakia frigida and Mrakia nivalis, the conspecificity of Mrakia gelida and Mrakia stokesii, and the conspecificity of Sterigmatomyces halophilus and Sterigmatomyces indicus were confirmed by RAPD analysis results . RAPD analysis was found to be a simple and highly sensitive method which can be used to differentiate species at the DNA level; it can replace nuclear DNA-nuclear DNA hybridization experiments for species identification, characterization, and delimitation.

Biochem Mol Biol Int, 1994 Oct, 34(3), 543 - 52
Ribosomal protein S1 in archaea; Ravi K et al.; Cell extracts and ribosomes of thermoacidophilic and halophilic archaebacteria were analysed by immunoblotting to detect ribosomal protein S1 homologues . Antisera to E . coli S1, the N-terminal ribosome binding domain (F2a) and the central and COOH-terminal nucleic acid binding domain (S1F1) of the protein were used . The results show that the thermoacidophilic archaebacterium sulfolobus acidocaldarius contains a protein with molecular weight of 66,000 that cross-reacts with anti-S1F1 . However, the halophilic archaebacteria Halobacterium halobium, H . cutirubrum and H . salinarum contain a protein of molecular weight of about 100,000 that cross-reacts only with anti-F2a . The results indicate a differential conservation of the structural and functional domains of protein S1 in archaebacteria.

Biophys J, 1994 Oct, 67(4), 1691 - 705
Measurement and global analysis of the absorbance changes in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila; Hoff WD et al.; The photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila was examined by time-resolved difference absorption spectroscopy in the wavelength range of 300-600 nm . Both time-gated spectra and single wavelength traces were measured . Global analysis of the data established that in the time domain between 5 ns and 2 s only two intermediates are involved in the room temperature photocycle of PYP, as has been proposed before (Meyer T.E., E . Yakali, M . A . Cusanovich, and G . Tollin . 1987 . Biochemistry . 26:418-423; Meyer, T . E., G . Tollin, T . P . Causgrove, P . Cheng, and R . E . Blankenship . 1991 . Biophys . J . 59:988-991) . The first, red-shifted intermediate decays biexponentially (60% with tau = 0.25 ms and 40% with tau = 1.2 ms) to a blue-shifted intermediate . The last step of the photocycle is the biexponential (93% with tau = 0.15 s and 7% with tau = 2.0 s) recovery to the ground state of the protein . Reconstruction of the absolute spectra of these photointermediates yielded absorbance maxima of about 465 and 355 nm for the red- and blue-shifted intermediate with an epsilon max at about 50% and 40% relative to the epsilon max of the ground state . The quantitative analysis of the photocycle in PYP described here paves the way to a detailed biophysical analysis of the processes occurring in this photoreceptor molecule.

Biochim Biophys Acta, 1994 Sep 28, 1201(1), 106 - 12
Characterization of 1-phosphofructokinase from halophilic archaebacterium Haloarcula vallismortis; Rangaswamy V et al.; 1-Phosphofructokinase (EC 2.7.1.56) (1PFK) was purified and characterized for the first time from an archaebacterial halophile Haloarcula vallismortis . The purification procedure involving (NH4)2SO4 fractionation, (NH4)2SO4-mediated chromatography on Sepharose 4B, CM-cellulose chromatography, hydrophobic chromatography on phenyl Sepharose and adsorption chromatography on hydroxylapatite yielded a preparation with a specific activity of 128 and 100-fold purification . From gel filtration and sucrose density gradient ultracentrifugation, the apparent molecular mass of halobacterial 1PFK was found as 76 +/- 5 kDa . The halobacterial 1PFK appears to be monomeric and the possibility of an unstable phosphoenzyme intermediate during its catalysis could not be ruled out . As in the case of many halobacterial enzymes, the 1PFK was found to be halophilic and thermostable . Other catalytic features of halobacterial 1PFK were similar to its counterparts from eubacterial sources.

Biochem Biophys Res Commun, 1994 Sep 15, 203(2), 972 - 8
Novel isoprenoid modified proteins in Halobacteria; Sagami H et al.; Incorporation of {3H}mevalonic acid-derived materials into proteins was studied with extremely halophilic archaebacteria, Halobacterium halobium and Halobacterium cutirubrum . Several labeled proteins were detected on SDS-polyacrylamide gel electrophoresis followed by fluorography . The majority of the radioactive materials released from the labeled proteins by sulfonium salt cleavage moved with a mobility similar to that of a C85 polyprenol on reverse-phase thin-layer chromatography, and no radioactive farnesol was found on the chromatography . However, a weak but significant protein farnesyltransferase activity was detected in in vitro experiments with a combination of {3H}farnesyl diphosphate and Ras precursor protein.

Ugeskr Laeger, 1994 Sep 12, 156(37), 5279 - 82
{Extraintestinal infections caused by Vibrio parahaemolyticus and Vibrio alginolyticus at the county of Funen 1987-1992}; Gahrn-Hansen B et al.; Over a six-year period we detected 30 clinical infections caused by halophilic vibrios in a restricted geographical area . Vibrio parahaemolyticus infections were found in 13 patients (three with wound infections, ten with ear infections), and Vibrio alginolyticus infections in 17 patients, all of whom had ear infections . From 1987 to 1990, infections caused by marine vibrios were found in at most four cases annually, in 1992 in six instances, whereas over a five-month period in 1991 we experienced 15 cases of extraintestinal infections . Fifteen of the infections found in 1991 and 1992 were ear infections, ten of which occurred shortly after exposure to Danish coastal seawater . The disease was mild in most of the patients and all recovered . Most of the patients showed predisposing conditions such as chronic otitis media, perforation of the tympanic membrane or ulcus cruris . The organisms were isolated in minor numbers from coastal water samples from five of 12 bathing areas around Funen at the end of July, but not at the end of November . This study indicates that halophilic marine vibrios may be pathogenic in Denmark in persons exposed to seawater.

J Bacteriol, 1994 Sep, 176(17), 5505 - 12
Ketohexokinase (ATP:D-fructose 1-phosphotransferase) from a halophilic archaebacterium, Haloarcula vallismortis: purification and properties; Rangaswamy V et al.; Ketohexokinase (ATP:D-fructose 1-phosphotransferase {EC 2.7.1.3}), detected for the first time in a prokaryote, i.e., the extreme halophile Haloarcula vallismortis, was isolated and characterized from the same archaebacterium . This enzyme was characterized with respect to its molecular mass, amino acid composition, salt dependency, immunological cross-reactivity, and kinetic properties . Gel filtration and sucrose density gradient centrifugation revealed a native molecular mass of 100 kDa for halobacterial ketohexokinase, which is larger than its mammalian counterpart . The enzyme could be labeled by UV irradiation in the presence of { gamma-32P}ATP, suggesting the involvement of a phosphoenzyme intermediate . Other catalytic features of the enzyme were similar to those of its mammalian counterparts . No antigenic cross-reactivity could be detected between the H . vallismortis ketohexokinase and the ketohexokinases from different rat tissues.

Microbiologia, 1994 Sep, 10(3), 217 - 28
Enzyme diversity in halophilic archaea; Oren A; The halophilic archaea display a considerable extent of enzyme diversity . The presence or absence of certain enzymatic activities is closely linked with the taxonomic status of the strains investigated . Thus, Halobacterium species such as Hb . salinarium, Hb . halobium, and Hb . cutirubrum differ from most other Halobacteriaceae tested by the possession of an NAD(+)-dependent glycerol dehydrogenase, by the absence of methylglyoxal synthase activity, and the ability of fermentative growth on arginine . Species such as Hb . saccharovorum and Hb . sodomense, which are still classified within the genus Halobacterium, have an enzymatic machinery greatly different from that of the Hb . salinarium-Hb . halobium group, confirming the need for a taxonomic reappraisal of these species . The presence of NAD(+)-dependent D-lactate dehydrogenase is characteristic of representatives of the genus Haloarcula, which possess only low activities of NAD(+)-independent L- and D-lactate dehydrogenases, if at all . Other enzymes which show considerable diversity are fructose 1,6-bisphosphate aldolase, of which two classes exist, and ribulose 1,6-bisphosphate carboxylase, which is present in a limited number of species.

Protein Eng, 1994 Sep, 7(9), 1145 - 50
Hyperexpression of a synthetic gene encoding a high potential iron sulfur protein; Eltis LD et al.; A gene encoding high potential iron sulfur protein (HiPIP) iso-1 from Ectothiorhodospira halophila was constructed in one step from long synthetic oligonucleotides . The gene was inserted into a phagemid vector from which the HiPIP was expressed as a fusion protein to > 10% of the soluble protein in Escherichia coli, demonstrating that a 4Fe-4S protein can be highly expressed in E . coli . The recombinant HiPIP was purified to apparent homogeneity by affinity chromatography followed by proteolytic removal of the leader sequence and anion exchange chromatography . Approximately 180 mg of HiPIP were purified from 10 l of cell culture . CD spectra of the oxidized and reduced forms of the protein and the 1H NMR spectrum of the oxidized protein are essentially identical to those of the wild type protein, indicating that the environment of the iron sulfur cluster in the two proteins is the same and thus that the recombinant protein is folded correctly . The reduction potential of the recombinant protein was determined to be 120 +/- 6 mV versus NHE (20 mM HEPES, 0.1 M sodium chloride, pH 7.0, 25 degrees C) . This efficient heterologous expression of an HiPIP enables a systematic investigation of structure-function relationships in this class of iron sulfur proteins.

Nippon Saikingaku Zasshi, 1994 Sep, 49(5-6), 769 - 77
{A simple method for differentiation of hydrogen sulfide-producing bacteria by the pH-dependent EDTA-sensitivity test}; Kida N et al.; The pH-dependent EDTA-sensitivity test was performed to differentiate several strains of bacteria forming black colonies by the production of hydrogen sulfide on TCBS (thiosulfate-citrate-bile salt-sucrose agar) medium (tentatively designated as hydrogen-sulfide production bacteria) . Two halotorelant strains of 16 hydrogen sulfide-producing strains showed the same bacteriological properties and isoprenoid quinone type as did a reference strain of Proteus mirabilis and were classified into the EDTA-insensitive group as were P . mirabilis and P . vulgaris . On the other hand, the other 14 halophilic strains, showing similar but not identical bacteriological properties or the isoprenoid quinone type to those of Shewanella putrefaciens IFO 3908, were classified into the "EDTA-sensitive (at pH 5)" group as were some species of the genus Vibrio . By the same sensitivity test, S . putrefaciens IFO 3908 was classified into the "EDTA-sensitive (at any pH)" group . These results indicate that the pH-dependent EDTA-sensitivity test is useful for differentiation of bacterial isolates producing hydrogen sulfide and having similar bacteriological properties.

Indian J Exp Biol, 1994 Sep, 32(9), 619 - 22
Plasmid-determined degradative metabolism and halophilism of Vibrio parahaemolyticus; Chakrabarti T et al.; A set of 25 Kanagawa(+) and Kanagawa(-) strains of V . parahaemolyticus was studied for their ability to degrade hydrocarbons in minimal media . All strains gave positive results with respect to crystal violet (CV), methyl violet, liquid paraffin, benzene, naphthalene and phenol . The CV double ring (CVDR) response had earlier appeared to be a significant pathogenic marker {Chakrabarti et al, Indian J Med Res, 85 (1987) 508} . The CVDR response was found also to be a biodegradative marker, and correlates perfectly well with polymyxin resistance and low level of halophilism (4% NaCl) . All these markers (characters) were found to be controlled by a single plasmid in the wild type . Elimination of the plasmid, as confirmed by gel electrophoresis studies, resulted in loss of CVDR response, polymyxin resistance, and acquisition of halophilism at a higher level (> 7%) . The massive drainage of industrial effluents, rich in hydrocarbons, in the estuarine areas in many countries might have altered the ecosystem in favour of V . parahaemolyticus and its emergence as a new biodegradative and enterotoxigenic pathogen, contaminating fauna and flora in the littoral sea regions, with increased changes of communicability to humans.

Biochem Biophys Res Commun, 1994 Aug 30, 203(1), 436 - 42
Influence of surface charges on redox properties in high potential iron-sulfur proteins; Luchinat C et al.; The pH-dependence of the reduction potential determined through differential pulse voltammetry for the high potential iron sulfur proteins (HiPIP) from R . globiformis, C . vinosum, R . gelatinosus, E . vacuolata (I and II), E . halophila (I and II) is reported . A decrease in reduction potential with pH is invariably observed in the pH range where deprotonation of the imidazolium nitrogen of histidine residue(s) occurs . No pH dependence is observed for the only protein lacking histidines . It appears that surface charges like the His imidazolium groups are capable of influencing the reduction potential despite the known quencing of the electrostatic interactions due to solvent effects.

