Application of the local-bulk partitioning and competitive binding models to interpret preferential interactions of glycine betaine and urea with protein surface

Two thermodynamic models have been developed to interpret the preferential accumulation or exclusion of solutes in the vicinity of biopolymer surface and the effects of these solutes on protein processes. The local-bulk partitioning model treats solute (and water) as partitioning between the region at/or near the protein surface (the local domain) and the bulk solution. The solvent exchange model analyzes a 1:1 competition between water and solute molecules for independent surface sites. Here we apply each of these models to interpret thermodynamic data for the interactions of urea and the osmoprotectant glycine betaine (N,N,N-trimethylglycine; GB) with the surface exposed in unfolding the marginally stable lacI HTH DNA binding domain. The partition coefficient K(P) quantifying accumulation of urea at this protein surface (K(P) approximately equal 1.1) is only weakly dependent on urea concentration up to 6 M urea. However, K(P) quantifying exclusion of GB from the vicinity of this protein surface increases from 0.83 (extrapolated to 0 M GB) to 1.0 (indicating that local and bulk GB concentrations are equal) at 4 M GB (activity > 40 M). We interpret the significant concentration dependence of K(P) for GB, predicted to be general for excluded, nonideal solutes such as GB, as a modest (8%) attenuation of the GB concentration dependence of solute nonideality in the local domain relative to that in the bulk solution. Above 4 M, K(P) for the interaction of GB with the surface exposed in protein unfolding is predicted to exceed unity, which explains the maximum in thermal stability observed for RNase and lysozyme at 4 M GB (Santoro, M. M., Liu, Y. F., Khan, S. M. A., Hou, L. X., and Bolen, D. W. (1992) Biochemistry 31, 5278-5283). Both thermodynamic models provide good two-parameter fits to GB and urea data for lacI HTH unfolding over a wide concentration range. The solute partitioning model allows for a full spectrum of attenuation effects in the local domain, encompasses the cases treated by the competitive binding model, and provides a somewhat better two-parameter fit of effects of high GB concentration on lacI HTH stability. Parameters of this fit should be applicable to isothermal and thermal unfolding data for all proteins with similar compositions of surface exposed in unfolding.     

Preferential interactions of glycine betaine and of urea with DNA: implications for DNA hydration and for effects of these solutes on DNA stability

Interactions of the solutes glycine betaine (GB) and urea with mononucleosomal calf thymus DNA in aqueous salt solutions are characterized by vapor pressure osmometry (VPO). Analysis of osmolality as a function of solute and DNA concentration yields the effect of the solute on the chemical potential, mu(2), of the DNA. Although both GB and urea generally are nucleic acid denaturants and therefore must interact favorably with the nucleic acid surface exposed upon melting, VPO demonstrates that neither interacts favorably with duplex DNA. Addition of GB greatly increases mu(2) of DNA, indicating that the average local concentration of GB in the vicinity of the double helix is much less than its bulk concentration. By contrast, addition of urea has almost no effect on mu(2) of duplex DNA, indicating that the average local concentration of urea in the vicinity of duplex DNA is almost the same as in bulk solution. Qualitatively, we conclude that the nonuniform distribution of GB occurs primarily because duplex DNA and GB prefer to interact with water rather than with each other. Comparison with thermodynamic data for the interaction of GB with various protein surfaces (Felitsky et al., Biochemistry, 43, 14732-14743) shows that GB is excluded primarily from anionic DNA surface and that the hydration of anionic DNA phosphate oxygen surface (>or approximately 17 H(2)O per nucleotide or >or approximately 0.22 H(2)O A(-)(2)) involves at least two layers of water. From analysis of literature data for effects of urea and of GB on DNA melting, we propose that urea is an effective nonspecific nucleic acid denaturant because of its favorable interactions with the polar amide-like surface of G, C, and especially T or U bases exposed in denaturation, whereas GB is a specific GC denaturant because of its favorable interaction with G and/or C surface in the single-stranded state.     

