Continuous synthesis and excretion of the compatible solute ectoine by a transgenic, nonhalophilic bacterium

The compatible solute 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) acts in microorganisms as an osmotic counterweight against halostress and has attracted commercial attention as a protecting agent. Its production and application are restricted by the drawbacks of the discontinuous harvesting procedure involving salt shocks, which reduces volumetric yield, increases reactor corrosion, and complicates downstream processing. In order to synthesize ectoine continuously in less-aggressive media, we introduced the ectoine genes ectABC of the halophilic bacterium Chromohalobacter salexigens into an Escherichia coli strain using the expression vector pASK-IBA7. Under the control of a tet promoter, the transgenic E. coli synthesized 6 g liter-1 ectoine with a space-time yield of 40 mg liter-1 h-1, with the vast majority of the ectoine being excreted.     

Unexpected property of ectoine synthase and its application for synthesis of the engineered compatible solute ADPC

A new cyclic amino acid was detected in a deletion mutant of the moderately halophilic bacterium Halomonas elongata deficient in ectoine synthesis. Using mass spectroscopy (MS) and nuclear magnetic resonance (NMR) techniques, the substance was identified as 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC). We were able to demonstrate that ADPC is the product of a side reaction of lone ectoine synthase (EC 4.2.1.108), which forms ADPC by cyclic condensation of glutamine. This reaction was shown to be reversible. Subsequently, a number of ectoine derivatives, in particular 4,5-dihydro-2-methylimidazole-4-carboxylate (DHMICA) and homoectoine, were also shown to be cleaved by ectoine synthase, which is classified as a hydro-lyase. This study thus reports for the first time that ectoine synthase accepts more than one substrate and is a reversible enzyme able to catalyze both the intramolecular condensation into and the hydrolytic cleavage of cyclic amino acid derivatives. As ADPC supports growth of bacteria under salt stress conditions and stabilizes enzymes against freeze-thaw denaturation, it displays typical properties of compatible solutes. As ADPC has not yet been described as a natural compound, it is presented here as the first man-made compatible solute created through genetic engineering.     

Kinetic stability of membrane proteins

Although membrane proteins constitute an important class of biomolecules involved in key cellular processes, study of the thermodynamic and kinetic stability of their structures is far behind that of soluble proteins. It is known that many membrane proteins become unstable when removed by detergent extraction from the lipid environment. In addition, most of them undergo irreversible denaturation, even under mild experimental conditions. This process was found to be associated with partial unfolding of the polypeptide chain exposing hydrophobic regions to water, and it was proposed that the formation of kinetically trapped conformations could be involved. In this review, we will describe some of the efforts toward understanding the irreversible inactivation of membrane proteins. Furthermore, its modulation by phospholipids, ligands, and temperature will be herein discussed.     

Regulation of compatible solute accumulation in bacteria

In their natural habitats, microorganisms are often exposed to osmolality changes in the environment. The osmotic stress must be sensed and converted into an activity change of specific enzymes and transport proteins and/or it must trigger their synthesis such that the osmotic imbalance can be rapidly restored. On the basis of the available literature, we conclude that representative Gram-negative and Gram-positive bacteria use different strategies to respond to osmotic stress. The main focus of this paper is on the initial response of bacteria to hyper- and hypo-osmotic conditions, and in particular the osmosensing devices that allow the cell to rapidly activate and/or to synthesize the transport systems necessary for uptake and excretion of compatible solutes. The experimental data allow us to discriminate the transport systems by the physicochemical parameter that is sensed, which can be a change in external osmotic pressure, turgor pressure, membrane strain, internal osmolality and/or concentration of specific signal mmicule. We also evaluate the mmicular basis for osmosensing by reviewing the unique structural features of known osmoregulated transport systems.     

Thermostabilization of proteins by diglycerol phosphate, a new compatible solute from the hyperthermophile Archaeoglobus fulgidus

Diglycerol phosphate accumulates under salt stress in the archaeon Archaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 63:896-902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianum rubredoxins. In contrast, the stability of D. desulfuricans rubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.     

The synthesis of compatible solute analogues-solvent effects on selective glycosylation

Ethyl 6-O-acetyl-2,3,4-tribenzyl-1-d-thioglucoside and ethyl 6-O-acetyl-2,3,4-tribenzyl-1-d-thiogalactoside, as a mixture of anomers, were employed in the study of the influence of solvent in the stereoselectivity of the glycosylation reaction with small and reactive acceptors. High α-selectivities were obtained in the glycosylation reactions using NIS/TfOH as activator and ethyl ether as the solvent at −60 °C. Other solvent mixtures such as dichloromethane, THF, THF/ethyl ether and toluene/dioxane were not nearly as selective. The corresponding thiogalactoside underwent similar glycosylations with the same solvents but with low anomer selectivity. These glycosides are key intermediates for the synthesis of new analogues of compatible solutes.     

