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.     

Regulation of envelope protein composition during adaptation to osmotic stress in Escherichia coli.

Adaptation to osmotic stress alters the amounts of several specific proteins in the Escherichia coli K-12 envelope. The most striking feature of the response to elevated osmolarity was the strong induction of a periplasmic protein with an Mr of 31,000. This protein was absent in mutants with lambda plac Mu insertions in an osmotically inducible locus mapping near 58 min. The insertions are likely to be in proU, a locus encoding a transport activity for the osmoprotectants glycine betaine and proline. Factors affecting the extent of proU induction were identified by direct examination of periplasmic proteins on sodium dodecyl sulfate gels and by measuring beta-galactosidase activity from proU-lac fusions. Expression was stimulated by increasing additions of salt or sucrose to minimal medium, up to a maximum at 0.5 M NaCl. Exogenous glycine betaine acted as an osmoregulatory signal; its addition to the high-osmolarity medium substantially repressed the expression of the 31,000-dalton periplasmic protein and the proU-lac+ fusions. Elevated osmolarity also caused the appearance of a second periplasmic protein (Mr = 16,000), and severe reduction in the amounts of two others. In the outer membrane, the well-characterized repression of OmpF by high osmolarity was observed and was reversed by glycine betaine. Additional changes in membrane composition were also responsive to glycine betaine regulation.     

Identification of bacterial periplasmic glycine betaine-binding protein after electrophoresis and affinity labeling

Antibodies were elicited in rabbits against periplasmic proteins obtained by cold osmotic shock from the Gram-negative eubacterium Rhizobium meliloti. When analyzed by crossed immunoelectrophoresis (CIE), the periplasmic proteins gave rise to 20 distinct immunoprecipitates corresponding to the same number of bands in polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions and in SDS-PAGE. The periplasmic glycine betaine-binding protein (GB-BP) was identified by autoradiography after affinity labeling with [14C]glycine betaine in PAGE and in CIE gels. The binding proved to be quite specific to glycine betaine, since the GB-BP was not labeled by choline (a metabolic precursor of glycine betaine in Escherichia coli and Rhizobium meliloti) and 15 distinct L-amino acids, including L-proline which, like glycine betaine is also an osmoprotectant. Affinity labeling of the GB-BP with [14C]glycine betaine after protein separation by PAGE or CIE is a simple and sensitive technique permitting the GB-BP to the unambiguously detected and identified in samples of complex protein mixtures containing down to 2 micrograms of GB-BP in PAGE and only 0.2 micrograms in CIE.     

Cation-pi interactions as determinants for binding of the compatible solutes glycine betaine and proline betaine by the periplasmic ligand-binding protein ProX from Escherichia coli

Compatible solutes such as glycine betaine and proline betaine are accumulated to exceedingly high intracellular levels by many organisms in response to high osmolarity to offset the loss of cell water. They are excluded from the immediate hydration shell of proteins and thereby stabilize their native structure. Despite their exclusion from protein surfaces, the periplasmic ligand-binding protein ProX from the Escherichia coli ATP-binding cassette transport system ProU binds the compatible solutes glycine betaine and proline betaine with high affinity and specificity. To understand the mechanism of compatible solute binding, we determined the high resolution structure of ProX in complex with its ligands glycine betaine and proline betaine. This crystallographic study revealed that cation-pi interactions between the positive charge of the quaternary amine of the ligands and three tryptophan residues forming a rectangular aromatic box are the key determinants of the high affinity binding of compatible solutes by ProX. The structural analysis was combined with site-directed mutagenesis of the ligand binding pocket to estimate the contributions of the tryptophan residues involved in binding.     

Purification, reconstitution, and characterization of the CpxRAP envelope stress system of Escherichia coli

In Escherichia coli the Cpx sensor regulator system senses different kinds of envelope stress and responds by triggering the expression of periplasmic folding factors and proteases. It consists of the membrane-anchored sensor kinase CpxA, the response regulator CpxR, and the periplasmic protein CpxP. The Cpx pathway is induced in vivo by a variety of signals including pH variation, osmotic stress, and misfolded envelope proteins and is inhibited by overproduced CpxP. Because it is not clear how the Cpx pathway is able to recognize and correspond to so many different signals we overproduced, solubilized, purified, and incorporated the complete membrane-integral CpxA protein into proteoliposomes to analyze its biochemical properties in more detail. Autokinase and phosphotransfer activities of the reconstituted CpxA-His6 protein were stimulated by KCl. NaCl also stimulated the activities but to a lesser extent. Other osmotic active solutes as glycine betaine, sucrose, and proline had no effect. The system was further characterized by testing for susceptibility to sensor kinase inhibitors. Among these, Closantel inhibited the activities of solubilized but not of the reconstituted CpxA-His6 protein. We further analyzed the effect of CpxP on CpxA activities. Purified tagless CpxP protein reduced the phosphorylation status of CpxA to 50% but had no effect on CpxA phosphotransfer or phosphatase activities. As the in vitro system excludes the involvement of other factors our finding is the first biochemical evidence for direct protein-protein interaction between the sensor kinase CpxA and the periplasmic protein CpxP resulting in a down-regulation of the autokinase activity of CpxA.     

