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E A Galinski

Publications and source records attributed to E A Galinski.

18 recordsLinked to original sources
PubMed2001

Near infra-red spectra of urea with glycine betaine or trimethylamine N-oxide are additive.

Glycine betaine and trimethylamine-N-oxide counteract urea denaturation in solutions containing urea and the methylamine in the mole ratio of 2:1. Near infra-red difference spectra (water spectrum subtracted) of solutions containing both urea with either glycine betaine or trimethylamine-N-oxide can be predicted from the spectra of the single solutes, with r(2)>0.999 both using the spectrum from 1200 to 2100 nm (where most absorbance is attributable to hydrogen bonding) and using an extended range 1000 to 2500 nm, which includes solute specific bands. Thus urea and the kosmotropes appear to interact with water independently and the counteraction cannot be attributed to specific interactions between them. The spectrum of aqueous glycine betaine can be predicted from tetramethylammonium and formate ions (r(2)=0.998), suggesting that independent interactions of the quaternary amine, and of the carboxyl function, with water are dominant. The exceptional properties of glycine betaine do not arise from specific intramolecular interactions between the charged groups.

Betaine↗
PubMed2001

Expression of the ATP-binding cassette transporter gene ABCG1 (ABC8) in Tangier disease.

Several members of the ATP-binding cassette (ABC) transporter family are involved in cholesterol efflux from cells. A defect in one member, ABCA1, results in Tangier disease, a condition characterized by cholesterol accumulation in macrophages and virtual absence of mature circulating high-density lipoproteins. Expression of a second member, ABCG1, is increased by cholesterol-loading in human macrophages. We now show that ABCG1, which we identified by differential display RT-PCR in foamy macrophages, is overexpressed in macrophages from patients with Tangier disease compared to control macrophages. On examination by confocal laser scanning microscopy, ABCG1 was present in perinuclear structures within the cell. In addition, a combination of in situ hybridization and indirect immunofluorescence microscopy revealed that ABCG1 is expressed in foamy macrophages within the atherosclerotic plaque. These data indicate that not only ABCA1 but also ABCG1 may play a role in the cholesterol metabolism of macrophages in vitro and in the atherosclerotic plaque.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2001

Genomic sequence and structure of the human ABCG1 (ABC8) gene.

The human ATP-binding cassette half transporter G1 (hABCG1) may play a role in cholesterol transport in macrophages. Using RACE assays we determined the structure of this gene. The hABCG1 gene spans more than 97 kb comprising 20 exons, 20 kb and 5 exons more than hitherto described. Four of the novel exons are upstream and one is downstream of previous exon 1, and they are predicted to encode at least five novel transcripts. We also detected two separate promoters, upstream of exons 1 and 5, respectively. The region 650 bp upstream of exon 1 was predicted to contain putative binding sites for SP1 and nuclear factor kappaB (NF-kappaB), but no sterol response elements (SREs) or retinoid X receptor (RXR) binding sites. The region 650 bp upstream of exon 5 contained 19 possible SP1 binding sites, one possible SRE, two possible NF-kappaB, and two putative RXR binding sites. Nevertheless, both promoters responded in macrophages to stimulation by hydroxycholesterol and retinoic acid.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2000

Salt adaptation in pseudomonads: characterization of glucosylglycerol-synthesizing isolates from brackish coastal waters and the rhizosphere.

The compatible solute glucosylglycerol (GG) is widespread among cyanobacteria, but, until now, has been reported for only two species of heterotrophic bacteria. About 120 bacterial isolates from coastal regions of the Baltic Sea were screened by HPLC for their ability to synthesize GG. Positive isolates (26) were grouped by SDS-PAGE of whole-cell proteins and representative strains of each group were investigated by sequencing their 16S rRNA genes and phenotypic characterization. All GG-synthesizing isolates were shown to belong to the genus Pseudomonas (sensu stricto) and were assigned to 4 distinct groups, although none of the GG-synthesizing isolates could be unambiguously assigned to described species. The identity of GG was verified by 13C NMR analysis and enzymatic digestion with alpha- and beta-glucosidases. Besides GG, salt adapted cultures of the aquatic isolates accumulated the dipeptide N-acetylglutaminylglutamine amide (NAGGN) and glutamate. The accumulation of noncharged compatible solutes was also tested in previously identified pseudomonads isolated from the rhizosphere of oilseed rape and potato. The majority of these strains were fluorescent species of the genus Pseudomonas and accumulated trehalose and NAGGN when grown under salt stress conditions. However, rhizosphere isolates of Stenotrophomonas maltophilia synthesized GG and trehalose or only trehalose in a strain-dependent manner. These data indicate that the ability to synthesize GG is widely distributed among slightly or moderately halotolerant pseudomonads.

Bacterial Proteins↗
PubMed2000

Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions.

The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coli under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.

Animals↗
PubMed1999

Extrinsic protein stabilization by the naturally occurring osmolytes beta-hydroxyectoine and betaine.

