Glycine Betaine Transport Research Articles

Proline betaine is a highly effective osmoprotectant for Staphylococcus aureus

Proline betaine is an osmoprotectant that is at least as effective as glycine betaine, and more effective than L-proline, for various strains of Staphylococcus aureus, and Staphylococcus epidermidis and Staphylococcus saprophyticus. 13C NMR studies revealed that proline betaine accumulated to high levels in osmotically stressed S. aureus, but was also detected in organisms grown in its presence in the absence of osmotic stress. Competition experiments indicated that proline betaine was taken up by the proline transport systems of S. aureus, but not by the high affinity glycine betaine transport system.

Osmoadaptation in rhizobia: ectoine-induced salt tolerance

After having shown that ectoine (a tetrahydropyrimidine) displays osmoprotective properties towards Escherichia coli (M. Jebbar, R. Talibart, K. Gloux, T. Bernard, and Blanco, J. Bacteriol. 174:5027-5035, 1992), we have investigated the involvement of this molecule in the osmotic adaptation of Rhizobium meliloti. Ectoine appeared almost as effective as glycine betaine in improving the growth of R. meliloti under adverse osmotic conditions (0.5 M NaCl). Moreover, improvement of growth of rhizobial strains insensitive to glycine betaine was also observed. Ectoine transport proved inducible, periplasmic protein dependent, and, as shown by competition experiments, distinct from the transport of glycine betaine. Medium osmolarity had little effect on the uptake characteristics, since the rate of influx increased from 12 to only 20 nmol min-1 mg of protein-1 when NaCl concentrations were raised from 0 to 0.3 or 0.5 M, with a constant of transport of 80 microM. Natural-abundance 13C-nuclear magnetic resonance and radiolabelling assays showed that ectoine, unlike glycine betaine, is not intracellularly accumulated and, as a consequence, does not repress the synthesis of endogenous compatible solutes (glutamate, N-acetylglutaminylglutamine amide, and trehalose). Furthermore, the strong rise in glutamate content in cells osmotically stressed in the presence of ectoine suggests that, instead of being involved in osmotic balance restoration, ectoine should play a key role in triggering the synthesis of endogenous osmolytes. Hence, we believe that there are at least two distinct classes of osmoprotectants: those such as glycine betaine or glutamate, which act as genuine osmolytes, and those such as ectoine, which act as chemical mediators.

Transport of glycine-betaine by Listeria monocytogenes

Transport of glycine-betaine by Listeria monocytogenes

Transport of glycine-betaine by Listeria monocytogenes.

Uptake of [14C]glycine-betaine by Listeria monocytogenes was stimulated by NaCl with optimal stimulation at 0.4-0.5 M. The glycine-betaine transport system had a Km of 22 microM and a Vmax of 11.7 nmol-1 min-1 mg-1 protein when grown in the absence of NaCl. When grown in the presence of 0.8 M NaCl the Vmax increased to 27.0 nmol-1 min-1 mg-1 protein in 0.8 M NaCl. At NaCl concentrations above 0.5 M the uptake rate of glycine-betaine was reduced. Measurement of intracellular K+ concentrations and fluorescent dye quenching indicated that higher NaCl concentrations also led to a decrease in the electrochemical potential difference across the cytoplasmic membrane. Uptake of glycine was also observed, but this was not stimulated by NaCl.

The Escherichia coli proU promoter element and its contribution to osmotically signaled transcription activation.

The proU operon of Escherichia coli encodes a high-affinity glycine betaine transport system which is osmotically inducible and enables the organism to recover from the deleterious effects of hyperosmotic shock. Regulation occurs at the transcriptional level. KMnO4 footprinting showed that the preponderance of transcription initiated at a single primary promoter region and that proU transcription activation did not occur differentially at alternate promoters in response to various levels of salt shock. Mutational analysis confirmed the location of the primary promoter and identified an extended -10 region required for promoter activity. Specific nucleotides within the spacer, between position -10 and position -35, were important for maximal expression, but every mutant which retained transcriptional activity remained responsive to osmotic signals. A chromosomal 90-bp minimal promoter fragment fused to lacZ was not significantly osmotically inducible. However, transcription from this fragment was resistant to inhibition by salt shock. A mutation in osmZ, which encodes the DNA-binding protein H-NS, derepressed wild-type proU expression by sevenfold but did not alter expression from the minimal promoter. The current data support a model in which the role of the proU promoter is to function efficiently at high ionic strength while other cis-acting elements receive and respond to the osmotic signal.