Use of urea and glycine betaine to quantify coupled folding and probe the burial of DNA phosphates in lac repressor-lac operator binding

Thermodynamic analysis of urea-biopolymer interactions and effects of urea on folding of proteins and alpha-helical peptides shows that urea interacts primarily with polar amide surface. Urea is therefore predicted to be a quantitative probe of coupled folding, remodeling, and other large-scale changes in the amount of water-accessible polar amide surface in protein processes. A parallel analysis indicates that glycine betaine [N,N,N-trimethylglycine (GB)] can be used to detect burial or exposure of anionic (carboxylate, phosphate) biopolymer surface. To test these predictions, we have investigated the effects of these solutes (0-3 m) on the formation of 1:1 complexes between lac repressor (LacI) and its symmetric operator site (SymL) at a constant KCl molality. Urea reduces the binding constant K(TO) [initial slope dlnK(TO)/dm(urea) = -1.7 +/- 0.2], and GB increases K(TO) [initial slope dlnK(TO)/dm(GB) = 2.1 +/- 0.2]. For both solutes, this derivative decreases with an increase in solute concentration. Analysis of these initial slopes predicts that (1.5 +/- 0.3) x 10(3) A2 of polar amide surface and (4.5 +/- 1.0) x 10(2) A2 of anionic surface are buried in the association process. Analysis of published structural data, together with modeling of unfolded regions of free LacI as extended chains, indicates that 1.5 x 10(3) A2 of polar amide surface and 6.3 x 10(2) A2 of anionic surface are buried in complexation. Quantitative agreement between structural and thermodynamic results is obtained for amide surface (urea); for anionic surface (GB), the experimental value is approximately 70% of the structural value. For LacI-SymL binding, two-thirds of the structurally predicted change in amide surface (1.0 x 10(3) A2) occurs outside the protein-DNA interface in protein-protein interfaces formed by folding of the hinge helices and interactions of the DNA binding domain (DBD) with the core of the repressor. Since urea interacts principally with amide surface, it is particularly well-suited to detect and quantify the extent of coupled folding and other large-scale remodeling events in the steps of protein-nucleic acid interactions and other protein associations.     

Thermal and urea-induced unfolding of the marginally stable lac repressor DNA-binding domain: a model system for analysis of solute effects on protein processes

Thermodynamic and structural evidence indicates that the DNA binding domains of lac repressor (lacI) exhibit significant conformational adaptability in operator binding, and that the marginally stable helix-turn-helix (HTH) recognition element is greatly stabilized by operator binding. Here we use circular dichroism at 222 nm to quantify the thermodynamics of the urea- and thermally induced unfolding of the marginally stable lacI HTH. Van't Hoff analysis of the two-state unfolding data, highly accurate because of the large transition breadth and experimental access to the temperature of maximum stability (T(S); 6-10 degrees C), yields standard-state thermodynamic functions (deltaG(o)(obs), deltaH(o)(obs), deltaS(o)(obs), deltaC(o)(P,obs)) over the temperature range 4-40 degrees C and urea concentration range 0 相似文献   

Roles of cytoplasmic osmolytes, water, and crowding in the response of Escherichia coli to osmotic stress: biophysical basis of osmoprotection by glycine betaine

To better understand the biophysical basis of osmoprotection by glycine betaine (GB) and the roles of cytoplasmic osmolytes, water, and macromolecular crowding in the growth of osmotically stressed Escherichia coli, we have determined growth rates and amounts of GB, K(+), trehalose, biopolymers, and water in the cytoplasm of E. coli K-12 grown over a wide range of high external osmolalities (1.02-2.17 Osm) in MOPS-buffered minimal medium (MBM) containing 1 mM betaine (MBM+GB). As osmolality increases, we observe that the amount of cytoplasmic GB increases, the amounts of K(+) (the other major cytoplasmic solute) and of biopolymers remain relatively constant, and the growth rate and the amount of cytoplasmic water decrease strongly, so concentrations of biopolymers and all solutes increase with increasing osmolality. We observe the same correlation between the growth rate and the amount of cytoplasmic water for cells grown in MBM+GB as in MBM, supporting our proposal that the amount of cytoplasmic water is a primary determinant of the growth rate of osmotically stressed cells. We also observe the same correlation between cytoplasmic concentrations of biopolymers and K(+) for cells grown in MBM and MBM+GB, consistent with our hypothesis of compensation between the anticipated large perturbing effects on cytoplasmic protein-DNA interactions of increases in cytoplasmic concentrations of K(+) and biopolymers (crowding) with increasing osmolality. For growth conditions where the amount of cytoplasmic water is relatively large, we find that cytoplasmic osmolality is adequately predicted by assuming that contributions of individual solutes to osmolality are additive and using in vitro osmotic data on osmolytes and a local bulk domain model for cytoplasmic water. At moderate growth osmolalities (up to 1 Osm), we conclude that GB is an efficient osmoprotectant because it is almost as excluded from the biopolymer surface in the cytoplasm as it is from native protein surface in vitro. At very high growth osmolalities where cells contain little cytoplasmic water, predicted cytoplasmic osmolalities greatly exceed observed osmolalities, and the efficiency of GB as an osmolality booster decreases as the amount of cytoplasmic water decreases.     