Thermodynamic aspects of protein stabilization by two widespread naturally occurring osmolytes, beta-hydroxyectoine and betaine, were studied using differential scanning calorimetry (DSC) and bovine ribonuclease A (RNase A) as a model protein. The osmolyte beta-hydroxyectoine purified from Marinococcus was found to be a very efficient stabilizer. At a concentration of 3M it increased the melting temperature of RNase A (Tm) by more than 12K and gave rise to a stability increase of 10.6kJ/mol at room temperature. The heat capacity difference between the folded and unfolded state (deltaC(p)) was found to be significantly increased. Betaine stabilized RNase A only at concentrations less than 3M. Also, here deltaCp was found to be increased. Calculation of the number of water molecules that additionally bind to unfolded RNase A resulted in surprisingly low numbers for both osmolytes. The significant stabilization of RNase A by beta-hydroxyectoine makes this osmolyte an interesting stabilizer in biotechnological processes in which enzymes are applied in the presence of denaturants or at high temperature.

Amino Acids, Diamino↗
PubMed1998

Construction and characterization of an NaCl-sensitive mutant of Halomonas elongata impaired in ectoine biosynthesis.

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

Amino Acids, Diamino↗
PubMed1998

Bacterial milking: A novel bioprocess for production of compatible solutes.

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

Amino Acids, Diamino↗
PubMed1998

An overview of the role and diversity of compatible solutes in Bacteria and Archaea.

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

Archaea↗
PubMed1997

Identification of plasmids in the genus Marinococcus and complete nucleotide sequence of plasmid pPL1 from Marinococcus halophilus.

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

Amino Acid Sequence↗
PubMed1996

Mannitol, a novel bacterial compatible solute in Pseudomonas putida S12.

The aim of this study was to identify the compatible solutes accumulated by Pseudomonas putida S12 subjected to osmotic stress. In response to reduced water activity, P. putida S12 accumulated Nalpha-acetylglutaminylglutamine amide (NAGGN) simultaneously with a novel compatible solute identified as mannitol (using 13C- and 1H-nuclear magnetic resonance, liquid chromatography-mass spectroscopy and high-performance liquid chromatography methods) to maximum concentrations of 74 and 258 micromol g (dry weight) of cells(-1), respectively. The intracellular amounts of each solute varied with both the type and amount of osmolyte applied to induce osmotic stress in the medium. Both solutes were synthesized de novo. Addition of betaine to the medium resulted in accumulation of this compound and depletion of both NAGGN and mannitol. Mannitol and NAGGN were accumulated when sucrose instead of salts was used to reduce the medium water activity. Furthermore, both compatible solutes were accumulated when glucose was substituted by other carbon sources. However, the intracellular quantities of mannitol decreased when fructose, succinate, or lactate were applied as a carbon source. Mannitol was also raised to high intracellular concentrations by other salt-stressed Pseudomonas putida strains. This is the first study demonstrating a principal role for the de novo-synthesized polyol mannitol in osmoadaptation of a heterotrophic eubacterium.

Betaine↗
PubMed1995

Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion of transposon Tn5 and Tn1732 in Halomonas elongata.

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.

Blotting, Southern↗
PubMed1995

Influence of salt concentration on the susceptibility of moderately halophilic bacteria to antimicrobials and its potential use for genetic transfer studies.

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.

Anti-Bacterial Agents↗
PubMed1993

Biosynthesis and function of trehalose in Ectothiorhodospira halochloris.

Trehalose 6-phosphate synthase, catalyzing the reaction between UDP-glucose and glucose 6-phosphate and forming trehalose 6-phosphate, was isolated and partially purified (30-fold) from the phototrophic, halo-alkaliphilic bacterium Ectothiorhodospira halochloris. The activity is stabilized by 20 mM MgCl2, 50 mM NaCe and 2M glycine betaine. The molecular weight was 63000. The enriched enzyme had a MgCl2 optimum at 3-6mM, a pH optimum at 7.5 (in Tris-HCl buffer) and a temperature optimum at 50 degrees C. The Km-values were 1.5 x 10(-3) M for UDP-glucose and 2 x 10(-3) M for glucose 6-phosphate. The enzyme showed a salinity dependence with optimal concentrations between 100 and 300mM salt. Higher concentrations of salt resulted in a decrease in activity. In the presence of inhibitory salt concentrations the compatible solute glycine betaine had a protective effect with a maximum between 0.5 and 2.0M.

Betaine↗
PubMed1991

Isolation and structure determination of a novel compatible solute from the moderately halophilic purple sulfur bacterium Ectothiorhodospira marismortui.

The halophilic phototrophic bacterium Ectothiorhodospira marismortui produces three organic osmolytes to counterbalance the osmotic pressure of the surrounding medium: glycine betaine, sucrose, and a novel compound. This new compound, which accounts for approximately 30% of the cells' compatible solutes, was isolated and identified by mass spectrometry and nuclear magnetic resonance. It was characterized as N alpha-carbamoyl-L-glutamine 1-amide, an unusual amino acid derivative with no previous reference in the chemical literature. The relatively high cytoplasmic concentration of this compound (approximately 0.5 M) observed at all growth conditions suggests that it may serve a vital function as an osmoticum and/or protectant for Ectothiorhodospira marismortui in a saline environment.

Chromatiaceae↗
PubMed1985

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira.

A novel cyclic amino acid was detected in and subsequently isolated from extremely halophilic species of the bacterial genus Ectothiorhodospira. The structure of this new compound was elucidated by a combination of nuclear magnetic resonance (NMR) techniques and mass spectrometry. 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid is only accumulated within the cytoplasm under certain growth conditions and seems to serve an osmoregulatory function. There is no previous reference to this molecule in the chemical literature and we, therefore, propose to use the trivial name 'ectoine', due to its discovery in members of the bacterial genus Ectothiorhodospira. (formula: see text).

Amino Acids, Diamino↗