Adaptation of Escherichia coli to high osmolarity environments: osmoregulation of the high-affinity glycine betaine transport system proU.

A sudden increase in the osmolarity of the environment is highly detrimental to the growth and survival of Escherichia coli and Salmonella typhimurium since it triggers a rapid efflux of water from the cell, resulting in a decreased turgor. Changes in the external osmolarity must therefore be sensed by the microorganisms and this information must be converted into an adaptation process that aims at the restoration of turgor. The physiological reaction of the cell to the changing environmental condition is a highly coordinated process. Loss of turgor triggers a rapid influx of K+ ions into the cell via specific transporters and the concomitant synthesis of counterions, such as glutamate. The increased intracellular concentration of K(+)-glutamate allows the adaptation of the cell to environments of moderately high osmolarities. At high osmolarity, K(+)-glutamate is insufficient to ensure cell growth, and the bacteria therefore replace the accumulated K+ ions with compounds that are less deleterious for the cell's physiology. These compatible solutes include polyoles such as trehalose, amino acids such as proline, and methyl-amines such as glycine betaine. One of the most important compatible solutes for bacteria is glycine betaine. This potent osmoprotectant is widespread in nature, and its intracellular accumulation is achieved through uptake from the environment or synthesis from its precursor choline. In this overview, we discuss the properties of the high-affinity glycine betaine transport system ProU and the osmotic regulation of its structural genes.

Adaptation of Escherichia coli to high osmolarity environments: Osmoregulation of the high-affinity glycine betaine transport system ProU

A sudden increase in the osmolarity of the environment is highly detrimental to the growth and survival of Fscherichia coli and Salmonella typhimurium since it triggers a rapid efflux of water from the cell, resulting in a decreased turgor. Changes in the external osmolarity must therefore be sensed by the microorganisms and this information must be converted into an adaptation process that aims at the restoration of turgor. The physiological reaction of the cell to the changing environmental condition is a highly coordinated process. Loss of turgor triggers a rapid influx of K+ ions into the cell via specific transporters and the concomitant synthesis of counterions, such as glutamate. The increased intracellular concentration of K+-glutamate allows the adaptation of the cell to environments of moderately high osmolarities. At high osmolarity, K+-glutamate is insufficient to ensure cell growth, and the bacteria therefore replace the accumulated K+ ions with compounds that are less d eleterious for the cell’s physiology. These compatible solutes include polyoles such as trehalose, amino acids such as proline, and methyl-amines such as glycine betaine. One of the most important compatible solutes for bacteria is glycine betaine. This potent osmoprotectant is widespread in nature, and its intracellular accumulation is achieved through uptake from the environment or synthesis from its precursor choline. In this overview, we discuss the properties of the high-affinity glycine betaine transport system ProU and the osmotic regulation of its structural genes.

Glycine betaine confers enhanced osmotolerance and cryotolerance on Listeria monocytogenes

Listeria monocytogenes is a gram-positive food-borne pathogen that is notably resistant to osmotic stress and can grow at refrigerator temperatures. These two characteristics make it an insidious threat to public health. Like several other organisms, L. monocytogenes accumulates glycine betaine, a ubiquitous and effective osmolyte, intracellularly when grown under osmotic stress. However, it also accumulates glycine betaine when grown under chill stress at refrigerator temperatures. Exogenously added glycine betaine enhances the growth rate of stressed but not unstressed cells, i.e., it confers both osmotolerance and cryotolerance. Both salt-stimulated and cold-stimulated accumulation of glycine betaine occur by transport from the medium rather than by biosynthesis. Direct measurement of glycine betaine uptake shows that cells transport betaine 200-fold faster at high salt concentration (4% NaCl) than without added salt and 15-fold faster at 7 than at 30 degrees C. The kinetics of glycine betaine transport suggest that the two transport systems are indistinguishable in terms of affinity for betaine and may be the same. Hyperosmotic shock and cold shock experiments suggest the transport system(s) to be constitutive; activation was not blocked by chloramphenicol. A cold-activated transport system is a novel observation and has intriguing implications concerning the physical state of the cell membrane at low temperature.