Characterization of a Snorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake

The symbiotic soil bacterium Sinorhizobium meliloti uses the compatible solutes glycine betaine and proline betaine for both protection against osmotic stress and, at low osmolarities, as an energy source. A PCR strategy based on conserved domains in components of the glycine betaine uptake systems from Escherichia coli (ProU) and Bacillus subtilis (OpuA and OpuC) allowed us to identify a highly homologous ATP-binding cassette (ABC) binding protein-dependent transporter in S. meliloti. This system was encoded by three genes (hutXWV) of an operon which also contained a fourth gene (hutH2) encoding a putative histidase, which is an enzyme involved in the first step of histidine catabolism. Site-directed mutagenesis of the gene encoding the periplasmic binding protein (hutX) and of the gene encoding the cytoplasmic ATPase (hutV) was done to study the substrate specificity of this transporter and its contribution in betaine uptake. These mutants showed a 50% reduction in high-affinity uptake of histidine, proline, and proline betaine and about a 30% reduction in low-affinity glycine betaine transport. When histidine was used as a nitrogen source, a 30% inhibition of growth was observed in hut mutants (hutX and hutH2). Expression analysis of the hut operon determined using a hutX-lacZ fusion revealed induction by histidine, but not by salt stress, suggesting this uptake system has a catabolic role rather than being involved in osmoprotection. To our knowledge, Hut is the first characterized histidine ABC transporter also involved in proline and betaine uptake.     

Glycine betaine, an osmotic effector in Klebsiella pneumoniae and other members of the Enterobacteriaceae

Osmoregulation was examined in members of the Enterobacteriaceae. Exogenous glycine betaine at a concentration as low as 1 mM was found to stimulate the growth rate of Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae in media of inhibitory osmotic strength. The stimulation was shown to be independent of any specific solutes, electrolytes, or nonelectrolytes. Therefore, the stimulatory effect of glycine betaine was a consequence of high osmotic potential. This effect was found to be far greater than the proline effect previously observed in S. typhimurium. Whereas nitrogen fixation by K. pneumoniae is completely inhibited under conditions of osmotic stress, nitrogenase activity could be partially restored by the addition of exogenous glycine betaine to the culture medium. Furthermore, glycine betaine in combination with proline, especially proline produced internally at a high level because of regulatory mutations affecting proline biosynthesis, strongly stimulated nitrogen fixation activity during osmotic stress. Glycine betaine was accumulated by the cells, and the amount taken up was correlated with the osmolarity of the medium. These findings are discussed in relation to the possible mechanisms by which glycine betaine might cause enhanced osmotolerance.     

Glycine betaine, an osmotic effector in Klebsiella pneumoniae and other members of the Enterobacteriaceae.

Osmoregulation was examined in members of the Enterobacteriaceae. Exogenous glycine betaine at a concentration as low as 1 mM was found to stimulate the growth rate of Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae in media of inhibitory osmotic strength. The stimulation was shown to be independent of any specific solutes, electrolytes, or nonelectrolytes. Therefore, the stimulatory effect of glycine betaine was a consequence of high osmotic potential. This effect was found to be far greater than the proline effect previously observed in S. typhimurium. Whereas nitrogen fixation by K. pneumoniae is completely inhibited under conditions of osmotic stress, nitrogenase activity could be partially restored by the addition of exogenous glycine betaine to the culture medium. Furthermore, glycine betaine in combination with proline, especially proline produced internally at a high level because of regulatory mutations affecting proline biosynthesis, strongly stimulated nitrogen fixation activity during osmotic stress. Glycine betaine was accumulated by the cells, and the amount taken up was correlated with the osmolarity of the medium. These findings are discussed in relation to the possible mechanisms by which glycine betaine might cause enhanced osmotolerance.     

Distribution of compatible solutes in the halophilic methanogenic archaebacteria.