Effect of solute hydrogen bonding capacity on osmotic stability of lysosomes

The effect of solute hydrogen bonding capacity on the osmotic stability of lysosomes was examined through measurement of free enzyme activity of lysosomes after their incubation in sucrose and poly(ethylene glycol) (PEG) (1500–6000 Da molecular mass) media. Free enzyme activity of the lysosomes was less in the PEG medium than that in the sucrose medium under the same hypotonic condition. The lysosomal enzyme latency loss decreased with increasing hydrogen bonding capacity of the solute. In addition, the lysosomes lost less latency at lower incubation temperature. The results indicate that solute hydrogen bonding capacity plays an important role in the osmotic protection of an incubation medium to lysosomes.     

Betaine is the main compatible solute of halophilic eubacteria.

A number of moderately halophilic bacteria of diverse taxonomic groups have been studied to determine the intracellular concentrations of organic compounds at various salt concentrations. Betaine was accumulated in all of these organisms in proportion to the salinity of the medium, suggesting that this compound plays a major role in osmoregulation.     

In planta function of compatible solute transporters of the AtProT family

The three proline transporters of Arabidopsis thaliana (AtProTs) transport the compatible solutes proline and glycine betaine and the stress-induced compound γ-aminobutyric acid when expressed in heterologous systems. The aim of the present study was to show transport and physiological relevance of these three AtProTs in planta. Using single, double, and triple knockout mutants and AtProT-overexpressing lines, proline content, growth on proline, transport of radiolabelled betaine, and expression of AtProT genes and enzymes of proline metabolism were analysed. AtProT2 was shown to facilitate uptake of L- and D-proline as well as [(14)C]glycine betaine in planta, indicating a role in the import of compatible solutes into the root. Toxic concentrations of L- and D-proline resulted in a drastic growth retardation of AtProT-overexpressing plants, demonstrating the need for a precise regulation of proline uptake and/or distribution. Furthermore evidence is provided that AtProT genes are highly expressed in tissues with elevated proline content--that is, pollen and leaf epidermis.     

The effect of compatible solutes on proteins

Summary The ability of the compatible solute, proline, to affect the behavior of proteins has been examined in many different systems by many researches. In the present study of protein solvation, proline has been shown to prevent or diminish, in a concentration-dependent manner, the glutamine synthetase-precipitating ability of polyethylene glycol (PEG). The effects of PEG concentration and molecular weight are reduced by proline, and the interaction is strongly affected by pH.PEG causes precipitation of many proteins, and the ability of proline to reduce the precipitation of two non-enzymatic conjugated proteins, alfalfa mosaic virus and an³H-testosterone/antiserum complex, was also examined. Proline was effective in reducing the PEG-induced precipitation of both proteins. Virus precipitation by PEG and its alleviation by proline are influenced by pH. The increased virus-precipitating effect of PEG in the presence of salt (NaCl) is also alleviated by proline. The precipitation of the radioimmune complex by PEG is diminished by proline and by a mixture of free amino acids.These results indicate the generality of the three-way interaction between proline, protein and PEG. They may be of importance for extraction of proteins from biological systems and in studies of enzyme inactivation or protein denaturation in a cytoplasmic milieu. The results suggest that the protective effects of some amino acids are at least additive and are consistent with the conclusion that the compatible solutes protect protein-containing systems against the unfavorable consequences of dehydration and other stresses, by increasing the tendency of the system to maintain thestatus quo.     

Effect of some proteins on the yeast cell membrane

Yeast cells, Candida utilis, in water suspension and in the absence of electrolytes were found to be very sensitive to several proteins of moderate size, including ribonuclease, protamine, lysozyme, bovine serum albumin, cytochrome c, and myoglobin. Viability ceases rapidly, and ultraviolet-absorbing compounds (260 mmu) and the amino acid pool are released into the medium. The ultraviolet-absorbing material appears to be the nucleotide and coenzyme fraction usually extracted by 0.2 n perchloric acid at low temperature. The ribonucleic acid fraction remains in the cell ghosts and can be released by ribonuclease. The enzymatic properties of some of these proteins have no relation to their damaging effect on the cell membrane. Poly-l-lysine shows the same activity.     