Biochim Biophys Acta, 1994 Aug 25, 1214(1), 97 - 108
Polar lipids of a non-alkaliphilic extremely halophilic archaebacterium strain 172: a novel bis-sulfated glycolipid; Matsubara T et al.; Extremely halophilic archaebacteria which require high salt concentrations for growth and survival contain glycerol diether analogues of phospholipids and sulfated glycolipids as major membrane polar lipids . A non-alkaliphilic, non-pigmented rod-shaped extreme halophile, isolated from sea sand in Japan and designated 'strain 172', was found to contain two phospholipids, phosphatidylglycerol (PG) and phosphatidylglyceromethylphosphate (PGP-Me), derived from both C20-C20- and C20-C25-glycerol diethers, and a novel major glycolipid (designated SGL-X) . This glycolipid has been identified as a bis-sulfated diglycosyl C20-C20- or C20-C25-glycerol diether, on the basis of its TLC mobility, positive-staining behavior with sugar and sulfate-staining reagents, its mole ratio sulfate/glycolipid = 2.2, and by spectrometric analysis (IR and FAB-MS) of the intact and the desulfated SGL-X . The sugars were identified as mannose and glucose, after acid hydrolysis of SGL-X, by paper chromatography of the free sugars and GC-MS of the derivatized sugars (alditol acetates) . Permethylation analysis and 1H- and 13C-NMR analysis established the position and configuration of the sugar linkages and the positions of the sulfate groups . The final structure of SGL-X (now designated S2-DGD-1) is proposed to be: 2,3-diphytanyl- or phytanyl-sesterterpenyl-1-{2,6-(HSO3)2-alpha-Manp-1--> 2- Glcp}-sn-glycerol . This lipid is the first bis-sulfated glycolipid to be reported in extremely halophilic archaebacteria, and is the first in the biosphere that possesses two sulfate groups attached to the same monosaccaride.

J Bacteriol, 1994 Aug, 176(16), 5131 - 4
Cloning, expression, and nucleotide sequence of the alpha-amylase gene from the haloalkaliphilic archaeon Natronococcus sp . strain Ah-36; Kobayashi T et al.; The alpha-amylase gene of a Natronococcus sp . (1,512 bp) contained a signal peptide of 43 amino acids . Haloferax volcanii expressed the gene and cleaved the signal peptide accurately . The signal peptide shared an extremely high amino acid sequence identity with that of a protease from the halophilic archaeon 172P1.

J Bacteriol, 1994 Aug, 176(16), 4966 - 73
Effects of salt and temperature on plasmid topology in the halophilic archaeon Haloferax volcanii; Mojica FJ et al.; We report here the effect of environmental parameters, salinity, temperature, and an intercalating drug on plasmid topology in the halophilic archaeon Haloferax volcanii . We first studied the topological state of the plasmid pHV11 in media of different salt compositions and concentrations . The superhelical density of plasmid PHV11 varies in a way that depends on the kind of salt and on the concentrations of individual salts . With respect to growth temperature, the plasmid linking number increased at higher temperature in a linear way, contrary to what has been reported for Escherichia coli, in which the plasmid linking number decreased at higher temperature . These results suggest that some of the mechanisms that control DNA supercoiling in halophilic Archaea may be different from those described for E . coli . However, homeostatic control of DNA supercoiling seems to occur in haloarchaea, as in Bacteria, since we found that relaxation of DNA by chloroquine triggers an increase in negative supercoiling.

Microbiology, 1994 Aug, 140 ( Pt 8), 1959 - 66
Lipids of extremely halophilic archaeobacteria from saline environments in India: a novel glycolipid in Natronobacterium strains; Upasani VN et al.; Several strains of extremely halophilic archaeobacteria, both non-alkaliphilic and alkaliphilic, including Halobacterium, Haloferax and Natronobacterium species, were isolated from salt locales in India . The major phospholipids in these strains were the C20-C20-glycerol diether analogues of phosphatidylglycerolmethylphosphate (PGP-Me), phosphatidylglycerol (PG) and phosphatidic acid (PA) . In addition, the Halobacterium strains possessed the characteristic glycolipids, sulfated triglycosyl and tetraglycosyl diethers (S-TGD-1 and S-TeGD, respectively) and the unsulfated triglycosyl diether (TGD-1); and the Haloferax strains had the characteristic sulfated and unsulfated diglycosyl glycerol diethers (S-DGD-1 and DGD-1, respectively) . The PGP-Me, and PG components of the haloalkaliphiles each occurred as two molecular species with C20-C20- and C20-C25-(isopranoid) glycerol diether lipid cores . In contrast to previous reports of the absence of glycolipids in natronobacteria, the Natronobacterium strains from India were found to contain small amounts of a novel glycolipid identified as glucopyranosyl-1-->6-glucopyranosyl-1-->1-glycerol diether (DGD-4) . The lipid cores of DGD-4 also contained mainly unhydroxylated or hydroxylated C20-C20, C20-C25 and C25-C25 molecular species with unsaturated (isoprenoid) chains . Hydroxylated lipid cores have previously been identified only in some methanogenic archaeobacteria.

J Bacteriol, 1994 Jul, 176(13), 3920 - 7
The photoactive yellow protein from Ectothiorhodospira halophila as studied with a highly specific polyclonal antiserum: (intra)cellular localization, regulation of expression, and taxonomic distribution of cross-reacting proteins; Hoff WD et al.; A rabbit antiserum was raised against the photoactive yellow protein (PYP) from Ectothiorhodospira halophila and purified by adsorption experiments to obtain a highly specific polyclonal antiserum . This antiserum was used to obtain the following results . (i) In E . halophila, PYP can be isolated from the fraction of soluble proteins . In the intact cell, however, PYP appeared to be associated with (intra)cytoplasmic membranes, as was concluded from analysis of immunogold-labelled thin sections of the organism . (ii) The regulation of expression of PYP was studied by using dot blot assays, Western blotting (immunoblotting), and rocket immunoelectrophoresis . Under all conditions investigated (light color, salt concentration, and growth phase), PYP was expressed constitutively in E . halophila . However, when Rhodospirillum salexigens was grown aerobically, the expression of PYP was suppressed . (iii) A large number of prokaryotic microorganisms contained a single protein, with an apparent size of approximately 15 kDa, that cross-reacted with the antiserum . Among the positively reacting organisms were both phototrophic and chemotrophic, as well as motile and nonmotile, organisms . After separation of cellular proteins into a membrane fraction and soluble proteins, it was established that organisms adapted to growth at higher salt concentrations tended to have the cross-reacting protein in the soluble fraction . In the cases of R . salexigens and Chromatium salexigens, we have shown that the cross-reacting protein involved is strongly homologous to PYP from E . halophila.

Biochem Mol Biol Int, 1994 Jul, 33(4), 785 - 92
Activity staining of halophilic enzymes: substitution of salt with a zwitterion in non-denaturing electrophoresis; Cadenas Q et al.; Several NAD(P)(+)-dependent dehydrogenases were partially purified from Halobacterium halobium . When salt (2M (NH4)2SO4) was replaced with glycine betaine (4M or 6M), the zwitterion stabilised activity less completely than the salt . Nevertheless most of the enzyme activity still remained after 90h, e.g . 70% for malate dehydrogenase . This level of stabilisation permitted non-denaturing gel electrophoresis in 4M betaine after dialysis to replace salt . Coomassie Blue staining showed good separation of the proteins, and activity staining, hitherto impossible for halophilic enzymes, readily identified the individual dehydrogenase bands . Transfer of activity-stained gels to Coomassie staining solution halted background formazan staining and showed up activity and other protein bands in contrasting colours.

Int J Syst Bacteriol, 1994 Jul, 44(3), 565 - 72
Haloanaerobium salsugo sp . nov., a moderately halophilic, anaerobic bacterium from a subterranean brine; Bhupathiraju VK et al.; A strictly anaerobic, moderately halophilic, gram-negative bacterium was isolated from a highly saline oil field brine . The bacterium was a non-spore-forming, nonmotile rod, appearing singly, in pairs, or occasionally as long chains, and measured 0.3 to 0.4 by 2.6 to 4 microns . The bacterium had a specific requirement for NaCl and grew at NaCl concentrations of between 6 and 24%, with optimal growth at 9% NaCl . The isolate grew at temperatures of between 22 and 51 degrees C and pH values of between 5.6 and 8.0 . The doubling time in a complex medium containing 10% NaCl was 9 h . Growth was inhibited by chloramphenicol, tetracycline, and penicillin but not by cycloheximide or azide . Fermentable substrates were predominantly carbohydrates . The end products of glucose fermentation were acetate, ethanol, CO2, and H2 . The major components of the cellular fatty acids were C14:0, C16:0, C16:1, and C17:0 cyc acids . The DNA base composition of the isolate was 34 mol% G+C . Oligonucleotide catalog and sequence analyses of the 16S rRNA showed that strain VS-752T was most closely related to Haloanaerobium praevalens GSLT (ATCC 33744), the sole member of the genus Haloanaerobium . We propose that strain VS-752 (ATCC 51327) be established as the type strain of a new species, Haloanaerobium salsugo, in the genus Haloanaerobium.

Int J Syst Bacteriol, 1994 Jul, 44(3), 534 - 40
Isolation and characterization of Halothermothrix orenii gen . nov., sp . nov., a halophilic, thermophilic, fermentative, strictly anaerobic bacterium; Cayol JL et al.; The occurrence of thermophilic, halophilic anaerobic bacteria in the sediment of a Tunisian salted lake was tested in samples collected at 20-cm intervals down to a depth of 1.20 m . A long rod, present only in the 40- to 60-cm layer, was isolated at 60 degrees C in a medium containing 100 g of NaCl per liter and designated strain H168 . This strain produced acetate, ethanol, H2, and CO2 from glucose metabolism . Fructose, xylose, ribose, cellobiose, and starch were also oxidized . The optimum temperature for growth was 60 degrees C . No growth was obtained at 42 or 70 degrees C . Strain H168 grew optimally in NaCl concentrations ranging from 50 to 100 g per liter, with the upper and lower limits of growth around 200 and 40 g per liter, respectively . The G+C ratio of the DNA was 39.6 mol% . Although halophilic, moderately thermophilic bacteria have been characterized among anaerobes, particularly within methanogens, strain H168 is the first true thermophilic (growing above 60 degrees C) halophilic anaerobic bacterium described so far . The phylogeny, physiology, morphology, lipid content, and high G+C content of strain H168 are sufficiently different from those of genera belonging to the family Haloanaerobiaceae to justify the definition of a new genus.

J Bacteriol, 1994 Jun, 176(12), 3820 - 3
Expression of a yeast intron-containing tRNA in the archaeon Haloferax volcanii; Palmer JR et al.; Expression of the yeast tRNAPro(UGG) gene in Haloferax volcanii resulted in the production of a single stable transcript that had not undergone intron processing or processing of 5' and 3' flanking sequences . Mutation of the exon-intron boundary region of this RNA to produce a precursor RNA with the preferred halobacterial consensus exon-intron boundary structure did not restore intron processing . Processing of 5' and 3' flanking sequences was restored when the acceptor stem U6-U67 pair was changed to A6-U67 . The significance of these results in defining the recognition requirements of tRNA maturation enzymes in the halophilic domain Archaea is discussed.

J Biochem (Tokyo), 1994 Jun, 115(6), 1162 - 5
Properties of the Na+/H+ antiporter in Vibrio parahaemolyticus; Kuroda T et al.; The properties of the Na+/H+ antiporter in Vibrio parahaemolyticus, a slightly halophilic bacterium, were investigated using everted membrane vesicles . It appears that at least two Na+/H+ antiporters are present, one that is pH-dependent and one that is pH-independent . These two antiporters appear to correspond to the NhaA and NhaB antiporters of Escherichia coli, respectively . It seems that amiloride strongly inhibits the pH-dependent antiporter . Na+ is the best substrate for both of the two V . parahaemolyticus antiporters . Li+ is a poorer substrate and K+ is not a substrate . No K+/H+ antiport activity was detected in membrane vesicles of this organism . The Na+(Li+)/H+ antiport activity greatly increased with an increase in pH of the assay medium . pH did not affect the Km value of the Na+/H+ antiport, but it did increase the Vmax.

J Mol Evol, 1994 Jun, 38(6), 566 - 76
Evolutionary relationships of bacterial and archaeal glutamine synthetase genes; Brown JR et al.; Glutamine synthetase (GS), an essential enzyme in ammonia assimilation and glutamine biosynthesis, has three distinctive types: GSI, GSII and GSIII . Genes for GSI have been found only in bacteria (eubacteria) and archaea (archaebacteria), while GSII genes only occur in eukaryotes and a few soil-dwelling bacteria . GSIII genes have been found in only a few bacterial species . Recently, it has been suggested that several lateral gene transfers of archaeal GSI genes to bacteria may have occurred . In order to study the evolution of GS, we cloned and sequenced GSI genes from two divergent archaeal species: the extreme thermophile Pyrococcus furiosus and the extreme halophile Haloferax volcanii . Our phylogenetic analysis, which included most available GS sequences, revealed two significant prokaryotic GSI subdivisions: GSI-alpha and GSI-beta . GSI-alpha-genes are found in the thermophilic bacterium, Thermotoga maritima, the low G+C Gram-positive bacteria, and the Euryarchaeota (includes methanogens, halophiles, and some thermophiles) . GSI-beta-type genes occur in all other bacteria . GSI-alpha- and GSI-beta-type genes also differ with respect to a specific 25-amino-acid insertion and adenylylation control of GS enzyme activity, both absent in the former but present in the latter . Cyanobacterial genes lack adenylylation regulation of GS and may have secondarily lost it . The GSI gene of Sulfolobus solfataricus, a member of the Crenarchaeota (extreme thermophiles), is exceptional and could not be definitely placed in either subdivision.