Identification of a high-affinity glycine betaine transport system in Staphylococcus aureus.

Staphylococcus aureus accumulates proline and glycine betaine when cells are grown at low water activity. In the present study, we have identified a high-affinity glycine betaine transport system in this bacterium. Optimal activity for this transport system was measured in the presence of high NaCl concentrations, but transport activity was not stimulated by high concentrations of other solutes.

Glycine betaine transport by Staphylococcus aureus: evidence for two transport systems and for their possible roles in osmoregulation

The transport of glycine betaine by Staphylococcus aureus was investigated. Two transport systems were found that could be differentiated on the basis of their affinity for glycine betaine and their activation by osmotic pressure. The high-affinity system was relatively independent of osmotic pressure and exhibited a Km of approximately 3 microM. This system was not inhibited by proline, for which a separate high-affinity transport system has been recently discovered. The low-affinity system was activated approximately 35-fold by an increase in osmotic pressure and exhibited a Km of approximately 130 microM for glycine betaine. This system is partially inhibited by excess proline and may be identical to the low-affinity system recently described for proline. Both glycine betaine transport systems are Na(+)-dependent.

Transport of glycine betaine in the extremely haloalkaliphilic sulphur bacterium Ectothiorhodospira halochloris

The effect of osmotic stress on the transport of the compatible solute glycine betaine was examined in Ectothiorhodospira halochloris, an extremely haloalkaliphilic, phototrophic sulphur bacterium. Kinetic data indicated that E. halochloris possesses an active transport system for glycine betaine which is saturable and exhibits Michaelis-Menten kinetics. Experiments with chloramphenicol-treated cells (50 μg per ml of cell suspension) indicated that the transport system is constitutive and might be activated by a change in osmotic pressure. The uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (CCFP) totally blocked uptake at concentrations of 25 μM and 100 μM, respectively. The system was insensitive to the cytochrome oxidase inhibitor sodium azide (1 mM), the respiratory chain inhibitor potassium cyanide (1 mM) and the glycolysis inhibitor iodoacetate (1 mM). The ionophore nigericin (50 μM) had the greatest inhibitory effect, completely abolishing uptake, while monensin (100 μM) caused 80% inhibition. Glycine betaine transport possessed considerable structural specifity: proline betaine was the most effective competitor and trigonelline and dimethylglycine exerted inhibition to a lesser extent. Transport in the dark was at a greatly reduced rate. These results collectively implied that the specific transport of glycine betaine might be driven by the electrochemical proton gradient generated by anaerobic photosynthesis.

Induction of glycinebetaine uptake into Xenopus oocytes by injection of poly(A)+ RNA from renal cells exposed to high extracellular NaCl

Madin-Darby canine kidney (MDCK) cells accumulate glycinebetaine via Na(+)-dependent transport in response to hypertonic stress. When extracellular tonicity is increased by the addition of NaCl, Vmax for glycinebetaine transport increases without an associated change in Km, consistent with an increase in the number of functioning transporters. To test whether increased transport activity results from increased gene expression, we injected poly(A)+ RNA (mRNA) from MDCK cells into Xenopus oocytes and assayed for glycinebetaine uptake in ovo. RNA-induced Na(+)-dependent uptake is observed in oocytes injected with mRNA from cells exposed to high extracellular NaCl, but not in oocytes injected with either water or mRNA from cells maintained in isotonic medium. Unfractionated mRNA induces glycinebetaine uptake in ovo at a rate which is approximately 3-fold higher than in water-injected controls. Size-fractionated mRNA (median size 2.8 kilobases) induces uptake at a rate which is approximately 7-fold higher than controls. Such RNA-induced transport activity in ovo is consistent with heterologous expression of Na(+)/glucinebetaine cotransporters encoded by renal mRNA. Increased transporter mRNA in cells exposed to hypertonicity probably underlies the pattern of expression observed in ovo. This can account for the observed rise in MDCK cell glycinebetaine transport during hypertonic stress.