Xerotolerance in fission yeasts and the role of glycerol as compatible solute

The effect on growth of reducing the water activity (a_w) of a medium with various solutes has been investigated for 27 strains of fission yeasts (Schizosaccharomyces). The minimum-tolerated a_w (MTA) was dependent on both the nature of the solute and the species. When the strains of each species were grouped together, the lowest mean MTA values were found with glucose, fructose or glycerol as stressing solutes, being in the range 0.89–0.90 for S. pombe, S. malidevorans, S. octosporus and S. slooffiae, but in the range 0.92–0.94 for S. japonicus. With the non-metabolizable sugars sorbose and xylose and the salts NH₄Cl, KCl, and NaCl, the mean MTA values were in the range 0.96–0.985, except for (1) the single strain of S. slooffiae, which was more tolerant of NH₄Cl and KCl with values of 0.95 and 0.94, respectively, and (2) the strains of S. pombe, S. malidevorans and S. japonicus, which were less tolerant of NaCl with mean values of about 0.99. One strain of each species was examined for intracellular solutes when actively growing in the presence of near-limiting concentrations of stressing solute. With glucose, fructose or glycerol, all five strains contained substantial amounts of glycerol but no other polyol; with the other solutes no glycerol or other polyol was found, except for small amounts of glycerol in strains of S. octosporus and S. slooffiae stressed with NH₄Cl, KCl, or NaCl.Abbreviations MTA Minimum-tolerated water activity - a_w water activity - YEPG yeast extract, phosphate, glucose medium     

Characterization of the synthetic compatible solute homoectoine as a potent PCR enhancer

Different substances such as dimethyl sulfoxide, tetramethylene sulfoxide, 2-pyrollidone, and the naturally occurring compatible solute betaine enhance PCR amplification of GC-rich DNA templates with high melting temperatures. In particular, cyclic compatible solutes outperform traditional PCR enhancers. We therefore investigated the effects that cyclic naturally occurring ectoine-type compatible solutes and their synthetic derivatives have on melting temperature of double-stranded DNA (dsDNA) and on PCR amplification of different templates. L-ectoine, betaine, and derivatives of L-ectoine decreased, whereas beta-hydroxyectoine increased, the melting temperature of dsDNA. The ability to decrease the melting temperature was greatest for homoectoine, a new synthetic derivative of l-ectoine. Furthermore, compatible solutes, especially homoectoine, enhanced PCR amplification of GC-rich DNA (72.6% GC content; effective range: 0.1-0.5M).     

Advantages of quantitative affinity chromatography for the analysis of solute interaction with membrane proteins.

The use of membrane proteins as chromatographic stationary phases for the quantitation of biospecific interaction between the proteins and solutes is reviewed. This method is one among the few where a membrane protein is immobilized for repeated analyses of solute binding. To our knowledge, five transmembrane proteins have been immobilized in chromatographic matrices: the glucose and nucleoside transporters from human red blood cells, the photosynthetic reaction center from Rhodobacter sphaeroides, the nicotinic acetylcholine receptor from rat brain and a recombinant P-glycoprotein. Proteoliposomes and membrane vesicles have thereby been entrapped in size-exclusion beads, such as Superdex 200, and membrane proteins have been adsorbed on 'immobilized artificial membrane' monolayers of lipid analogs grafted to silica beads. Encouragingly, immobilized glucose transporter and P-glycoprotein showed constant interactant affinities for months. Analysis is done in the frontal mode at equilibrium because there is no separation between bound and free ligand. Both the affinity constant, which generally coincides with the corresponding constant determined by use of nonchromatographic methods, and the amount of active binding sites are obtained. The method has been successfully applied to functional analysis of membrane proteins in cells or reconstituted in lipid mono- or bilayers, screening of low-molecular interactants, investigation of protein-protein interaction and studies of effects of physico-chemical parameters on solute-protein interaction. The analyses require sensitive detection of the analyte and matching between amount of binding sites and affinity.     

Microscale fluorescent thermal stability assay for membrane proteins

Systematic efforts to understand membrane protein stability under a variety of different solution conditions are not widely available for membrane proteins, mainly due to technical problems stemming from the presence of detergents necessary to keep the proteins in the solubilized state and the background that such detergents usually generate during biophysical characterization. In this report, we introduce an efficient microscale fluorescent stability screen using the thiol-specific fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) for stability profiling of membrane proteins under different solution and ligand conditions. The screen uses the chemical reactivity of the native cysteines embedded in the protein interior as a sensor for the overall integrity of the folded state. The thermal information gained by thorough investigation of the protein stability landscape can be effectively used to guide purification and biophysical characterization efforts including crystallization. To evaluate the method, three different protein families were analyzed, including the Apelin G protein-coupled receptor (APJ).     

Folding and stability of membrane transport proteins in vitro

Transmembrane transporters are responsible for maintaining a correct internal cellular environment. The inherent flexibility of transporters together with their hydrophobic environment means that they are challenging to study in vitro, but recently significant progress been made. This review will focus on in vitro stability and folding studies of transmembrane alpha helical transporters, including reversible folding systems and thermal denaturation. The successful re-assembly of a small number of ATP binding cassette transporters is also described as this is a significant step forward in terms of understanding the folding and assembly of these more complex, multi-subunit proteins. The studies on transporters discussed here represent substantial advances for membrane protein studies as well as for research into protein folding. The work demonstrates that large flexible hydrophobic proteins are within reach of in vitro folding studies, thus holding promise for furthering knowledge on the structure, function and biogenesis of ubiquitous membrane transporter families. This article is part of a Special Issue entitled: Protein Folding in Membranes.