FEBS Lett, 1994 May 16, 344(2-3), 125 - 8
Transcription of the halophage phi H repressor gene is abolished by transcription from an inversely oriented lytic promoter; Stolt P et al.; The temperate phage phi H of the extremely halophilic archaebacterium Halobacterium salinarium encodes a repressor, Rep, which in the immune state represses the production of an early lytic transcript, denoted T4 . Rep acts at the transcriptional level by blocking the promoter for T4 . The promoter for the rep gene itself is positioned back to back to the promoter for T4, in a manner analogous to that of the cI/cro genes in bacteriophage lambda . Transcription of the rep gene does not occur when the phase is growing lytically . We show that this repressor of rep transcription during lytic growth is due to the transcription per se from the stronger, oppositely oriented promoter for T4, without the need of a phage gene product.

Can J Microbiol, 1994 May, 40(5), 369 - 74
Effect of ionizing dose rate on the radioresistance of some food pathogenic bacteria; Dion P et al.; Food pathogenic bacteria including Listeria monocytogenes (1A1 and ATCC 19111), Staphylococcus aureus (GD13 and ATCC 13565), Escherichia coli O157:H7 (ATCC 35150), Salmonella typhimurium, Yersinia enterocolitica, Vibrio parahaemolyticus, and Campylobacter jejuni were exposed to various rates of ionizing radiation (0.78, 2.6, and 22 kGy/h) emitted by three different 60Co irradiators . D10 values (D10 is the radiation dose required to eliminate 90% of a bacterial population (one logarithmic cycle reduction)) were calculated for the various strains and growth conditions tested . A covariance analysis of these results revealed that the dose rates studied had no significant influence on the radiosensitivity of these bacteria . At all dose rates, the bacteria were more radiosensitive when irradiated in a saline solution (0.85% NaCl) than in a chicken breast meat suspension . The growth phase of the bacterial population had a variable influence on its radioresistance . For L . monocytogenes 1A1, Staphylococcus aureus ATCC 13565, E . coli O157:H7, Y . enterocolitica, and V . parahaemolyticus, radioresistance was not significantly different in the exponential and stationary phases . Populations of L . monocytogenes ATCC 19111 and Staphylococcus aureus GD13 were significantly more resistant in the stationary phase (D10 = 0.23 and 0.12 kGy, respectively) than in the exponential phase (D10 = 0.17 and 0.09 kGy, respectively) . Among the pathogenic bacteria investigated in this study, the most radioresistant was L . monocytogenes (D10 = 0.16-0.38 kGy, Gram-positive bacilli) and the most radiosensitive was V . parahaemolyticus (D10 = 0.03-0.04 kGy, halophilic Gram-negative bacilli).

Biophys Chem, 1994 May, 50(1-2), 3 - 16
Crystallography of halophilic ribosome: the isolation of an internal ribonucleoprotein complex; Evers U et al.; Crystals of 50S ribosomal subunits from Haloarcula marismortui diffracting to 2.9 A resolution were grown . Because of their large unit cells and the extremely weak diffracting power, almost all X-ray crystallographic analysis of these crystals must be performed with intense synchrotron radiation . At ambient temperature, all ribosomal crystals decay upon the first instance of X-irradiation . To overcome this severe sensitivity, procedures for data collection at cryo temperature were developed . Under these conditions the crystals can be irradiated for periods sufficient for the collection of more than one data set from an individual crystal (days or weeks) with no observable damage . They also can be stored for months, to resume interrupted measurements . To assist the interpretation of the anticipated electron density map, a specific internal nucleoprotein complex of protein HmaL1 and a stretch of H23S rRNA was isolated from the halophilic ribosome . The fragments of the 23S rRNA protected by the protein from nuclease digestion were sequenced . Alignment of the sequences of some archaebacterial L1-specific RNA fragments to the corresponding parts of eubacterial and eukaryotic rDNAs, localized the sequence identities to two distinct regions . Chimeric complexes were reconstituted with the corresponding E . coli ribosomal components, indicating a rather high homology, despite the evolution distance . A feasible secondary structure of the rRNA stretch participating in this complex was found to be compatible with the one proposed for the corresponding part in the E . coli ribosomal RNA.

Eur J Biochem, 1994 Apr 15, 221(2), 863 - 9
Characterisation and purification of ribulose-bisphosphate carboxylase from heterotrophically grown halophilic archaebacterium, Haloferax mediterranei; Rajagopalan R et al.; The CO2-fixing enzyme of Calvin cycle ribulose-1,5-bisphosphate-carboxylase/oxygenase has been isolated from a halophilic bacterium, Haloferax mediterranei grown heterotrophically . A homogeneous preparation was obtained from sonicated extract of the cells by three steps, resulting in a specific activity of 52 nmol.min-1.mg protein-1 . The physicochemical and catalytic properties of the enzyme were studied . The halobacterial ribulose-bisphosphate carboxylase is an oligomer of 54-kDa and 14-kDa subunits as detected by SDS/PAGE . By sucrose-density-gradient centrifugation, the molecular mass of the enzyme was estimated as approximately 500 kDa indicating a hexadecameric nature . No evidence for an additional form of the enzyme devoid of small subunits was obtained . The enzyme required Mg2+ for activity, KCl for activity and stability, and an optimal pH of 7.8 . In contrast to many halophilic proteins, ribulose-bisphosphate carboxylase from H . mediterranei is not an acidic protein . From the comparison of amino acid composition of halobacterial enzyme with its counterparts from a few eukaryotic and eubacterial sources, the S delta Q values showed that these proteins share some compositional similarities.

Eur J Biochem, 1994 Apr 15, 221(2), 779 - 85
Comparative analysis of the protein components from 5S rRNA.protein complexes of halophilic archaebacteria; McDougall J et al.; The 5S RNA.protein complexes have been isolated from the 50S subunit of the halophilic archaebacteria Halobacterium cutirubrum, Halobacterium halobium, Halobacterium salinarium, Haloferax mediterranei, Haloferax volcanii and Haloarcula marismortui . The 50S subunits from most of the halophiles released a multiprotein ribonucleoprotein particle similar to that previously observed with the H . cutirubrum 5S RNA.protein complex, which contained proteins from the L5 and L18 ribosomal protein families . Ribosomes from H . marismortui, however, released an RNA.protein complex containing a single protein (L18) that is homologous to the single protein found in the eukaryotic 5S ribonucleoprotein complexes . N-terminal sequence analyses of the halophilic 5S RNA-binding proteins suggest that the L18 protein primary structure is highly conserved, with only the H . marismortui protein having a sequence difference in at least the first twenty amino acids . Although the L5 group of ribosomal proteins also shows a high conservation, it appears that the proteins may have had more freedom to diverge throughout evolution.

Biochem Mol Biol Int, 1994 Apr, 32(6), 1075 - 83
Aminoacyl-tRNA synthetases from Haloarcula marismortui: an evidence for a multienzyme complex in a procaryotic system; Goldgur Y et al.; Aminoacyl-tRNA synthetases in halophilic archaebacterium Haloarcula marismortui form a high molecular weight multienzyme complex which is resistant to dissociation when subjected to gel filtration, ion exchange, ammonium sulfate mediated and hydroxyapatite chromatography . Functional and structural aspects of the aminoacyl-tRNA synthetase complex formation are discussed.

J Mol Evol, 1994 Apr, 38(4), 420 - 32
DNA-dependent RNA polymerase subunit B as a tool for phylogenetic reconstructions: branching topology of the archaeal domain; Klenk HP et al.; The branching topology of the archaeal (archaebacterial) domain was inferred from sequence comparisons of the largest subunit (B) of DNA-dependent RNA polymerases (RNAP) . Both the nucleic acid sequences of the genes coding for RNAP subunit B and the amino acid sequences of the derived gene products were used for phylogenetic reconstructions . Individual analysis of the three nucleotide positions of codons revealed significant inequalities with respect to guanosine and cytosine (GC) content and evolutionary rates . Only the nucleotides at the second codon positions were found to be unbiased by varied GC contents and sufficiently conserved for reliable phylogenetic reconstructions . A decision matrix was used for the combination of the results of distance matrix, maximum parsimony, and maximum likelihood methods . For this purpose the original results (sums of squares, steps, and logarithms of likelihoods) were transformed into comparable effective values and analyzed with methods known from the theory of statistical decisions . Phylogenetic invariants and statistical analysis with resampling techniques (bootstrap and jackknife) confirmed the preferred branching topology, which is significantly different from the topology known from phylogenetic trees based on 16S rRNA sequences . The preferred topology reconstructed by this analysis shows a common stem for the Methanococcales and Methanobacteriales and a separation of the thermophilic sulfur archaea from the methanogens and halophiles . The latter coincides with a unique phylogenetic location of a characteristic splitting event replacing the largest RNAP subunit of thermophilic sulfur archaea by two fragments in methanogens and halophiles . This topology is in good agreement with physiological and structural differences between the various archaea and demonstrates RNAP to be a suitable phylogenetic marker molecule.

Enzyme Microb Technol, 1994 Apr, 16(4), 266 - 75
Catalytic properties and potential of an extracellular protease from an extreme halophile; Ryu K et al.; An extracellular protease has been isolated and partially purified from the extreme halophile Halobacterium halobium (ATCC 43214) . The major enzyme component has a M(r) of 66,000 and is highly dependent upon salt concentrations near saturation for catalytic activity and stability . In aqueous solutions, a decrease in the NaCl concentration from 4 to 1 M results in an increase of nearly three orders of magnitude in the first-order rate constant of inactivation at 30 degrees C . Salt effects the stability of the enzyme in a cooperative manner, with a Hill coefficient of 4.1, which is similar to that of other enzymes from extreme halophiles . The enzyme activity is dramatically affected by the salt concentration, with a loss of 2.5 orders of magnitude in kcat/Km in going from 4 to 0 M NaCl . This loss in catalytic efficiency is primarily due to a dramatic increase in the Km for the substrate in low-salt media . Thermodynamic analysis revealed that this Km increase was mainly the result of increased solubility of the synthetic peptide substrate in low-salt media, which dramatically increases the ground-state stability of the substrate . This results in an effectively reduced substrate partitioning from the bulk solution into the enzyme's active site and an increased value of Km . The halophilic protease is also active in DMF/water mixtures, albeit with novel catalytic properties . In 33% (v/v) DMF in aqueous buffer, the esterase activity of the enzyme is ca . 80-fold higher than the corresponding amidase activity . This contrasts to the situation in pure aqueous buffer, in which the esterase activity is only fourfold higher than the amidase activity . The increased esterase activity relative to amidase activity prompted us to investigate the use of the protease in kinetically controlled peptide synthesis . The enzyme has a broad acyl donor substrate specificity and can effectively use amino acid esters of Phe, Tyr, Trp, Ser, Gly, and Ala . The enzyme is significantly more selective for the amino acid amide, preferring Gly in the P'1 site . A series of glycine-containing oligopeptides have been prepared in yields up to 76% without degradation due to secondary hydrolysis.

Gene, 1994 Mar 11, 140(1), 17 - 24
PCR-mediated cloning and sequencing of the gene encoding glutamate dehydrogenase from the archaeon Sulfolobus shibatae: identification of putative amino-acid signatures for extremophilic adaptation; Benachenhou-Lahfa N et al.; Highly degenerate oligodeoxyribonucleotides (oligos) were used to PCR amplify the most conserved region of the glutamate dehydrogenase (GDH)-encoding gene from the extreme thermophilic archaeon, Sulfolobus shibatae . The amplified fragment was cloned and sequenced, and then used as a homologous probe to clone a genomic restriction fragment containing the near-complete gdhA gene . The deduced amino acid (aa) sequence shows a very high degree of similarity with the aa sequence previously determined by direct sequencing of the purified enzyme from Sulfolobus solfataricus {Maras et al., Eur . J . Biochem . 203 (1992) 81-87} . The introduction of this new sequence into our GDH phylogenetic trees {Benachenhou-Lahfa et al., J . Mol . Evol . 35 (1993) 335-346} showed that it is a new member of hexameric GDH family II, and did not modify the topology of the trees . Comparison of the primary structures of extremophilic GDH enzymes (halophilic, thermophilic and hyperthermophilic) with those of their non-halophilic and mesophilic counterparts in this family II led us to identify a few aa changes which seem to be specific either to hyperthermophilic or halophilic adaptation.