Osmoregulation in Azospirillum brasilense: glycine betaine transport enhances growth and nitrogen fixation under salt stress

Addition of glycine betaine (1 mM) stimulated aerobic growth of Azospirillum brasilense Sp 7 in the presence of 0.3 M-NaC1. The nitrogenase activity of whole cells was particularly sensitive to salt stress, being almost totally inhibited in the presence of the same concentration of salt. Added glycine betaine strongly enhanced nitrogen fixation activity under salt stress. Under such conditions, maximal nitrogenase activity was obtained at a p(02),value (1 kPa) that inhibits nitrogen fixation activity in the absence of salt. We demonstrated the presence of a high affinity transport system for glycine betaine, with an apparent K(m), of 10 μM. The osmolarity of the medium regulated the activity of the transport system. The maximal transport rates were 4 and 20 nmol min(-l) (mg protein)(-l) in cells grown in low-salt and high-salt medium, respectively. A high intracellular concentration of glycine betaine (480 mM) was observed only at a high osmolarity (0.3 M-NaC1). Glycine betaine uptake was significantly reduced in osmotically shocked cells and a glycine betaine binding activity was detected in the crude periplasmic shock fluid. This suggests a transport mechanism involving a periplasmic glycine betaine binding protein. A. brasilense was unable to use the transported glycine betaine as a carbon- or nitrogen-source, in low- or high-salt medium. Intracellular glycine betaine was not catabolized.

Activation of a new proline transport system in Salmonella typhimurium

Proline uptake can be mediated by three different transport systems in wild-type Salmonella typhimurium: a high-affinity proline transport system encoded by the putP gene and two glycine-betaine transport systems with a low affinity for proline encoded by the proP and proU genes. However, only the PutP permease transports proline well enough t allow growth on proline as a sole carbon or nitrogen source. By selecting for mutations that allow a putP mutant to grow on proline as a sole nitrogen source, we isolated mutants (designated proZ) that appeared to activate a cryptic proline transport system. These mutants enhanced the transport of proline and proline analogs but did not require the function of any of the known proline transport genes. The mutations mapped between 75 and 77.5 min on the S. typhimurium linkage map. Proline transport by the proZ mutants was competitively inhibited by isoleucine and leucine, which suggests that the ProZ phenotype may be due to unusual mutations that alter the substrate specificity of the branched-chain amino acid transport system encoded by the liv genes.

Uptake of glycine betaine and its analogues by bacteroids of Rhizobium meliloti

Bacteroids isolated from alfalfa nodules induced by Rhizobium meliloti 102F34 transported glycine betaine at a constant rate for up to 30 min. Addition of sodium salts greatly increased the uptake activity, whereas other salts or non-electrolytes had less effect. The apparent Km for glycine betaine uptake was 8.3 microM and V was about 0.84 nmol min-1 (mg protein)-1 in the presence of 200 mM-NaCl which gave maximum stimulation of the transport. Supplementing bacteroid suspensions with various energy-yielding substrates, or ATP, did not increase glycine betaine uptake rates. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), and the respiratory inhibitor potassium cyanide strongly inhibited glycine betaine uptake, but arsenate was totally inactive. Glycine betaine transport showed considerable structural specificity: choline, proline betaine, gamma-butyrobetaine and trigonelline did not competitively inhibit the system, although choline and proline betaine were transported by bacteroids. Both a high-affinity activity and a low-affinity activity were found for choline uptake. These osmoprotective compounds might have a significant role in the maintenance of nitrogenase activity in bacteroids subjected to salt stress.

