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PubMed2004

BetP of Corynebacterium glutamicum, a transporter with three different functions: betaine transport, osmosensing, and osmoregulation.

In order to circumvent deleterious effects of hypo- and hyperosmotic conditions in its environment, Corynebacterium glutamicum has developed a number of mechanisms to counteract osmotic stress. The first response to an osmotic upshift is the activation of uptake mechanisms for the compatible solutes betaine, proline, or ectoine, namely BetP, EctP, ProP, LcoP and PutP. BetP, the most important uptake system responds to osmotic stress by regulation at the level of both protein activity and gene expression. BetP was shown to harbor three different properties, i.e. catalytic activity (betaine transport), sensing of appropriate stimuli (osmosensing) and signal transduction to the catalytic part of the carrier protein which adapts its activity to the extent of osmotic stress (osmoregulation). BetP is comprised of 12 transmembrane segments and carries N- and C-terminal domains, which are involved in osmosensing and/or osmoregulation. Recent results on molecular properties of these domains indicate the significance of particular amino acids within the terminal 25 amino acids of the C-terminal domain of BetP for the process of osmosensing and osmoregulation.

Bacterial Proteins↗
PubMed2000

Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane.

An osmoregulated ABC transporter (OpuA) with novel structural features has been identified that responds to water stress. This glycine betaine transport system consists of an ATP-binding/hydrolyzing subunit (OpuAA) and a protein (OpuABC) that contains both the translocator and the substrate-binding domain. The components of OpuA have been overexpressed, purified, and functionally incorporated into liposomes with an ATP-regenerating system in the vesicle lumen. A transmembrane osmotic gradient (outside hyperosmotic relative to the inside) of both ionic and nonionic compounds was able to osmotically activate OpuA in the proteoliposomal system. Hypoosmotic medium conditions inhibited the basal activity of the system. The data show that OpuAA and OpuABC are sufficient for osmoregulated transport, indicating that OpuA can act both as osmosensor and osmoregulator. Strikingly, OpuA could also be activated by low concentrations of cationic and anionic amphipaths, which interact with the membrane. This result indicates that activation by a transmembrane osmotic gradient is mediated by changes in membrane properties/protein-lipid interactions.

ATP-Binding Cassette Transporters↗
PubMed2000

Osmoregulated periplasmic glucans in Proteobacteria.

Large amounts of osmoregulated periplasmic glucans (OPGs) are found in the periplasmic space of Proteobacteria. Four families of OPGs are described on the basis of structural features of the polyglucose backbone. Depending on the species considered, OPGs can be modified to various extent by a variety of substituents. Genes governing the backbone synthesis are identified in a limited number of species. They belong to three unrelated families. OPG synthesis is subject to osmoregulation and feedback control. Osmoregulation can occur at the level of gene expression and/or at the level of enzyme activity. Mutants defective in OPG synthesis have a highly pleiotropic phenotype, indicative of an overall alteration of their envelope properties. Mutants of this kind were obtained as attenuated or avirulent derivatives of plant or animals pathogen. Thus, OPGs appear to be important intrinsic components of the Gram-negative bacterial envelope, which can be essential in extreme conditions found in nature, and especially when bacteria must interact with an eukaryotic host.

Gene Expression Regulation, Bacterial↗
PubMed2006

Cyclic AMP-dependent osmoregulation of crp gene expression in Escherichia coli.

We have found that the cyclic AMP (cAMP) receptor protein (CRP)-cAMP regulatory complex in Escherichia coli is subject to osmoregulation at the level of crp gene expression. This osmoregulation was lost in a cya mutant strain but could be restored by external addition of cAMP, suggesting that the intracellular level of cAMP is a key factor in the osmoregulation of CRP. The ability of the cell to maintain optimal CRP activity was essential for the growth and survival of the bacteria under low-osmolarity conditions as shown by studies with different crp mutant alleles. A suppressor mutant with a novel amino acid substitution (L124R) in CRP showed restored growth at low osmolarity. CRP(L124R) was not activated by cAMP and was shown to be dominant negative over the wild type. Our findings suggest that the fine-tuning of the CRP activity may be critical for bacterial viability and adaptability to changing osmotic conditions.

Cyclic AMP↗
PubMed1984

Promoter exchange between ompF and ompC, genes for osmoregulated major outer membrane proteins of Escherichia coli K-12.