Biochemistry, 1994 Mar 8, 33(9), 2476 - 83
Molecular structure of the oxidized high-potential iron-sulfur protein isolated from Ectothiorhodospira vacuolata; Benning MM et al.; The high-potential iron-sulfur protein (iso-form II) isolated from Ectothiorhodospira vacuolata has been crystallized and its three-dimensional structure determined by molecular replacement procedures and refined to 1.8-A resolution with a crystallographic R factor of 16.3% . Crystals employed in the investigation belonged to the space group C222(1) with unit cell dimensions of a = 58.4 A, b = 64.7 A, and c = 39.3 A and one molecule per asymmetric unit . Like those HiPIPs structurally characterized thus far, the E . vacuolata molecule contains mostly reverse turns that wrap around the iron-sulfur cluster with cysteine residues 34, 37, 51, and 65 ligating the metal center to the polypeptide chain . There are 57 ordered solvent molecules, most of which lie at the surface of the protein . Two of these water molecules play important structural roles by stabilizing the loops located between Asp 42 and Lys 57 . The metal center binding pocket is decidedly hydrophobic with the closest solvent molecule being 6.9 A from S2 of the {4Fe-4S} cluster . The E . vacuolata HiPIP molecules pack in the crystalline lattice as dimers with their iron-sulfur centers approximately 17.5 A apart . On the basis of biochemical properties, it was anticipated that the E . vacuolata HiPIP would be structurally more similar to the HiPIP isolated from Ectothiorhodospira halophila than to the protein obtained from Chromatium vinosum . In fact, the E . vacuolata molecule is as structurally close to the C . vinosum HiPIP as it is to the E . halophila protein due to the presence of various insertions and deletions that disrupt local folding.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbiol Rev, 1994 Mar, 58(1), 27 - 38
Anaerobic bacteria from hypersaline environments; Ollivier B et al.; Strictly anaerobic halophiles, namely fermentative, sulfate-reducing, homoacetogenic, phototrophic, and methanogenic bacteria are involved in the oxidation of organic carbon in hypersaline environments . To date, six anaerobic fermentative genera, containing nine species, have been described . Two of them are homoacetogens . Six species belong to the family Haloanaerobiaceae, as indicated by their unique 16S rRNA oligonucleotide sequences . Desulfohalobium retbaense and Desulfovibrio halophilus represent the only two moderately halophilic sulfate reducers so far reported . Among anoxygenic phototrophic anaerobes, a few purple bacteria with optimal growth at salinities between 6 and 11% NaCl have been isolated from hypersaline habitats . They belong to the genera Rhodospirillum, Chromatium, Thiocapsa, and Ectothiorhodospira . The commonest organisms isolated so far are Chromatium salexigens, Thiocapsa halophila, and Rhodospirillum salinarum . Extremely halophilic purple bacteria have most commonly been isolated from alkaline brines and require about 20 to 25% NaCl for optimal growth . They belong to the family Ectothiorodhospiraceae . Their osmoregulation involves synthesis or uptake of compatible solutes such as glycine-betaine that accumulate in their cytoplasm . The existence of methanogens in hypersaline environments is related to the presence of noncompetitive substrates such as methylamines, which originate mainly from the breakdown of osmoregulatory amines . Methanogenesis probably does not contribute to the mineralization of carbohydrates at NaCl concentrations higher than 15% . Above this concentration, sulfate reduction is probably the main way to oxidize H2 (although at rates too low to use up all the H2 formed) and occupies a terminal function kn the degradation of carbohydrates . Three genera and five species of halophilic methylotrophic methanogens have been reported . A bloom of phototrophic bacteria in the marine salterns of Salins-de-Giraud, located on the Mediterranean French coast in the Rhone Delta, is also described.

Biochem J, 1994 Mar 1, 298 ( Pt 2), 465 - 70
Further thermal characterization of an aspartate aminotransferase from a halophilic organism; Muriana FJ et al.; Aspartate aminotransferase (AspAT, EC 2.6.1.1) from the halophilic archaebacterium Haloferax mediterranei was purified {Muriana, Alvarez-Ossorio and Relimpio (1991) Biochem . J . 278, 149-154} and further characterization of the effects of temperature on the activity and stability of the halophilic AspAT were carried out . The halophilic transaminase is most active at 65 degrees C and stable at high temperatures, under physiological or nearly physiological conditions (3.5 M KCl, pH 7.8) . Thermal inactivation (60-85 degrees C) of the halophilic AspAT followed first-order kinetics, 2-oxoglutarate causing a shift of the thermal inactivation curves to higher temperatures . The salt concentration affected the thermal stability of the halophilic transaminase at 60 degrees C, suggesting that disruption of hydrophobic interactions may play an important role in the decreased thermal stability of the enzyme.

Clin Infect Dis, 1994 Mar, 18(3), 310 - 2
Severe gastroenteritis associated with Vibrio hollisae infection: report of two cases and review; Abbott SL et al.; Vibrio hollisae, one of the more recently described halophilic Vibrio species, is infrequently associated with gastrointestinal disease and only rarely recovered from individuals presenting with gram-negative sepsis . In this report we describe two cases of severe gastrointestinal disease associated with V . hollisae in otherwise healthy individuals . In one of these individuals, severe epigastric pain was apparently associated with signs of pseudoappendicitis, necessitating exploratory surgery . In both individuals, infection was associated with the ingestion of raw shellfish . These cases are discussed in light of previous reports on the epidemiology, pathogenesis, and spectrum of disease caused by this unusual pathogen.

Trends Biotechnol, 1994 Mar, 12(3), 81 - 8
Retinal-protein complexes as optoelectronic components; Vsevolodov NN et al.; Naturally occurring retinal-protein complexes (RPCs) have recently received much attention with regard to their potential use as light-sensitive elements for optical recording . The best-known RPC is bacteriorhodopsin (BR), a photosensitive protein from the membrane of extreme halophilic bacteria, which has been studied in great detail . The remarkably robust nature of BR, coupled with its ability to reversibly change color upon illumination and its high cyclicity of ground-to-photoinduced state transitions, makes BR a promising material for optical information processing.

Protein Eng, 1994 Feb, 7(2), 213 - 20
A structure-based model for the halophilic adaptation of dihydrofolate reductase from Halobacterium volcanii; Bohm G et al.; 'Halophilic adaptation' of proteins, i.e . the requirement for high concentrations of monovalent ions for thermodynamic stability of proteins from halophilic organisms, is not fully understood . In this work, an explanation for the halophilic behavior of dihydrofolate reductase (h-DHFR) from Halobacterium volcanii is attempted, based on a model structure derived from comparative modeling to dihydrofolate reductase from Escherichia coli . The model structure of h-DHFR shows an unique asymmetrical charge distribution over the protein surface, with positively charged amino acids centered around the active site and negative charges on the opposite side of the enzyme . This particular charge distribution and the correlated molecular dipole are functionally relevant . The negative charges on the surface form clusters which are shielded at high salt concentrations; at low salt, they repulse each other, thus destabilizing the protein . Results are in accordance with denaturation data and, thus, provide an explanation for the exceptional stability properties of h-DHFR.

Biochem Biophys Res Commun, 1994 Jan 28, 198(2), 466 - 72
Cyclosporin A sensitive peptidyl-prolyl cis-trans isomerase in a halophilic archaeum, Halobacterium cutirubrum; Nagashima K et al.; A cyclophilin type peptidyl-prolyl cis-trans isomerase was purified from a halophilic archaeum, Halobacterium cutirubrum DSM 669 . The activity increased with an increase in KCl concentration up to 4 M . Sensitivity to cyclosporin A was comparable to that of eukaryotic cyclophilin and was also affected by KCl . IC50 for Cyclosporin A was 1.5 x 10(-8) M in 2.9 M KCl but 1.4 x 10(-7) M in 1.4 M KCl . The apparent molecular weight was 31 K by SDS-PAGE and 22 K in 2.9 M KCl and 150 K in 0 M KCl by gel filtration chromatography . The N-terminal amino acid sequence (33 residues) sheared 4 completely and 5 highly conserved amino acid residues with other reported cyclophilin family PPIases.

J Exp Biol, 1994 Dec, 197(1), 165 - 78
IS WALKING COSTLY FOR ANURANS? THE ENERGETIC COST OF WALKING IN THE NORTHERN TOAD BUFO BOREAS HALOPHILUS
Walton B, Peterson C, Bennett A.
Locomotor mode and the maximal capacity for aerobic metabolism are thought to be co-adapted in anuran amphibians . Species that rely heavily on walking often have high capacities for aerobic metabolism relative to species that rely primarily on saltation . We tested the hypothesis of co-adaptation of gait and aerobic metabolism by investigating the locomotor energetics of Bufo boreas halophilus, a toad that walks, but does not hop . Rates of oxygen consumption during locomotion were measured in an enclosed variable-speed treadmill . The steady-state rate of oxygen consumption (V(dot)O2ss) increased linearly within a range of sustainable speeds {V(dot)O2ss (ml O2 g-1 h-1) = 0.93 x speed (km h-1) + 0.28} . The minimum cost of transport, Cmin (the slope of this relationship), varied significantly among individual toads . When expressed in units of oxygen consumed per distance travelled (ml O2 km-1), Cmin scaled isometrically with body mass: Cmin = 0.69mass1.07 . Consequently, mass-specific Cmin (ml O2 g-1 km-1) was uncorrelated with body mass . Variation in Cmin was also unrelated to experimental temperature . Mass-specific Cmin estimates were similar to previous allometric predictions for terrestrial animals of similar size, which contrasts with previous findings for another toad species . Maximum rates of oxygen consumption measured in closed, rotating respirometers were significantly higher than the maximum rates achieved on the treadmill, but lower than those measured previously in other Bufo species . Our results indicate that walking is not necessarily a costly gait for toads and that high maximum rates of oxygen consumption are not associated with reliance on walking within the genus Bufo.

Arch Biochem Biophys, 1994 Jan, 308(1), 78 - 81
Amino acid sequences of two high-potential iron sulfur proteins (HiPIPs) from the moderately halophilic purple phototrophic bacterium Ectothiorhodospira vacuolata; Ambler RP et al.; There are two equally abundant high-potential iron sulfur protein (HiPIP) isozymes present in the purple sulfur bacterium Ectothiorhodospira vacuolata . We have determined the amino acid sequences, which contain 71 and 72 residues . The two HiPIPs can be aligned without any internal insertions or deletions and are 65% identical to one another . The E . vacuolata HiPIPs are most similar to the HiPIP isozymes from Ectothiorhodospira halophila (32-36% identity) and require at least one internal gap for alignment . Other HiPIPs require greater numbers of insertions and deletions for alignment with those of E . vacuolata and E . halophila, and the percentage similarities are slightly smaller (19-40% identity) . The E . vacuolata HiPIP isozymes appear to be slightly closer to other species than are the E . halophila isozymes . The E . vacuolata and E . halophila HiPIPs also show slightly greater similarity to the five species of Chromatiaceae, which have been studied, and less similarity to the non-sulfur purple species . These results are in agreement with other studies, which indicate that the two purple sulfur bacterial families, Ectothiorhodospiraceae and Chromatiaceae, are more closely related to one another than to the Rhodospirillaceae.

Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi, 1994 Jan-Feb, 35(1), 84 - 9
{Beta-thalassemia major complicated with Vibrio vulnificus septicemia: report of one case}; Shih YT et al.; Vibrio vulnificus is a halophilic Vibrio that has been isolated repeatedly from sea-water and shellfish during the warm months of the year . It's a virulent pathogen for men and is frequently associated with overwhelming infections including sepsis, gangrene of extremities and high mortality rate . We encountered a 13-year-old boy who had a history of beta-thalassemia major with secondary hemochromatosis, suffering from vomiting, diarrhea, fever and hypotension . Physical examination revealed that ecchymosis, bullae and ulceration were noted over the left leg . Vibrio vulnificus was isolated from the blood . Initially, the patient did not respond to the appropriate antibiotics treatment, subsequently surgical debridement was performed . After that, the patient recovered gradually, and discharged home after 17 days of admission . In conclusion, when patients present with sepsis and/or characteristic skin lesion-hemorrhagic bullae, particularly those with thalassemia major, hemochromatosis or underlying liver disease and a history of marine exposure, clinicians should be alerted to this potentially fatal infection and should commence appropriate assessment and treatment immediately.

Int J Pept Protein Res, 1994 Jan, 43(1), 97 - 106
Relevance of sequence statistics for the properties of extremophilic proteins; Bohm G et al.; The amino acid composition of proteins from mesophilic and extremophilic organisms is commonly assumed to reflect the mechanisms of molecular adaptation to extremes of physical conditions . In this context, halophilic behaviour has been attributed to significantly increased numbers of aspartic and glutamic acid residues . However, extending the analysis to a statistically relevant set of related proteins, dihydrofolate reductase from Halobacterium volcanii, as an example, shows that the increase in negative charge is found to be less significant than other exchanges of amino acids (e.g., Ala, Asn, Arg, Lys, Phe, Ser) . Thus, the high water binding capacity of negatively charged residues cannot be unambiguously correlated with the anomalous stability of halophilic proteins . A similar caveat holds for generalizations regarding the thermal stability of proteins . In this case, D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was compared with a number of mesophilic and moderately thermophilic homologs . Again, 'traffic rules of stabilization', in terms of amino acid changes in going from mesophilic to thermophilic proteins, cannot be given.