Molecular characterization of the proU loci of Salmonella typhimurium and Escherichia coli encoding osmoregulated glycine betaine transport systems.

The proU loci of Salmonella typhimurium and Escherichia coli encode high-affinity glycine betaine transport systems which play an important role in survival under osmotic stress. Transcription of the proU locus is tightly regulated by osmolarity and this regulation appears to be mediated by osmotically induced changes in DNA supercoiling. In order to study the regulatory mechanisms involved we have cloned and characterized the proU locus of S. typhimurium by an in vivo transductional procedure. The locus is shown to consist of at least three genes, designated proVWX, cotranscribed as a single operon. The first gene in the operon encodes a protein sharing considerable sequence identity with ATP-binding proteins from other periplasmic transport systems. Unexpectedly, the highly expressed periplasmic glycine betaine binding protein was found to be encoded by a distal gene, proX, in the operon. The operon has no significant internal promoters but is expressed from a single osmoregulated promoter whose transcription start site has been mapped. The proU promoter of E. coli has also been sequenced and the transcription start site shown to be similar to that of S. typhimurium. Evidence is presented which suggests that, besides de novo glycine betaine uptake, an important function of ProU may be the recapture and recycling of other osmolytes that leak from the cell.

Cloned structural genes for the osmotically regulated binding-protein-dependent glycine betaine transport system (ProU) of Escherichia coli K-12.

The proU locus of Escherichia coli encodes a high-affinity, binding-protein-dependent transport system (ProU) for the osmoprotectant glycine betaine. We cloned this locus into both low-copy-number lambda vectors and multicopy plasmids and demonstrated that these clones restore osmotically controlled synthesis of the periplasmic glycine betaine binding protein (GBBP) and the transport of glycine betaine in a delta (proU) strain. These clones allowed us to investigate the influence of osmolarity on ProU transport activity independent of the osmotically controlled expression of proU. ProU activity was strongly stimulated by a moderate increase in osmolarity and was partially inhibited by high osmolarity. This activity profile differs from the profile of the osmotically regulated proU expression. The proU locus is organized in an operon and the position of the structural gene (proV) for GBBP is defined using a minicell system. We determined that at least three proteins (in addition to GBBP) are encoded by the proU locus. We also investigated the permeation of glycine betaine across the outer membrane. At low substrate concentration (0.7 microM), permeation of glycine betaine was entirely dependent on the OmpF and OmpC porins.

A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli

The proU locus encodes an osmotically inducible glycine betaine transport system that is important in the adaptation to osmotic stress. We present evidence that DNA supercoiling plays a key role in the osmotic induction of proU transcription. An increase in extracellular osmolarity increases in vivo DNA supercoiling, and the expression of proU is highly sensitive to these changes. Furthermore, topA mutations can mimic an increase in osmolarity, facilitating proU expression even in media of low osmolarity in which it is not normally expressed. Selection for trans-acting mutations that affect proU expression has yielded only mutations that alter DNA supercoiling, either in topA or a new genetic locus, osmZ, which strongly influences in vivo supercoiling. Mutations in osmZ are highly pleiotropic, affecting expression of a variety of chromosomal genes including ompF, ompC, fimA, and the bgl operon, as well as increasing the frequency of site-specific DNA inversions that mediate fimbrial phase variation.

Glycine betaine reverses the effects of osmotic stress on DNA replication and cellular division in Escherichia coli

The accumulation of glycine betaine to a high internal concentration by Escherichia coli cells in high osmolarity medium restores, within 1 h, a subnormal growth rate. The experimental results support the view that cell adaptation to high osmolarity involves a decrease in the initiation frequency of DNA replication via a stringent response; in contrast, glycine betaine transport and accumulation could suppress the stringent response within 1-2 min and restore a higher initiation frequency. High osmolarity also triggers the cells to lengthen, perhaps via an inhibition of cellular division; glycine betaine also reverses this process. It is inferred that turgor could control DNA replication and cell division in two separate ways. Glycine betaine action is not mediated by K+ ions as the internal level of K+ ions is not modified significantly following glycine betaine accumulation.

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.