Molecular determinants for substrate specificity of the ligand-binding protein OpuAC from Bacillus subtilis for the compatible solutes glycine betaine and proline betaine

Compatible solutes play a decisive role in the defense of microorganisms against changes in temperature and increases in osmolarity in their natural habitats. In Bacillus subtilis, the substrate-binding protein (SBP)-dependent ABC-transporter OpuA serves for the uptake of the compatible solutes glycine betaine (GB) and proline betaine (PB). Here, we report the determinants of compatible solute binding by OpuAC, the SBP of the OpuA transporter, by equilibrium binding studies and X-ray crystallography. The affinity of OpuAC/GB and OpuAC/PB complexes were analyzed by intrinsic tryptophan fluorescence and the K(D) values were determined to be 17(+/-1)microM for GB and 295(+/-27)microM for PB, respectively. The structures of OpuAC in complex with GB or PB were solved at 2.0 A and 2.8 A, respectively, and show an SBP-typical class II fold. The ligand-binding pocket is formed by three tryptophan residues arranged in a prism-like geometry suitable to coordinate the positive charge of the trimethyl ammonium group of GB and the dimethyl ammonium group of PB by cation-pi interactions and by hydrogen bonds with the carboxylate moiety of the ligand. Structural differences between the OpuAC/GB and OpuAC/PB complexes occur within the ligand-binding pocket as well as across the domain-domain interface. These differences provide a structural framework to explain the drastic differences in affinity of the OpuAC/GB and OpuAC/PB complexes. A sequence comparison with putative SBP specific for compatible solutes reveals the presence of three distinct families for which the crystal structure of OpuAC might serve as a suitable template to predict the structures of these putative compatible solute-binding proteins.     

Purification and characterization of a glycine betaine binding protein from Escherichia coli

A major component of the Escherichia coli response to elevated medium osmolarity is the synthesis of a periplasmic protein with an Mr of 31,000. The protein was absent in mutants with lambda placMu insertions in the proU region, a locus involved in transport of the osmoprotectant glycine betaine. This periplasmic protein has now been purified to homogeneity. Antibody directed against the purified periplasmic protein crossreacts with the fusion protein produced as a result of the lambda placMu insertion, indicating that proU is the structural gene specifying the 31-kDa protein. The purified protein binds glycine betaine with high affinity but has no affinity for either proline or choline, clarifying the role of proU in osmoprotectant transport. The amino-terminal sequence of the mature glycine betaine binding protein is Ala-Asp-Leu-Pro-Gly-Lys-Gly-Ile-Thr-Val-Asn-Pro.     

Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD.

The accumulation of the osmoprotectant glycine betaine from exogenous sources provides a high degree of osmotic tolerance to Bacillus subtilis. We have identified, through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, a new glycine betaine transport system from B. subtilis. The DNA sequence of a 2,310-bp segment of the cloned region revealed a single gene (opuD) whose product (OpuD) was essential for glycine betaine uptake and osmoprotection in E. coli. The opuD gene encodes a hydrophobic 56.13-kDa protein (512 amino acid residues). OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporter CaiT from E. coli and a BetT-like protein from Haemophilus influenzae. These membrane proteins form a family of transporters involved in the uptake of trimethylammonium compounds. The OpuD-mediated glycine betaine transport activity in B. subtilis is controlled by the environmental osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine betaine uptake activity. An opuD mutant was constructed by marker replacement, and the OpuD-mediated glycine betaine uptake activity was compared with that of the previously identified multicomponent OpuA and OpuC (ProU) glycine betaine uptake systems. In addition, a set of mutants was constructed, each of which synthesized only one of the three glycine betaine uptake systems. These mutants were used to determine the kinetic parameters for glycine betaine transport through OpuA, OpuC, and OpuD. Each of these uptake systems shows high substrate affinity, with Km values in the low micromolar range, which should allow B. subtilis to efficiently acquire the osmoprotectant from the environment. The systems differed in their contribution to the overall glycine betaine accumulation and osmoprotection. A triple opuA, opuC, and opuD mutant strain was isolated, and it showed no glycine betaine uptake activity, demonstrating that three transport systems for this osmoprotectant operate in B. subtilis.     