Biochimie, 1994, 76(7), 583 - 91
Amino acid sequences of cytochromes c2 and c' from the moderately halophilic purple phototrophic bacterium Rhodospirillum salexigens; Ambler RP et al.; Rhodospirillum salexigens is a moderately halophilic purple phototrophic bacterium which grows optimally in 8% NaCl . The amino acid sequences of the two principal soluble cytochromes c have been determined . One of these is a cytochrome c2, similar in size to mitochondrial cytochrome c . While clearly of the same sequence class as mitochondrial cytochrome c and the proteins from several other Gram-negative bacteria, it does not show particular affinity to any already known sequence in terms of the percentage sequence identity . The other protein is a cytochrome c', but is also a divergent member of this widespread group . The lack of appreciable sequence identity to other species is probably due to a limit of divergence which has been reached for the majority of purple bacterial species . However, the numbers of insertions and deletions and their locations in cytochromes c2 and c' suggest that R salexigens may be related to Rhodospirillum molischianum . Like other electron transport proteins from halophiles, both of these cytochromes are notable for their high content of arginine as compared with lysine and both are acidic . However, they do not show any particular sequence homology to electron transport proteins that have been characterized from the extremely halophilic phototrophes of the genus Ectothiorhodospira . Thus, it appears that adaptation to halophilic habitats has independently occurred more than once in purple bacteria.

Antonie Van Leeuwenhoek, 1994, 65(4), 331 - 47
Photobiology of bacteria; Hellingwerf KJ et al.; The field of photobiology is concerned with the interactions between light and living matter . For Bacteria this interaction serves three recognisable physiological functions: provision of energy, protection against excess radiation and signalling (for motility and gene expression) . The chemical structure of the primary light-absorbing components in biology (the chromophores of photoactive proteins) is surprisingly simple: tetrapyrroles, polyenes and derivatised aromats are the most abundant ones . The same is true for the photochemistry that is catalysed by these chromophores: this is limited to light-induced exciton- or electron-transfer and photoisomerization . The apoproteins surrounding the chromophores provide them with the required specificity to function in various aspects of photosynthesis, photorepair, photoprotection and photosignalling . Particularly in photosynthesis several of these processes have been resolved in great detail, for others at best only a physiological description can be given . In this contribution we discuss selected examples from various parts of the field of photobiology of Bacteria . Most examples have been taken from the purple bacteria and the cyanobacteria, with special emphasis on recently characterised signalling photoreceptors in Ectothiorhodospira halophila and in Fremyella diplosiphon.

Antonie Van Leeuwenhoek, 1994, 65(4), 271 - 84
Bioenergetics: the evolution of molecular mechanisms and the development of bioenergetic concepts; Skulachev VP; Possible routes for the evolution of cell energetics are considered . It is assumed that u.v . light was the primary energy source for the precursors of the primordial living cell and that primitive energetics might have been based on the use of the adenine moiety of ADP as the u.v . chromophore . It is proposed that the excitation of the adenine residue facilitated phosphorylation of its amino group with subsequent transfer of a phosphoryl group to the terminal phosphate of ADP to form ATP . ATP-driven carbohydrate synthesis is considered as a mechanism for storing u.v.-derived energy, which was then used in the dark . Glycolysis presumably produced compounds like ethanol and CO2, which easily penetrate the membrane and therefore were lost by the cell . Later lactate-producing glycolysis appeared, the end product being non-penetrant and, hence, retained inside the cell to be utilized to regenerate carbohydrates when light energy became available . Production of lactate was accompanied by accumulation of equimolar H+ . To avoid acidification of the cell interior, an F0-type H+ channel was employed . Later it was supplemented with F1 . This allowed the ATP energy to be used for 'uphill' H+ pumping to the medium, which was acidified due to glycolytic activity of the cells . In the subsequent course of evolution, u.v . light was replaced by visible light, which has lower energy but is less dangerous for the cell . It is assumed that bacteriorhodopsin, a simple and very stable light-driven H+ pump which still exists in halophilic and thermophilic Archaea, was the primary system utilizing visible light . The delta mu-H+ formed was used to reverse the H(+)-ATPase, which began to function as H(+)-ATP-synthase . Later, bacteriorhodopsin photosynthesis was substituted by a more efficient chlorophyll photosynthesis, producing not only ATP, but also carbohydrates . O2, a side product of this process, was consumed by the H(+)-motive respiratory chain to form delta mu-H+ in the dark . At the next stage of evolution, a parallel energy-transducing mechanism appeared which employed Na+ instead of H+ as the coupling ion (the Na+ cycle).(ABSTRACT TRUNCATED AT 400 WORDS)

Microbios, 1994, 80(325), 209 - 14
Effect of osmotic variation on the outer membrane proteins of Vibrio vulnificus: identification of a major heat-modifiable protein; Simpson LM et al.; Cells of the moderately halophilic bacterium Vibrio vulnificus showed marked morphological changes, manifested primarily by pronounced cell elongation when grown in a defined medium with increased salt concentrations of 0.5 to 2.0% . These morphological changes were not accompanied by changes in outer membrane proteins (Omp) . A similar lack of Omp variation was observed when cells were grown in heart infusion broth containing 1-2% NaCl . Osmotic upshock during growth also had no effect on Omp profiles . A heat modifiable protein with a molecular weight of 38 kD similar to Omp A of Escherichia coli, was identified in cells grown in heart infusion broth . The results suggest that under these osmotic conditions V . vulnificus may not need to regulate transport through modification of its outer membrane proteins.

Gene, 1993 Dec 22, 136(1-2), 373 - 4
Sequence of the gene encoding ribosomal protein L11 from Thermus thermophilus HB8; Heinrich T et al.; The complete nucleotide sequence of the gene encoding ribosomal protein L11 from the extreme thermophilic eubacterium, Thermus thermophilus HB8, was determined . L11 amino acid (aa) sequences from mesophilic and halophilic organisms, as well as from another thermophiles, were compared with the T . thermophilus L11 aa sequence.

Experientia, 1993 Dec 15, 49(12), 1027 - 36
Biology of halophilic bacteria, Part II . Membrane lipids of extreme halophiles: biosynthesis, function and evolutionary significance; Kates M; Archaebacteria (archaea) are comprised of three groups of prokaryotes: extreme halophiles, methanogens and thermoacidophiles (extreme thermophiles) . Their membrane phospholipids and glycolipids are derived entirely from a saturated, isopranoid glycerol diether, sn-2,3-diphytanylglycerol ('archaeol') and/or its dimer, dibiphytanyldiglyceroltetraether ('caldarchaeol') . In extreme halophiles, the major phospholipid is the archaeol analogue of phosphatidylglycerolmethylphosphate (PGP-Me); the glycolipids are sulfated and/or unsulfated glycosyl archaeols with diverse carbohydrate structure characteristic of taxons on the generic level . Biosynthesis of these archaeol-derived polar lipids occurs in a multienzyme, membrane-bound system that is absolutely dependent on high salt concentration (4 M) . The highly complex biosynthetic pathways involve intermediates containing glycerol ether-linked C20-isoprenyl groups which are reduced to phytanyl groups to give the final saturated polar lipids . In methanogens, polar lipids are derived both from archaeol and caldarchaeol, and thermoacidophiles contain essentially only caldarchaeol-derived polar lipids . The function of these membrane polar lipids in maintaining the stability, fluidity and ionic properties of the cell membrane of extreme halophiles, as well as the evolutionary implications of the archaeol and caldarchaeol-derived structures will be discussed.

Nucleic Acids Res, 1993 Dec 11, 21(24), 5595 - 9
Structure specific ds/ss-RNase activity in the extreme halophile Halobacterium salinarium; Stolt P et al.; A ds/ss-RNA processing activity involved in antisense-RNA mediated gene regulation in the extremely halophilic archaebacterium Halobacterium salinarium was investigated in vivo . H.salinarium cells were transformed with DNA encoding an RNA species complementary to a part of the major lytic transcript, termed T4, of the H.salinarium phage phi H . The transformants transcribing this construct, when infected by phage were able to process T4 in a similar way to the processing of the lytic transcript denoted T1, in the natural sense-antisense system . Processing of T4 was not observed under normal phage growth on wild-type cells . Thus the antisense-RNA mediated processing activity earlier reported is dependent on the presence of an RNA duplex and is not sequence specific.

Biochim Biophys Acta, 1993 Dec 2, 1210(1), 35 - 40
The glycolipid of Halobacterium trapanicum; Trincone A et al.; The structural elucidation of the polar lipids in Halobacterium trapanicum is reported with particular emphasis on a new sulfated disaccharide derivative of 2,3-di-O-phytanyl-sn-glycerol . The full structural designation of this glycolipid is 2,3-di-O-phytanyl-1-O- (mannopyranosyl-(2-sulfate)-alpha-D-1-2-glucopyranosyl-alpha-D)-sn-glyce rol . The value of glycolipid structures in the taxonomy of halophilic Archaea is also discussed.

Virology, 1993 Dec, 197(2), 678 - 84
HF1 and HF2: novel bacteriophages of halophilic archaea; Nuttall SD et al.; Two novel halophilic archaebacterial bacteriophages, HF1 and HF2, were isolated from an Australian solar saltern . They were morphologically identical with icosahedral-shaped heads (diameter 58 nm) and contractile tails (length 94 nm) . Other similarities included sensitivity to reduced ionic conditions, similar protein profiles by SDS-PAGE, and dsDNA genomes of identical size (73.5 kbp) with analogous restriction patterns . DNA-DNA hybridization data showed the two phages to be closely related . HF1 has a broad host-range, infecting members of three halobacterial genera including Halobacterium salinarium and the genetically well-characterized strain Haloferax volcanii WFD11 . Mutants showing increased plating efficiency on alternative hosts were readily selectable . By contrast, HF2 showed a limited host range, confined to the closely related dam-methylated strains Ch2 and H . saccharovorum.

Eur J Cell Biol, 1993 Dec, 62(2), 248 - 58
Immunoelectron microscopic location of tryptophanyl-tRNA synthetase in mammalian, prokaryotic and archaebacterial cells; Popenko VI et al.; Monoclonal antibody Am1 against conservative epitope of tryptophanyl-tRNA synthetase (WRS) was labeled with colloidal gold particles and used to localize the enzyme on ultrathin sections of eubacteria (Escherichia coli), archaebacteria (Methanococcus halophilus), rat pancreas tissue and rat fibroblasts (cell line RAT1) . In all cell types immunoelectron microscopy revealed predominant cytoplasmic location of gold particles, as this could be expected from known biochemical data . In particular, in mammalian cells intensive labeling was observed in cytoplasmic regions rich in polysomes and free ribosomes . At the same time, the label was virtually absent in cytoplasmic regions where microfilament bundles were present . Significant concentrations of gold particles were found in mitochondria and nuclei . In the latter case, gold particles were located over diffuse chromatin regions and were virtually absent over compact chromatin . The density of diffuse chromatin in labeling may amount to about 50% of that found in the cytoplasm . Distribution of labeled antibodies over E . coli cells looks rather similar to that found for M . halophilus: gold particles are preferably concentrated over the cytoplasm and "boundary zone", i.e., a 30 nm wide cytoplasmic zone adjacent to the nucleoid border, while the label over nucleoid is virtually absent . Two main conclusions are drawn: (i) although in the animal cell homogenates WRS is recovered mainly as a soluble cytosolic enzyme, in intact cells it is associated with defined cellular organelles and compartments; this may be an evolutionarily acquired feature probably typical for multicellular organisms; (ii) the considerable density of labeling in diffuse (not compact) chromatin regions may be indicative of WRS involvement in the active chromatin functions (transcription, processing, transfer of gene products, etc.).

Biochim Biophys Acta, 1993 Nov 16, 1216(2), 335 - 8
Nucleotide sequence of the genes encoding the L3, L4, and L23 equivalent ribosomal proteins from the archaebacterium Halobacterium halobium; Yuki Y et al.; A lambda EMBL clone containing a gene cluster coding for the ribosomal proteins L3, L4, L23 and 5' region of L2 was identified in a genomic library for the halophilic archaebacterium Halobacterium halobium using a heterologous hybridization probe from the related organism Halobacterium marismortui . The clone also contains two conserved open reading frames found in H . marismortui, although with still unknown function . Its gene organization is very similar to that of 'S10 operon' of H . marismortui . The deduced amino acid sequence of these ribosomal proteins (HhaL3, HhaL4, HhaL23 and 5' region of HhaL2) shows high similarity (64-71%) to those of the archaebacterium H . marismortui and a lesser degree of similarity to their eukaryotic (31-42%) and eubacterial (17-33%) counterparts.