Accumulation of compatible solutes, by uptake or de novo synthesis, enables bacteria to reduce the difference between osmotic potentials of the cell cytoplasm and the extracellular environment. To examine this process in the halophilic and halotolerant methanogenic archaebacteria, 14 strains were tested for the accumulation of compatible solutes in response to growth in various extracellular concentrations of NaCl. In external NaCl concentrations of 0.7 to 3.4 M, the halophilic methanogens accumulated K+ ion and low-molecular-weight organic compounds. beta-Glutamate was detected in two halotolerant strains that grew below 1.5 M NaCl. Two unusual beta-amino acids, N epsilon-acetyl-beta-lysine and beta-glutamine (3-aminoglutaramic acid), as well as L-alpha-glutamate were compatible solutes among all of these strains. De novo synthesis of glycine betaine was also detected in several strains of moderately and extremely halophilic methanogens. The zwitterionic compounds (beta-glutamine, N epsilon-acetyl-beta-lysine, and glycine betaine) and potassium were the predominant compatible solutes among the moderately and extremely halophilic methanogens. This is the first report of beta-glutamine as a compatible solute and de novo biosynthesis of glycine betaine in the methanogenic archaebacteria.     

Identification of an osmotically induced periplasmic glycine betaine-binding protein from Rhizobium meliloti.

The effect of salt stress on glycine betaine-binding activity has been investigated in periplasmic fractions released from Rhizobium meliloti 102F34 by cold osmotic shock. Binding activity was monitored by three techniques: equilibrium dialysis, filter procedure, and detection of 14C ligand-protein binding by direct non-denaturing polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. The three methods demonstrated the existence of a strong glycine betaine-binding activity, but only in periplasmic fractions from cells grown at high osmolarity. The non-denaturing PAGE of such periplasmic shock fluids mixed with [methyl-14C]glycine betaine showed only one radioactive band, indicating the involvement of one glycine betaine-binding protein. To determine the possible implication of this binding protein in glycine betaine uptake, transport activity was measured with cells submitted to cold osmotic shock. No significant decrease of transport activity was noticed. This lack of effect could be explained by the small quantity of periplasmic proteins released as judged by the low activity of phosphodiesterase, a periplasmic marker enzyme, observed in the shock fluid. The specificity of binding was analysed with different potential competitors: other betaines such as gamma-butyrobetaine, proline betaine, pipecolate betaine, trigonelline and homarine, or amino acids like glycine and proline, did not bind to the glycine betaine-binding protein, whereas glycine betaine aldehyde and choline were weak competitors. Optimum pH for binding was around 7.0, but approx. 90% of the glycine betaine-binding activity remained at pH 6.0 or 8.0. The calculated binding affinity (KD) was 2.5 microM. Both glycine betaine-binding activity and affinity were not significantly modified whether or not the binding assays were done at high osmolarity. A 32 kDa osmotically inducible periplasmic protein, identified by SDS-PAGE, apparently corresponds to the glycine betaine-binding protein.     

Characterization of an osmoregulated periplasmic glycine betaine-binding protein in Azospirillum brasilense sp7.

Azospirillum brasilense is able to use glycine betaine as a powerful osmoprotectant; the uptake of this compound is strongly stimulated by salt stress, but significantly reduced by cold osmotic shock. Non-denaturing PAGE in the presence of [methyl-14C] glycine betaine and autoradiography demonstrated the presence of one glycine betaine-binding protein (GBBP) in periplasmic shock fluid obtained from high-osmolarity-grown cells. The binding activity was absent in periplasmic fractions from cells grown at low osmolarity. SDS-PAGE analysis showed that the osmotically inducible GBBP has an apparent molecular weight of 32,000. The isoelectric point was between 5.9 and 6.6, as determined by isoelectric focusing. This protein bound glycine betaine with high affinity (KD of 3 microM), but had no affinity for either other betaines (proline betaine, gamma-butyrobetaine, pipecolate betaine, trigonelline, homarine) or related compounds (choline, glycine betaine aldehyde, glycine and proline). Optimum binding activity occurred at pH 7.0 to 7.5, and was not altered whether or not the binding assays were done at low or high osmolarity. Immunoprecipitation and Western blotting showed that immunoadsorbed anti-GBBP antibody from E coli cross-reacted with the GBBP produced by A brasilense cells grown at high osmolarity.     

Isolation, characterization, and expression of the Corynebacterium glutamicum betP gene, encoding the transport system for the compatible solute glycine betaine.