Sodium-driven, osmotically activated glycine betaine transport in Listeria monocytogenes membrane vesicles.

Transport of the osmoprotectant and cryoprotectant glycine betaine was investigated in membrane vesicles of Listeria monocytogenes. Uptake-driving transmembrane potentials ranging from 111 to 122 mV within the pH range of 5.5 to 7.5 could be generated by the electron donor system ascorbate-phenazine methosulfate but not by the electron donor system ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine. Transport was dependent on both high concentrations of sodium ion and the presence of a hypertonic solute gradient. Arrhenius-type temperature activation was observed. Lineweaver-Burk plots indicated a Km of 4.4 microM for glycine betaine and a Vmax of 700 pmol/min x mg of protein. The Michaelis constant for NaCl depended on the solute used to maintain a constant hyperosmotic pressure, and the Km values were 200 and 75 mM when KCl and sucrose were employed, respectively. Transport was 65% lower in vesicles derived from cells grown under stress provided by KCI rather than NaCl and approximately 94% lower in vesicles derived from cells that were not grown under osmotic stress. This porter appears to be specific for glycine betaine, since neither proline, carnitine, nor choline inhibited uptake effectively. Kinetic studies using ionophores and artificial gradients indicate that glycine betaine is cotransported with sodium ion.     

Human telomerase RNA pseudoknot and hairpin thermal stability with glycine betaine and urea: preferential interactions with RNA secondary and tertiary structures

Thermal denaturation of the human telomerase RNA (hTR) DeltaU177 pseudoknot and hTR p2b hairpin was investigated by dual UV-wavelength absorbance spectroscopy in aqueous glycine betaine and urea solutions. The hTR DeltaU177 pseudoknot contains two helix-loop interactions that comprise the tertiary structure, as well as a GC-rich 6 bp stem (stem 1) and an AU-rich 9 bp stem (stem 2). The p2b hairpin also contains GC-rich stem 1 and a unique uridine-rich helix with a pentaloop. Glycine betaine stabilizes the pseudoknot tertiary structure in 135 mm NaCl and facilitates only a minor destabilization of tertiary structure in 40 mm NaCl. As with double-helical DNA, glycine betaine interacts more strongly with the surface area exposed upon unfolding of GC-rich stem 1 than either AU-rich stem 2 or the hairpin uridine-rich helix. Urea was shown to destabilize all RNA pseudoknot and hairpin secondary and tertiary structures but exhibits a stronger preferential interaction with AU-rich stem 2. Correlating these interactions with water-accessible surface area calculations indicates that the extent of interaction of glycine betaine with the surface area exposed upon RNA unfolding decreases as the nonpolar character of the unfolded RNA surface increases. As expected, the extent of interaction of urea with the surface area exposed for unfolding RNA increases as the fraction of amide functional groups increases. However, interaction of urea with amide functional groups alone cannot explain the stronger preferential interaction of urea with AU-rich stem 2. Interaction of urea with adenine relative to guanine and cytosine bases or sequence-dependent hydration is proposed for the stronger preferential interaction of urea with AU-rich duplexes.     

Vapor pressure osmometry studies of osmolyte-protein interactions: implications for the action of osmoprotectants in vivo and for the interpretation of "osmotic stress" experiments in vitro