Zhonghua Yi Xue Za Zhi (Taipei), 1993 Nov, 52(5), 351 - 4
Septicemia caused by Vibrio parahemolyticus: a case report; Hsu GJ et al.; Vibrio parahemolyticus is a halophilic marine vibrio commonly associated with outbreaks of acute gastroenteritis which also sometimes causes serious wound infection . It is an uncommon cause of septicemia . A few reports suggest that patients with chronic liver disease and leukemia are more susceptible . A case of liver cirrhosis with septicemia caused by this organism is discussed . The patient's condition rapidly deteriorated, and he died 12 hours after admission.

Arch Biochem Biophys, 1993 Nov 1, 306(2), 515 - 7
Transient proton uptake and release is associated with the photocycle of the photoactive yellow protein from the purple phototrophic bacterium Ectothiorhodospira halophila; Meyer TE et al.; Upon excitation by a laser flash at 445 nm, the photoactive yellow protein (PYP) undergoes a bleach and red-shift occurring in less than 10 ns, undergoes a further bleach in approximately 200 microseconds, and then recolors in approximately 200 ms . A conformational change occurs during photobleaching which exposes a hydrophobic site . We have now shown that this photocycle also involves a net uptake of one proton during formation of the fully bleached second intermediate, followed by an equivalent proton release upon return of PYP to the ground state . Proton uptake lags slightly behind PYP bleaching and is first-order, indicating that the protein conformational change occurs in two steps . The results suggest that a basic residue which is normally buried in the protein interior is transiently exposed to solvent during the PYP photocycle and, as a consequence, undergoes a change in pK . On the basis of the crystal structure of PYP, we propose that this basic residue is lysine 111.

Arch Biochem Biophys, 1993 Oct, 306(1), 83 - 93
Amino acid sequences of cytochromes c-551 from the halophilic purple phototrophic bacteria, Ectothiorhodospira halophila and E . halochloris; Ambler RP et al.; The cytochromes c-551 from Ectothiorhodospira halophila and E . halochloris contain 78 and 79 residues, respectively . The sequences can be aligned without the need to postulate any internal deletions or insertions to give 63% identity . They are apparently distantly related to the class I cytochromes c, based on the location of the heme attachment site near the N-terminus and the sixth ligand methionine near the C-terminus . Alignment with cytochromes c5 from Azotobacter and Pseudomonas, with cytochromes c6 from cyanobacteria and algae, and with cytochromes c-555 from the green phototrophic bacteria suggests that residues which occupy important positions in the three-dimensional structures of these proteins have their equivalents in the Ectothiorhodospira cytochromes c-551, but the levels of overall identity are very low, around 30% . Although the Ectothiorhodospira cytochromes c-551 are apparently distantly related to the above, they should be regarded as representative of a new subclass of type I bacterial cytochromes c . Homologs of all of the cytochromes c normally found in Pseudomonas and Azotobacter have now been found in one or more purple bacterial species . Among these, cytochrome c5 homologs are the most widely occurring in purple, green, and cyanobacteria . For the first time, all families of phototrophic bacteria plus Pseudomonas can be related to one another at the molecular level.

Int J Syst Bacteriol, 1993 Oct, 43(4), 729 - 34
Ch2, a novel halophilic archaeon from an Australian solar saltern; Nuttall SD et al.; A novel halophilic archaeon, strain Ch2, was isolated from a marine solar saltern in Geelong, Australia . The fact that this organism had a dam-methylated genome suggested that it is closely related to the taxon that includes Halobacterium saccharovorum, Halobacterium sodomense, and Halobacterium trapanicum . A sequence analysis of the 16S rRNA gene (Ch2 has three copies of this gene) showed that Ch2 is phylogenetically equidistant from the genera Haloarcula and Haloferax and closely related to H . saccharovorum . The susceptibility of both Ch2 and H . saccharovorum to the recently isolated halophage HF2 supported the hypothesis that these two organisms are closely related.

Antibiot Khimioter, 1993 Oct-Nov, 38(10-11), 60 - 7
{The microbiology of halophilic vibrios}; Ved'mina EA et al.; The current literature data and results of the authors' studies on the taxonomy and biological characteristics of halophilic Vibrio such as V . parahaemolyticus and V . alginolyticus are presented . The morphological, cultural, biochemical and fermentative properties, phages, virulence, antigenic structure, resistance to the environment and ecology of the microbes and their susceptibility to antibiotics of various chemical groups are described.

Biochemistry, 1993 Sep 14, 32(36), 9387 - 97
The electronic structure of {Fe4S4}3+ clusters in proteins . An investigation of the oxidized high-potential iron-sulfur protein II from Ectothiorhodospira vacuolata; Banci L et al.; Within the framework of an investigation of the electronic structure of oxidized high-potential iron-sulfur proteins (HiPIP), we have studied the HiPIP II from Ectothiorhodospira vacuolata, which was known to have a peculiar temperature dependence of the 1H NMR isotropic hyperfine shifts . The signals of the cysteine ligand protons have been sequence specifically assigned through NOE, NOESY, and TOCSY experiments . Nine hyperfine-shifted signals are observed: seven in the downfield and two in the upfield region . They have been assigned to the eight beta-CH2 protons of the four coordinated cysteines and to one alpha-CH cysteine proton . The two most downfield-shifted signals belong to the beta-CH2 protons of Cys 63 (Chromatium vinosum numbering) and the two upfield protons to those of Cys 43 . These two pairs of protons show a Curie-type temperature dependence of the hyperfine shifts . Among the remaining five downfield-shifted signals, three show a Curie-type temperature dependence and two have an anti-Curie temperature dependence . The former are assigned to the beta-CH2 and alpha-CH protons of Cys 77 and the latter to the beta-CH2 protons of Cys 46 . The shift patterns are thus similar, in a sequence-specific sense, to those of the analogous proteins from C . vinosum and Rhodocyclus gelatinosus, whereas they differ from those of Rhodocyclus globiformis HiPIP and even more from those of Ectothiorhodospira halophila HiPIP II . Oxidized HiPIPs can be formally viewed as containing a cluster of four ferric ions plus one extra electron . We present here a model based on a chemical equilibrium, fast on the NMR time scale, between two species, both of which contain a pair of iron(III) ions and a mixed-valence pair but are differently oriented within the protein frame . The EPR data are also discussed in the light of the debate on the nature of the different species detected at low temperature . The interpretation of the whole set of data on HiPIPs in the light of the present model is compared with that based on previous models.

Biochem Biophys Res Commun, 1993 Aug 16, 194(3), 1330 - 5
A protein-serine phosphatase from the halophilic archaeon Haloferax volcanii; Oxenrider KA et al.; We have detected a protein phosphatase activity in soluble extracts from the halophilic archaeon Haloferax volcanii . This activity was markedly stimulated by the divalent metal ions Mn2+ and Cd2+ . It dephosphorylated phosphoseryl residues in casein, mixed histones, and phosphorylase a, but not phosphotyrosyl residues in reduced, carboxyamidomethylated and maleylated lysozyme . This protein phosphatase activity was inhibited by NaF, Zn2+, vanadate, molybdate, inorganic phosphate, inorganic pyrophosphate, or p-nitrophenyl phosphate, or by treatment with diethylpyrocarbonate . Activity was unaffected by other potential inhibitors or activators such as polyamines, heparin, cyclic nucleotides, Ca2+/calmodulin, tartrate, tetramisole, okadaic acid, microcystin LR, or sulfhydryl-modifying agents . The functional similarities between this protein-serine phosphatase and that previously identified in another archaeon, the extreme acidothermophile Sulfolobus solfataricus, suggest the existence of a family of divalent metal ion-stimulated protein-serine phosphatases of extremely ancient origin in the Archaea.

Eur J Biochem, 1993 Aug 15, 216(1), 199 - 203
High expression in Escherichia coli of the gene coding for dihydrofolate reductase of the extremely halophilic archaebacterium Haloferax volcanii . Reconstitution of the active enzyme and mutation studies; Blecher O et al.; The gene coding for the enzyme dihydrofolate reductase of the extremely halophilic archaebacterium Haloferax volcanii was recombined into the Escherichia coli expression vector pET11d . Following induction, the enzyme was produced in large quantities and accumulated in the cells in an insoluble form . The enzymic activity could be efficiently reconstituted by dissolving the aggregate in 6 M guanidine hydrochloride followed by dilution into salt solutions . Mutants were produced in which Lys30 was converted to Leu (K30L), Lys31 was converted to Ala (K31A) and a double mutant in which both lysines were converted (K30L, K31A) . The mutated enzymes were produced in E . coli, activated and purified to homogeneity . The effect of the salt concentration on the steady-state kinetic parameters was determined . It was found that the salt concentration affects the Km but not kcat of the various mutants.

Mol Microbiol, 1993 Aug, 9(3), 613 - 21
Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites; Mojica FJ et al.; Two genomic sequences from the halophilic archaeon Haloferax mediterranei, where we had found PstI restriction-pattern modifications depending on the salinity of the growth medium, have been studied . A markedly salt-dependent differential expression has been detected in the nearby regions . Two of the open reading frames characterized correspond to two of the differentially expressed transcripts . In both cases the PstI sites were included in purine-pyrimidine alternancies suggestive of Z-DNA structures and located in non-coding regions with frequent repetitive motifs . A long alternating adenine-thymine tract also appears in the upstream regions of one of these open reading frames . A possible role of local DNA configuration in osmoregulation in this organism is discussed.

J Bioenerg Biomembr, 1993 Aug, 25(4), 385 - 91
Na(+)-translocating NADH-quinone reductase of marine and halophilic bacteria; Unemoto T et al.; The respiratory chain of marine and moderately halophilic bacteria requires Na+ for maximum activity, and the site of Na(+)-dependent activation is located in the NADH-quinone reductase segment . The Na(+)-dependent NADH-quinone reductase purified from marine bacterium Vibrio alginolyticus is composed of three subunits, alpha, beta, and gamma, with apparent M(r) of 52, 46, and 32 kDa, respectively . The FAD-containing beta-subunit reacts with NADH and reduces ubiquinone-1 (Q-1) by a one-electron transfer pathway to produce ubisemiquinones . In the presence of the FMN-containing alpha-subunit and the gamma-subunit, Q-1 is converted to ubiquinol-1 without the accumulation of free radicals . The reaction catalyzed by the alpha-subunit is strictly dependent on Na+ and is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), which is tightly coupled to the electrogenic extrusion of Na+ . A similar type of Na(+)-translocating NADH-quinone reductase is widely distributed among marine and moderately halophilic bacteria . The respiratory chain of V . alginolyticus contains another NADH-quinone reductase which is Na+ independent and has no energy-transducing capacity . These two types of NADH-quinone reductase are quite different with respect to their mode of quinone reduction and their sensitivity toward NADH preincubation.

Biochim Biophys Acta, 1993 Jul 21, 1169(1), 46 - 53
On the revised structure of the major phospholipid of Halobacterium salinarium; Kates M et al.; Recent fast atom bombardment-mass spectrometry (FABMS) studies (Tsujimoto, K., Yorimitsu, S., Takahashi, T . and Ohashi, M . (1989) J . Chem . Commun . 668-670; Frederickson, H.L., De Leeuw, J.W., Tas, A.C., Van der Greef, J., LaVos, G.F . and Boon, J.J . (1989) Biomed . Environ . Mass . Spectrom . 18, 96-105; Kloppel, K.D . and Fredrickson, H.L . (1991) J . Chromatogr . 562, 369-376) have indicated that the structure of the major phospholipid of Halobacterium salinarium (formerly Halobacterium cutirubrum) is not 2,3-diphytanyl-sn-glycerol-1-phospho-3'-sn-glycerol-1'- phosphate (PGP), but the monomethylated derivative, 2,3-diphytanyl-sn-glycerol-1-phospho-3'-sn-glycerol-1'-methylphosphate (PGP-Me) . We have now confirmed the structure of the major phospholipid of extremely halophilic archaebacteria as being this methylated structure (PGP-Me) by 1H- and 13C-NMR, FABMS and TLC of the native phospholipid and its product of mild acid hydrolysis PGP . The methylated structure (PGP-Me), rather than PGP itself, is also the major phospholipid in species of other genera of extreme halophiles examined so far, such as, Haloferax, Haloarcula, Halococcus, Natronobacterium and Natronococcus.

J Mol Biol, 1993 Jul 20, 232(2), 693 - 700
Localization of proteins HL29 and HL31 from Haloarcula marismortui within the 50 S ribosomal subunit by chemical crosslinking; Bergmann U et al.; Isolated 50 S ribosomal subunits from the halophilic archaebacterium Haloarcula marismortui were treated in situ with the homobifunctional and cleavable crosslinking reagent dithiobis(succinimidyl propionate) (12 A) . Several crosslinked complexes were obtained . Among these were the protein pairs HmaL4-HL29 and HmaL18-HL31; HL29 and HL31 are ribosomal proteins without any equivalent in eubacterial ribosomes . The crosslinked protein pairs were isolated on a preparative scale by combining conventional ion-exchange chromatography and reverse phase high-pressure liquid chromatography . The monomeric proteins involved in crosslink formation were unambiguously identified by two-dimensional gel electrophoresis and N-terminal or internal protein sequencing . Due to the homology between HmaL4 and HmaL18 and their Escherichia coli counterparts, and the roughly known location of these proteins within the 50 S subunit, our results demonstrate that HL29 is probably located in the centre of the large subunit in the vicinity of the peptidyltransferase domain, whereas HL31 must be situated within the central protuberance close to the region of the 5 S RNA.