Corynebacterium glutamicum accumulates glycine betaine under conditions of high osmolarity. Previous work revealed the existence of a high-affinity glycine betaine permease which is osmotically regulated. In the present study, the corresponding gene was cloned. The betP gene, encoding the glycine betaine uptake carrier, was isolated by heterologous complementation of mutant strain Escherichia coli MKH13. From sequence analysis it is predicted to encode a protein of 595 amino acids. This protein shares 36% identity with the choline transport system BetT and 28% identity with the carnitine transport system CaiT of E. coli, as well as 38% identity with a protein with an unknown function from Haemophilus influenzae. Analysis of hydropathy indicated a common structure for all four transport proteins. After heterologous expression of betP in E. coli MKH13, the measured Km values for glycine betaine and the cotransported Na+ were similar to those found in C. glutamicum, whereas the modulation of activity by osmotic gradients was shifted to lower osmotic values.     

Protection of Bacillus subtilis against cold stress via compatible-solute acquisition

Accumulation of compatible solutes is a strategy widely employed by bacteria to achieve cellular protection against high osmolarity. These compounds are also used in some microorganisms as thermostress protectants. We found that Bacillus subtilis uses the compatible solute glycine betaine as an effective cold stress protectant. Glycine betaine strongly stimulated growth at 15°C and permitted cell proliferation at the growth-inhibiting temperature of 13°C. Initial uptake of glycine betaine at 15°C was low but led eventually to the buildup of an intracellular pool whose size was double that found in cells grown at 35°C. Each of the three glycine betaine transporters (OpuA, OpuC, and OpuD) contributed to glycine betaine accumulation in the cold. Protection against cold stress was also accomplished when glycine betaine was synthesized from its precursor choline. Growth of a mutant defective in the osmoadaptive biosynthesis for the compatible solute proline was not impaired at low temperature (15°C). In addition to glycine betaine, the compatible solutes and osmoprotectants l-carnitine, crotonobetaine, butyrobetaine, homobetaine, dimethylsulfonioactetate, and proline betaine all served as cold stress protectants as well and were accumulated via known Opu transport systems. In contrast, the compatible solutes and osmoprotectants choline-O-sulfate, ectoine, proline, and glutamate were not cold protective. Our data highlight an underappreciated facet of the acclimatization of B. subtilis to cold environments and allow a comparison of the characteristics of compatible solutes with respect to their osmotic, heat, and cold stress-protective properties for B. subtilis cells.     

Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.     

Identification of organic solutes accumulated by purple and green sulphur bacteria during osmotic stress using natural abundance 13C nuclear magnetic resonance spectroscopy

Abstract Natural abundance ¹³C NMR spectroscopy has identified sucrose and trehalose as the principle compatible solutes accumulated by non-halophilic purple and green sulphur bacteria respectively, in response to osmotic stress. Synthesis of glycine betaine as a compatible solute was rare in non-halophilic phototrophic sulphur bacteria and appears to be limited almost exclusively to halotolerant isolates, although all isolates tested were able to accumulate exogenous glycine betaine from the growth medium in response to osmotic stress. These data support the hypothesis that the degree of halotolerance of a microorganism may be due, at least in part, to the metabolic effects of the compatible solute(s) accumulated.     

Osmoregulation in Agrobacterium tumefaciens: accumulation of a novel disaccharide is controlled by osmotic strength and glycine betaine.

We have investigated the mechanism of osmotic stress adaptation (osmoregulation) in Agrobacterium tumefaciens biotype I (salt-tolerant) and biotype II (salt-sensitive) strains. Using natural-abundance 13C nuclear magnetic resonance spectroscopy, we identified all organic solutes that accumulated to significant levels in osmotically stressed cultures. When stressed, biotype I strains (C58, NT1, and A348) accumulated glutamate and a novel disaccharide, beta-fructofuranosyl-alpha-mannopyranoside, commonly known as mannosucrose. In the salt-sensitive biotype II strain K84, glutamate was observed but mannosucrose was not. We speculate that mannosucrose confers the extra osmotic tolerance observed in the biotype I strains. In addition to identifying the osmoregulated solutes that this species synthesizes, we investigated the ability of A. tumefaciens to utilize the powerful osmotic stress protectant glycine betaine when it is supplied in the medium. Results from growth experiments, nuclear magnetic resonance spectroscopy, and a 14C labeling experiment demonstrated that in the absence of osmotic stress, glycine betaine was metabolized, while in stressed cultures, glycine betaine accumulated intracellularly and conferred enhanced osmotic stress tolerance. Furthermore, when glycine betaine was taken up in stressed cells, its accumulation caused the intracellular concentration of mannosucrose to drop significantly. The possible role of osmoregulation of A. tumefaciens in the transformation of plants is discussed.