To interpret or to predict the responses of biopolymer processes in vivo and in vitro to changes in solute concentration and to coupled changes in water activity (osmotic stress), a quantitative understanding of the thermodynamic consequences of interactions of solutes and water with biopolymer surfaces is required. To this end, we report isoosmolal preferential interaction coefficients (Gamma(mu1) determined by vapor pressure osmometry (VPO) over a wide range of concentrations for interactions between native bovine serum albumin (BSA) and six small solutes. These include Escherichia coli cytoplasmic osmolytes [potassium glutamate (K(+)Glu(-)), trehalose], E. coli osmoprotectants (proline, glycine betaine), and also glycerol and trimethylamine N-oxide (TMAO). For all six solutes, Gamma(mu1) and the corresponding dialysis preferential interaction coefficient Gamma(mu1),(mu3) (both calculated from the VPO data) are negative; Gamma(mu1), (mu3) is proportional to bulk solute molality (m(bulk)3) at least up to 1 m (molal). Negative values of Gamma(mu1),(mu3) indicate preferential exclusion of these solutes from a BSA solution at dialysis equilibrium and correspond to local concentrations of these solutes in the vicinity of BSA which are lower than their bulk concentrations. Of the solutes investigated, betaine is the most excluded (Gamma(mu1),(mu3)/m(bulk)3 = -49 +/- 1 m(-1)); glycerol is the least excluded (Gamma(mu1),(mu3)/m(bulk)3 = -10 +/- 1 m(-1)). Between these extremes, the magnitude of Gamma(mu1),(mu3)/m(bulk)3 decreases in the order glycine betaine > proline >TMAO > trehalose approximately K(+)Glu(-) > glycerol. The order of exclusion of E. coli osmolytes from BSA surface correlates with their effectiveness as osmoprotectants, which increase the growth rate of E. coli at high external osmolality. For the most excluded solute (betaine), Gamma(mu1),(mu3) provides a minimum estimate of the hydration of native BSA of approximately 2.8 x 10(3) H(2)O/BSA, which corresponds to slightly less than a monolayer (estimated to be approximately 3.2 x 10(3) H(2)O). Consequently, of the solutes investigated here, only betaine might be suitable for use in osmotic stress experiments in vitro as a direct probe to quantify changes in hydration of protein surface in biopolymer processes. More generally, however, our results and analysis lead to the proposal that any of these solutes can be used to quantify changes in water-accessible surface area (ASA) in biopolymer processes once preferential interactions of the solute with biopolymer surface are properly taken into account.     

Isolation and characterization of ButA, a secondary glycine betaine transport system operating in Tetragenococcus halophila

Through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, we identified a single-component glycine betaine transporter from Tetragenococcus halophila, a moderate halophilic lactic acid bacterium. DNA sequence analysis characterized the ButA protein as a member of the betaine choline carnitine transporter (BCCT) family, that includes a variety of previously characterized compatible solute transporters such as OpuD from Bacillus subtilis, EctP and BetP from Corynebacterium glutamicum, and BetL from Listeria monocytogenes. When expressed in the heterologous host E. coli, the permease is specific for glycine betaine and does not transport the other osmoprotectants previously described for T. halophila (i.e. carnitine, choline, dimethylsulfonioacetate, dimethylsulfoniopropionate, and ectoine). In E. coli, statement of ButA is mainly constitutive and maximal uptake activity may result from a weak osmotic induction. This is the first study demonstrating a role for a permease in osmoregulation, and GB uptake, of a lactic acid bacterium.     

Effect of the drug cyclophosphamide on the activity of porcine kidney betaine aldehyde dehydrogenase

Molecular and Cellular Biochemistry - The enzyme betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the synthesis of glycine betaine (GB), an osmolyte and osmoprotectant. Also, it...     

Toxicity and osmoprotective activities of analogues of glycine betaine obtained by solid phase organic synthesis towards Sinorhizobium meliloti

Seven analogues of the bacterial osmoprotectant glycine betaine (GB, trimethylammonioacetate), in which the methyl groups of the Me₃N⁺ moiety are replaced by various substituents, were obtained by SPOS using Wang resin. Their biological activities (osmoprotection vs toxicity), appeared closely related to their uptake efficiency and their catabolism in the betaine-demethylating model bacterium Sinorhizobium meliloti.     

Glycine betaine transport in the obligate halophilic archaeon Methanohalophilus portucalensis

Transport of the osmoprotectant glycine betaine was investigated using the glycine betaine-synthesizing microbe Methanohalophilus portucalensis (strain FDF1), since solute uptake for this class of obligate halophilic methanogenic Archaea has not been examined. Betaine uptake followed a Michaelis-Menten relationship, with an observed K(t) of 23 microM and a V(max) of 8 nmol per min per mg of protein. The transport system was highly specific for betaine: choline, proline, and dimethylglycine did not significantly compete for [(14)C]betaine uptake. The proton-conducting uncoupler 2, 4-dinitrophenol and the ATPase inhibitor N, N-dicyclohexylcarbodiimide both inhibited glycine betaine uptake. Growth of cells in the presence of 500 microM betaine resulted in faster cell growth due to the suppression of the de novo synthesis of the other compatible solutes, alpha-glutamate, beta-glutamine, and N(epsilon)-acetyl-beta-lysine. These investigations demonstrate that this model halophilic methanogen, M. portucalensis strain FDF1, possesses a high-affinity and highly specific betaine transport system that allows it to accumulate this osmoprotectant from the environment in lieu of synthesizing this or other osmoprotectants under high-salt growth conditions.     