Experientia, 1993 Jul 5, 49(6-7), 503 - 13
Partial sequence of the gene for a serine protease from a halophilic archaeum Haloferax mediterranei R4, and nucleotide sequences of 16S rRNA encoding genes from several halophilic archaea; Kamekura M et al.; A part of the gene coding for a halophilic serine protease from a halophilic archaeum Haloferax mediterranei R4 was amplified by PCR and its 672 nucleotide sequence was determined . Tentative translation to the amino acid sequence suggested that the enzyme was quite similar to halolysin produced by another halophilic archaeum strain 172P1 . Nucleotide sequences of 16S rRNA encoding genes from 9 halophilic archaea were determined . Alignment of 19 sequences known so far showed that there are more than 20 positions carrying bases or deletions specific for each halobacterial genus: Halobacterium, Haloarcula, Haloferax, and Halococcus.

Protein Sci, 1993 Jul, 2(7), 1114 - 25
Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila, with evidence for the mass and the binding site of the chromophore; Van Beeumen JJ et al.; The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV . This is the first sequence to be reported for this class of proteins . There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc . Natl . Acad . Sci . USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules . The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%) . The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second . Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis . The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching . The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein . The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent . The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.

Int J Syst Bacteriol, 1993 Jul, 43(3), 514 - 20
Arhodomonas aquaeolei gen . nov., sp . nov., an aerobic, halophilic bacterium isolated from a subterranean brine; Adkins JP et al.; Arhodomonas aquaeolei gen . nov., sp . nov., isolated from a petroleum reservoir production fluid, is described . The single isolate was an obligately halophilic, aerobic, gram-negative, oval rod-shaped bacterium that was actively motile by means of a single polar flagellum . It was catalase and oxidase positive . The isolate had a specific requirement for NaCl; growth occurred at NaCl concentrations between 6 and 20%, and optimal growth occurred in the presence of 15% NaCl . This species metabolized primarily organic acids and required biotin for growth . The name Arhodomonas is proposed for the new genus, which was placed in the gamma subclass of the Proteobacteria on the basis of the results of a 16S rRNA sequence analysis . Although A . aquaeolei is most closely related to purple sulfur bacteria (the genera Ectothiorhodospira and Chromatium), it is not a phototrophic microorganism, which is consistent with its isolation from a subterranean environment . The major components of its cellular fatty acids were C16:0, C18:1, C19:0, C16:1, and C18:0 acids . The DNA base composition of the type strain is 67 mol% G+C . The type and only strain is strain HA-1 (= ATCC 49307).

J Bacteriol, 1993 Jul, 175(13), 4197 - 202
Purification of a catalase-peroxidase from Halobacterium halobium: characterization of some unique properties of the halophilic enzyme; Brown-Peterson NJ et al.; A hydroperoxidase purified from the halophilic archaeon Halobacterium halobium exhibited both catalase and peroxidase activities, which were greatly diminished in a low-salt environment . Therefore, the purification was carried out in 2 M NaCl . Purified protein exhibited catalase activity over the narrow pH range of 6.0 to 7.5 and exhibited peroxidase activity between pH 6.5 and 8.0 . Peroxidase activity was maximal at NaCl concentrations above 1 M, although catalase activity required 2 M NaCl for optimal function . Catalase activity was greatest at 50 degrees C; at 90 degrees C, the enzymatic activity was 20% greater than at 25 degrees C . Peroxidase activity decreased rapidly above its maximum at 40 degrees C . An activation energy of 2.5 kcal (ca . 10 kJ)/mol was calculated for catalase, and an activation energy of 4.0 kcal (ca . 17 kJ)/mol was calculated for peroxidase . Catalase activity was not inhibited by 3-amino-1,2,4-triazole but was inhibited by KCN and NaN3 (apparent Ki {KiApp} of 50 and 67.5 microM, respectively) . Peroxidative activity was inhibited equally by KCN and NaN3 (KiApp for both, approximately 30 microM) . The absorption spectrum showed a Soret peak at 404 nm, and there was no apparent reduction by dithionite . A heme content of 1.43 per tetramer was determined . The protein has a pI of 3.8 and an M(r) of 240,000 and consists of four subunits of 60,300 each.

Rev Latinoam Microbiol, 1993 Jul-Sep, 35(3), 267 - 72
{Incidence of Vibrio parahaemolyticus in raw fish, oysters, and shrimp}; Torres Vitela MR et al.; The incidence of Vibrio parahaemolyticus in fresh seafood sold in Guadalajara, was studied by two procedures . These two procedures were compared to choose a reliable technique when outbreaks of V . parahaemolyticus illness occur . For one year, 57 samples of fresh oysters, fish and shrimp were analyzed for mesophilic aerobic bacteria (MAB) content, V . parahaemolyticus and pH . Total volatile nitrogen (TVN) was also determined in samples of fish and shrimp . MAB were counted by the pour plate method, and TVN was determined by modified Conway's micro diffusion technique . V . parahaemolyticus was investigated in 20-g samples by enrichment in lauryl dextrose salt broth (LDSB), isolating on plates of thiosulfate citrate bile sucrose agar (TCBS) and bile salts No . 3 agar (BS No . 3), and by direct isolation on TCBS and BS No.3 agar plates . Vibrio was characterized by tests described in standard methods, based upon the halophilism of the organism . Global percent of Vibrio parahaemolyticus positive samples was 45.6%, being 71.4% in fish, 44.0% in oysters, and 27.6% in shrimp . The use of two techniques enhanced the ability to recover the vibrio . There was a greater number of positives during the warm months (p = 0.0038) . Means of TVN and pH in both positive and negative samples were not significantly different . Means of MAB counts were similar either in positive or negative samples.

Chem Biol Interact, 1993 Jun, 87(1-3), 141 - 8
Screening of halophilic bacteria and Alteromonas species for organophosphorus hydrolyzing enzyme activity; DeFrank JJ et al.; Previously, a G-type nerve agent degrading enzyme activity was found in a halophilic bacterial isolate designated JD6.5 . This organism was tentatively identified as an unknown species of the genus Alteromonas . In order to determine whether this type of enzyme activity was common in other species of Alteromonas, a screening program was initiated . A number of Alteromonas species and five halophilic bacterial isolates were cultured and their crude cell extracts screened for hydrolytic activity against several organophosphorus chemical agents and other related compounds . The samples were also screened for cross-reactivity with a monoclonal antibody raised against the purified enzyme from JD6.5 and for hybridization with a DNA probe based on its N-terminal amino acid sequence A wide spectrum of activities and reactivities were seen, suggesting a significant heterogeneity between the functionally similar enzymes that are present in these bacterial species . Enzymes of the type described here have considerable potential for the decontamination and demilitarization of chemical warfare agents.

Glycoconj J, 1993 Jun, 10(3), 235 - 9
Synthesis of 2,3-di-O-phytanyl-1-O-(alpha-D-glucopyranosyl)-sn-glycerol derivatives, analogues of polar lipids isolated from a halophilic bacterial strain; Kamikawa T et al.; 2,3-Di-O-phytanyl-1-O-glucopyranosylglycerol and polar derivatives of its 6'-glucose moiety have been synthesized . The target molecule contains the diphytanyl-sn-glycerol moiety which is alpha-linked to glucose . The key step in its synthesis involves the coupling of phytanyl bromide and isopropylidene threitol . We also demonstrated that the 6'-hydroxyl group of glycolipids can be functionalized without protection of the sugar moiety.

J Biol Chem, 1993 May 5, 268(13), 9316 - 22
Identity elements of tRNA(Trp) . Identification and evolutionary conservation; Xue H et al.; In this study, the varying reactivities of Bacillus subtilis tryptophanyl-tRNA synthetase toward prokaryotic, eukaryotic, and halophile tRNAs were employed to define the potential identity elements on tRNA(Trp) . On this basis mutagenesis was performed to obtain, through in vivo heterologous expression in Escherichia coli and in vitro transcription with T7 RNA polymerase, mutant B . subtilis tRNA(Trp) for comparison with the wild-type . These comparisons served to establish G73 and the anticodon as major identity elements, and A1-U72, G5-C68, and A9 as minor identity elements . While the tryptophanyl-tRNA synthetase from B . subtilis and E . coli require G73 to function, replacement of G73 by A73 favors the enzyme from yeast . This change points to the variation of the identity elements for the same amino acid among different organisms . The similarity in these elements between B . subtilis and E . coli tryptophanyl-tRNA synthetase, however, suggests that identity elements on tRNA, like the active centers on enzymes, undergo evolutionary change at slower rates than less essential portions of the macromolecule.

J Bacteriol, 1993 May, 175(10), 3096 - 104
The eubacterium Ectothiorhodospira halophila is negatively phototactic, with a wavelength dependence that fits the absorption spectrum of the photoactive yellow protein; Sprenger WW et al.; The motile, alkalophilic, and extremely halophilic purple sulfur bacterium Ectothiorhodospira halophila is positively photophobotactic . This response results in the accumulation of bacteria in light spots (E . Hustede, M . Liebergesell, and H . G . Schlegel, Photochem . Photobiol . 50:809-815, 1989; D . E . McRee, J . A . Tainer, T . E . Meyer, J . Van Beeumen, M . A . Cusanovich, and E . D . Getzoff, Proc . Natl . Acad . Sci . USA 86:6533-6537, 1989; also, this work) . In this study, we demonstrated that E . halophila is also negatively phototactic . Video analysis of free-swimming bacteria and the formation of cell distribution patterns as a result of light-color boundaries in an anaerobic suspension of cells revealed the existence of a repellent response toward intense (but nondamaging) blue light . In the presence of saturating background photosynthetic light, an increase in the intensity of blue light induced directional switches, whereas a decrease in intense blue light gave rise to suppression of these reversals . To our knowledge, this is the first report of a true repellent response to light in a free-swimming eubacterium, since the blue light response in Escherichia coli and Salmonella typhimurium (B . L . Taylor and D . E . Koshland, Jr., J . Bacteriol . 123:557-569, 1975), which requires an extremely high light intensity, is unlikely to be a sensory process . The wavelength dependence of this negative photoresponse was determined with narrow band pass interference filters . It showed similarity to the absorption spectrum of the photoactive yellow protein from E . halophila.

Ann Surg, 1993 May, 217(5), 525 - 30; discussion 530-1
Infections caused by halophilic marine Vibrio bacteria; Howard RJ et al.; OBJECTIVE: The authors reviewed patients who developed sepsis or soft tissue infections caused by marine Vibrio bacteria in Florida . SUMMARY BACKGROUND DATA: Marine Vibrio bacteria are the most common bacteria found in seawater . They are concentrated in marine animals that feed by filtration such as oysters and clams . These bacteria can cause gastroenteritis, sepsis, cellulitis leading to necrotizing soft tissue infection after exposure to seawater or consumption of raw seafood . METHODS: The authors received 182 systemic infections that occurred in Florida between January 1, 1979, and December 31, 1991, which were treated by the authors or were reported to the Florida Department of Health and Rehabilitative Services . Patients were divided into two groups depending on whether they presented with primary bacteremia or soft tissue infection . RESULTS: Seventy-one patients had been exposed to these bacteria by eating raw seafood, 94 had direct exposure to seawater, and exposure was uncertain in 27 patients . Vibrio species were cultured from the blood of 103 patients and from wounds or soft tissues of 113 . An additional 5 patients had cellulitis but bacteria were not cultured from these sites . In patients in whom it could be determined, 93 had primary soft tissue infections and 82 had primary bacteremia . Twenty-four patients had necrotizing soft tissue infections and required surgical debridement . Three of these 24 patients required amputation . Thirty-seven (20.3%) patients died . Severe liver disease occurred in 54 patients and 25 of these patients died . CONCLUSIONS: Marine Vibrio bacteria can cause sepsis and soft tissue infections, especially in individuals with severe liver disease and other chronic illnesses such as diabetes mellitus . The authors believe all individuals, especially those with systemic illness, should be warned against eating raw seafood.

Curr Genet, 1993 May-Jun, 23(5-6), 443 - 9
Osmophilic linear plasmids from the salt-tolerant yeast Debaryomyces hansenii; Gunge N et al.; Three novel linear plasmids, pDHL1 (8.4 kb), pDHL2 (9.2 kb) and pDHL3 (15.0 kb), were discovered in the halophilic (salt-tolerant) yeast Debaryomyces hansenii . Exonuclease treatment indicated that all three plasmids were blocked at their 5' ends, presumably, by analogy with most other eukaryotic linear plasmids which involved protein attachment . The Debaryomyces plasmids were entirely cured simply by growing cells in normal culture medium, but were stably maintained in culture medium containing salts, sorbitol or glycerol at suitable concentrations . This suggested that the pDHL plasmids required an osmotic pressure for stable replication and maintenance . The Debaryomyces yeast secreted a killer toxin against various yeasts species . Toxin activity was demonstrated only in the presence of salts such as NaCl or KCl, but this killer phenotype was not associated with the pDHL plasmids . Analysis of the plasmid-curing pattern suggested that pDHL3 may play a key role in the replication of the Debaryomyces plasmids . Southern hybridization showed that an extensive homology exists between specific regions of pDHL1 and pDHL2, whereas pDHL3 is unique.

Biol Chem Hoppe Seyler, 1993 May, 374(5), 305 - 12
N-terminal modification and amino-acid sequence of the ribosomal protein HmaS7 from Haloarcula marismortui and homology studies to other ribosomal proteins; Klussmann S et al.; The ribosomal protein HmaS7 from the 30S subunit of the extreme halophilic archaeum Haloarcula marismortui was isolated by semi-preparative RP-HPLC . The complete amino-acid sequence of this protein was determined by automated microsequence analysis of appropriate peptide fragments from several proteinase digests . The entire protein consists of 205 amino acids with a corresponding molecular mass of 22580 Da . The modification at the amino-terminal amino acid was deblocked so that the N-terminal amino acids could be sequenced and the type of the modification was identified as an acetyl group by electrospray mass spectrometry of suitable peptides . Homology studies of HmaS7 showed similarities to ribosomal proteins derived from organisms of all three urkingdoms, such as to EcoS7, HmoS7, MvaS7, SacS7 and RatS7; due to the strong sequence homologies found within the archaebacterial ribosomal proteins we conclude that the protein sequence which was determined for S7 from Methanococcus vannielii by nucleotide sequencing of the gene should be about 20 or 30 amino acids longer than previously published (Lechner, K., Heller, G . & Bock, A . (1989) J . Mol . Evol . 29, 20-27).

Mol Biol (Mosk), 1993 May-Jun, 27(3), 485 - 99
{Instability of the halophilic archaebacteria genome}; Derkacheva NI et al.; The phenomenon of high genetic variability in the extremely halophilic archaebacterium Hb . salinarium is reviewed . The role of IS elements and homologous recombination in frequent genetic rearrangements in this organism is discussed . A possible cause of high genetic variability of Hb . salinarium is considered.

Biochemistry, 1993 Apr 27, 32(16), 4308 - 13
Cloning, sequencing, and expression in Escherichia coli of the gene coding for malate dehydrogenase of the extremely halophilic archaebacterium Haloarcula marismortui; Cendrin F et al.; The gene coding for the enzyme malate dehydrogenase (MDH) of the extremely halophilic archaebacterium Haloarcula marismortui was isolated and sequenced . The enzyme is composed of 303 amino acids, and its molecular mass is 32,638 Da . The deduced amino acid sequence of the enzyme was found to be more similar to the sequence of L-lactate dehydrogenase (L-LDH) from various sources than to the sequence of other MDHs . The structural gene was cloned in the Escherichia coli expression vector pET11a, and large amounts of a soluble but inactive form of the enzyme were produced upon its induction . Activation of the enzyme was obtained by increasing the salt concentration to 3 M NaCl . The recombinant protein was purified to homogeneity and shown to be indistinguishable from the native enzyme isolated from halobacteria . These findings present the first example of the successful expression of a halobacterial gene coding for a soluble protein in Escherichia coli and its recovery as a functional enzyme . Site-directed mutagenesis was employed to modify Arg100 on the enzyme to Gln . This modification produced an enzyme that has considerably higher specificity for pyruvate (the substrate of L-LDH) than for oxaloacetate (the substrate of MDH) . The mutation also caused a modification in the relative activities of the enzyme at different salt concentrations . The greater similarity of the amino acid sequence of the halobacterial MDH to that of L-LDHs than to that of MDHs sheds light on the molecular evolution of these enzymes.

J Mol Evol, 1993 Apr, 36(4), 335 - 46
Evolution of glutamate dehydrogenase genes: evidence for two paralogous protein families and unusual branching patterns of the archaebacteria in the universal tree of life; Benachenhou-Lahfa N et al.; The existence of two families of genes coding for hexameric glutamate dehydrogenases has been deduced from the alignment of 21 primary sequences and the determination of the percentages of similarity between each pair of proteins . Each family could also be characterized by specific motifs . One family (Family I) was composed of gdh genes from six eubacteria and six lower eukaryotes (the primitive protozoan Giardia lamblia, the green alga Chlorella sorokiniana, and several fungi and yeasts) . The other one (Family II) was composed of gdh genes from two eubacteria, two archaebacteria, and five higher eukaryotes (vertebrates) . Reconstruction of phylogenetic trees using several parsimony and distance methods confirmed the existence of these two families . Therefore, these results reinforced our previously proposed hypothesis that two close but already different gdh genes were present in the last common ancestor to the three Ur-kingdoms (eubacteria, archaebacteria, and eukaryotes) . The branching order of the different species of Family I was found to be the same whatever the method of tree reconstruction although it varied slightly according the region analyzed . Similarly, the topological positions of eubacteria and eukaryotes of Family II were independent of the method used . However, the branching of the two archaebacteria in Family II appeared to be unexpected: (1) the thermoacidophilic Sulfolobus solfataricus was found clustered with the two eubacteria of this family both in parsimony and distance trees, a situation not predicted by either one of the contradictory trees recently proposed; and (2) the branching of the halophilic Halobacterium salinarium varied according to the method of tree construction: it was closer to the eubacteria in the maximum parsimony tree and to eukaryotes in distance trees . Therefore, whatever the actual position of the halophilic species, archaebacteria did not appear to be monophyletic in these gdh gene trees . This result questions the firmness of the presently accepted interpretation of previous protein trees which were supposed to root unambiguously the universal tree of life and place the archaebacteria in this tree.

Biochemistry, 1993 Mar 23, 32(11), 2880 - 7
Identification of cross-linked amino acids in the protein pair HmaL23-HmaL29 from the 50S ribosomal subunit of the archaebacterium Haloarcula marismortui; Bergmann U et al.; 50S ribosomal subunits from the extreme halophilic archaebacterium Haloarcula marismortui were treated with the homobifunctional protein-protein cross-linking reagents diepoxybutane (4 A) and dithiobis(succinimidyl propionate) (12 A) . The dominant product with both cross-linking reagents was identified on the protein level as HmaL23-HmaL29, which is homologous to the protein pair L23-L29 from Escherichia coli {Walleczek, J., Martin, T., Redl, B., Stoffler-Meilicke, M., & Stoffler, G . (1989) Biochemistry 28, 4099-4105} and from Bacillus stearothermophilus {Brockmoller, J., & Kamp, R . M . (1986) Biol . Chem . Hoppe-Seyler 367, 925-935} . To reveal the exact cross-linking site in HmaL23-HmaL29, the cross-linked complex was purified on a preparative scale by conventional and high-performance liquid chromatography . After endoproteolytic fragmentation of the protein pair, the amino acids engaged in cross-link formation were unambiguously identified by N-terminal sequence analysis and mass spectrometry of the cross-linked peptides . The cross-link is formed between lysine-57 in the C-terminal region of HmaL29 and the alpha-amino group of the N-terminal serine in protein HmaL23, irrespective of the cross-linking reagent . This result demonstrates that the N-terminal region of protein HmaL23 and the C-terminal domain of HmaL29 are highly flexible so that the distance between the two polypeptide chains can vary by at least 8 A . Comparison of our cross-linking results with those obtained with B . stearothermophilus revealed that the fine structure within this ribosomal domain is at least partially conserved.

J Bacteriol, 1993 Mar, 175(6), 1572 - 9
Structure, function, and evolution of the family of superoxide dismutase proteins from halophilic archaebacteria; Joshi P et al.; The protein sequences of seven members of the superoxide dismutase (SOD) family from halophilic archaebacteria have been aligned and compared with each other and with the homologous Mn and Fe SOD sequences from eubacteria and the methanogenic archaebacterium Methanobacterium thermoautotrophicum . Of 199 common residues in the SOD proteins from halophilic archaebacteria, 125 are conserved in all seven sequences, and 64 of these are encoded by single unique triplets . The 74 remaining positions exhibit a high degree of variability, and for almost half of these, the encoding triplets are connected by at least two nonsynonymous nucleotide substitutions . The majority of nucleotide substitutions within the seven genes are nonsynonymous and result in amino acid replacement in the respective protein; silent third-codon-position (synonymous) substitutions are unexpectedly rare . Halophilic SODs contain 30 specific residues that are not found at the corresponding positions of the methanogenic or eubacterial SOD proteins . Seven of these are replacements of highly conserved amino acids in eubacterial SODs that are believed to play an important role in the three-dimensional structure of the protein . Residues implicated in formation of the active site, catalysis, and metal ion binding are conserved in all Mn and Fe SODs . Molecular phylogenies based on parsimony and neighbor-joining methods coherently group the halophile sequences but surprisingly fail to distinguish between the Mn SOD of Escherichia coli and the Fe SOD of M . thermoautotrophicum as the outgroup . These comparisons indicate that as a group, the SODs of halophilic archaebacteria have many unique and characteristic features . At the same time, the patterns of nucleotide substitution and amino acid replacement indicate that these genes and the proteins that they encode continue to be subject to strong and changing selection . This selection may be related to the presence of oxygen radicals and the inter- and intracellular composition and concentration of metal cations.

J Bacteriol, 1993 Mar, 175(6), 1561 - 71
Characterization of paralogous and orthologous members of the superoxide dismutase gene family from genera of the halophilic archaebacteria; Joshi P et al.; Four species representing three genera of halophilic archaebacteria were examined for the presence of genomic sequences that encode proteins of the superoxide dismutase family . Three species, Halobacterium cutirubrum, Halobacterium sp . strain GRB, and Haloferax volcanii, contain duplicated (paralogous) genes of the sod family; a fourth species, Haloarcula marismortui, contains only a single gene . These seven genes were cloned and sequenced, and their transcripts were characterized by Northern (RNA) hybridization, S1 nuclease protection, and primer extension . The expression of one of the two genes in H . cutirubrum, Halobacterium sp . strain GRB, and Haloferax volcanii was shown to be elevated in the presence of paraquat, a generator of superoxide radicals . The other genes, including the single gene from Haloarcula marismortui, exhibited no elevated expression in the presence of paraquat . The 5' and 3' flanking regions of all the genes contain recognizable promoter and terminator elements that are appropriately positioned relative to the 5' and 3' transcript end sites . Between genera, the orthologous paraquat-responsive genes exhibit no sequence similarity in either their 5' or 3' flanking regions, whereas the orthologous nonresponsive genes exhibit limited sequence similarity but only in the 5' flanking region . Within the coding region, the two paralogous genes of Haloferax volcanii are virtually identical (99.5%) despite the absence of similarity in the flanking regions . In contrast, the paralogous genes of H . cutirubrum and Halobacterium sp . strain GRB are only about 87% identical . In the alignment of all seven sequences, there are nine codon positions where both the TCN and AGY serine codons are utilized; some or all of these may well be examples of convergent evolution.

J Bacteriol, 1993 Mar, 175(6), 1629 - 36
Abundance, subunit composition, redox properties, and catalytic activity of the cytochrome bc1 complex from alkaliphilic and halophilic, photosynthetic members of the family Ectothiorhodospiraceae; Leguijt T et al.; Ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complexes were demonstrated to be present in the membranes of the alkaliphilic and halophilic purple sulfur bacteria Ectothiorhodospira halophila, Ectothiorhodospira mobilis, and Ectothiorhodospira shaposhnikovii by protoheme extraction, immunoblotting, and electron paramagnetic resonance spectroscopy . The gy values of the Rieske {2Fe-2S} clusters observed in membranes of E . mobilis and E . halophila were 1.895 and 1.910, respectively . In E . mobilis membranes, the cytochrome bc1 complex was present in a stoichiometry of approximately 0.2 per reaction center . This complex was isolated and characterized . It contained four prosthetic groups: low-potential cytochrome b (cytochrome bL; Em = -142 mV), high-potential cytochrome b (cytochrome bH; Em = 116 mV), cytochrome c1 (Em = 341 mV), and a Rieske iron-sulfur cluster . The absorbance spectrum of cytochrome bL displayed an asymmetric alpha-band with a maximum at 564 nm and a shoulder at 559 nm . The alpha bands of cytochrome bH and cytochrome c1 peaked at 559.5 and 553 nm, respectively . These prosthetic groups were associated with three different polypeptides: cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein, with apparent molecular masses of 43, 30, and 21 kDa, respectively . No evidence for the presence of a fourth subunit was obtained . Maximal ubiquinol-cytochrome c oxidoreductase activity of the purified complex was observed at pH 8; the turnover rate was 57 mol of cytochrome c reduced.(mol of cytochrome c1)-1.s-1 . The complex showed a strikingly low sensitivity towards typical inhibitors of cytochrome bc1 complexes.

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