Osmotic adaptation in Brevibacterium linens: differential effect of proline and glycine betaine on cytoplasmic osmolyte pool

In the coryneform Brevibacterium linens, ectoine constitutes the major intracellular solute accumulated under elevated medium osmolarity. Here we report that exogenously supplied proline, choline, glycine betaine, and even ectoine, protected bacterial cells against deleterious effects of a hyperosmotic constraint (i.e. 1.5 M NaCl). In all cases, a significant improvement of growth was observed; in parallel, intracellular osmolyte pools composed mainly of glutamate and ectoine substantially increased, either with added glycine betaine (under limiting supply) or with proline. However, these two osmoprotectants behaved differently: glycine betaine acted as a genuine osmoprotectant, whereas proline was accumulated only transiently and participated actively in the biosynthesis of glutamate, ectoine, and trehalose. The strategy developed by B. linens cells allows the proposal of a novel role for proline in the osmoprotection process through its conversion to the apparently preferred endogenous osmolyte ectoine.     

Isolation and molecular characterization of theSinorhizobium meliloti bet locus encoding glycine betaine biosynthesis

To cope with osmotic stress,Sinorhizobium meliloti accumulates organic compatible solutes such as glutamate, trehalose, N-acetylglutaminylglutamine amide, and the most potent osmoprotectant glycine betaine. In order to study the regulation of the glycine betaine biosynthetic pathway, a genetic and molecular analysis was performed. We have selected a Tn5 mutant ofS. meliloti which was deficient in choline dehydrogenase activity. The mutation was complemented using a genomic bank ofS. meliloti. Subcloning and DNA sequencing of a 8-6 kb region from the complemented plasmid showed four open reading frames with an original structural organization of thebet locus compared to that described inE. coli. (i) ThebetB and thebetA genes which encode a glycine betaine aldehyde dehydrogenase, and a choline dehydrogenase, respectively, are separated from thebetI gene (regulatory protein) by an additional gene namedbetC. The BetC protein shares about 30% identity with various sulphatases and is involved in the conversion of choline-O-sulphate into choline. Choline-O-sulphate is used as an osmoprotectant, or as a carbon or sulphur source and this utilization is dependent on a functionalbet locus. (ii) No sequence homologous tobetT (encoding a high-affinity choline transport system inE. coli) was found in the vicinity of thebet locus. (iii) ThebetB and thebetA genes, as well as thebetI and thebetC genes are, respectively, separated by 211 and 167 bp sequences containing inverted repeats. Southern blot analysis indicated that thebet locus is located on the chromosome, and not on the megaplasmids.     

Isolation and characterization of adenylate kinase (adk) mutations in Salmonella typhimurium which block the ability of glycine betaine to function as an osmoprotectant.

Mutants of Salmonella typhimurium that were not protected by glycine betaine (GB) but could still use proline as an osmoprotectant in media of high osmolality were isolated. The mutations responsible for this phenotype proved to be alleles of the adenylate kinase (adk) gene, as shown by genetic mapping, sequencing of the cloned mutant alleles, complementation with the Escherichia coli adk gene, and assay of Adk enzyme activity in crude extracts. One of the mutations was in the untranslated leader of the adk mRNA, a second was in the putative Shine-Dalgarno sequence, and a third was in the coding region of the gene. The loss of osmoprotection by GB was shown to be due to the fact that the accumulation of this solute actually resulted in a severe inhibition of growth in the adk mutants. The addition of GB in the presence of 0.5 M NaCl resulted in a rapid decline in the ATP pool and a dramatic increase in the AMP pool in the mutants. Proline, which is not toxic to the adk mutants, did not have any significant effects on the cellular levels of ATP and AMP. The mutants exhibited two different phenotypes with respect to the utilization of other osmoprotectants: they were also inhibited by propiothiobetaine, L-carnitine, and gamma-butyrobetaine, but they were stimulated normally in media of high osmolality by proline, choline-O-sulfate, and stachydrine.