Expression of the ompF and ompC genes coding for major outer membrane proteins OmpF and OmpC is regulated in opposite directions by medium osmolarity. Chimera genes were constructed by a reciprocal exchange of the promoter-signal sequence region between the two genes. The chimera gene construction was designed so that the proteins synthesized by these genes were essentially the same as the OmpC and OmpF proteins. Studies with the chimera genes demonstrated that the osmoregulation of the OmpF-OmpC synthesis was promoter dependent. They also showed that cells grew normally even when the osmoregulation took place in opposite directions. The effects of the ompR2 and envZ mutations, which suppress ompC and ompF expression, respectively, also became reversed. The reduced expression was still subject to the promoter-controlled osmoregulation. Based on these observations, the mechanism of regulation of the ompF-ompC gene expression and its physiological importance are discussed.

Bacterial Outer Membrane Proteins↗
PubMed1984

Mutation causing reverse osmoregulation of synthesis of OmpF, a major outer membrane protein of Escherichia coli.

Supplementation of growth media with high concentrations of substances like sucrose results in the induction of OmpC synthesis and the suppression of OmpF synthesis. We isolated a novel mutant in which OmpF synthesis is in the opposite direction from normal osmoregulation. By transductional mapping, the mutation was localized at 75 min between malA and aroB on the Escherichia coli chromosome map where the ompR-envZ region is. The mutation was suppressed by a plasmid carrying the ompR gene but not by a plasmid carrying the envZ gene alone. The mutation also resulted in the almost complete suppression of OmpC synthesis. However, the remaining OmpC synthesis was osmoregulated normally. Based on these observations, the mechanism of osmoregulation of OmpF-OmpC synthesis is discussed.

Bacterial Outer Membrane Proteins↗
PubMed1985

Uncoupling of osmoregulation of the Escherichia coli K-12 ompF gene from ompB-dependent transcription.

The expression of the genes for the Escherichia coli K-12 outer membrane proteins, ompF and ompC, is subject to osmoregulation and responds to changes in the osmolarity of the growth medium. The transcription of these genes is dependent on the products of the regulatory locus ompB (comprising the genes ompR and envZ). The native promoter of ompF was replaced with an inducible lpp promoter to eliminate this transcriptional dependence of ompF expression on ompB. As a result, it was possible for the OmpF protein to be produced in an ompB mutant strain that does not normally express ompF. Surprisingly, the expression of ompF under the lpp promoter was still osmoregulated not only in the ompB+ strain but also in two ompB strains tested. These results indicate the involvement of a factor(s) besides the ompR and envZ gene products in the osmoregulation of OmpF production. This factor may interact with a sequence downstream of the ompF promoter. In addition, we show that the expression of ompF under the lpp promoter has no direct effect on ompC expression.

Bacterial Outer Membrane Proteins↗
PubMed1990

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.

Betaine↗
PubMed1991

Characterization of mutations affecting the osmoregulated proU promoter of Escherichia coli and identification of 5' sequences required for high-level expression.

Expression of the Escherichia coli proU operon, which encodes an efficient uptake system for the osmoprotectant glycine betaine, is strongly increased in cells grown at high osmolarity. We isolated 182 independent spontaneous mutants with elevated expression of the chromosomal phi(proV-lacZ) (Hyb2) fusion at low osmolarity. Genetic analysis demonstrated that eight of these mutant strains carried mutations closely linked to the fusion, whereas all others carried mutations that appeared to be in osmZ. All of the mutations resulted in increased but still osmoregulated expression of the phi(proV-lacZ)(Hyb2) fusion. The proU-linked mutants carried an identical point mutation (proU603) which changes the -35 sequence of the proU promoter from TTGCCT to TTGACT and thereby increases the homology of the -35 region to the consensus sequence (TTGACA) of E. coli promoters. We also selected for mutants with decreased expression of the plasmid pOS7-encoded phi(proV-lacZ)(Hyb2) fusion and isolated a plasmid with an IS1 insertion (proU607) between the proU -10 and -35 regions. This insertion creates a hybrid promoter and drastically reduces expression of the fusion but does not abolish its osmotic regulation. Deletion analysis of chromosomal sequences 5' to the proU promoter revealed that sequences located approximately 200 bp upstream of the -35 region were required for high-level expression. Removal of these sequences resulted in a 10-fold decline of phi(proV-lacZ)(Hyb2) expression. Osmotic regulation was retained in deletion constructs carrying just 19 bp of chromosomal DNA 5' of the promoter, showing that no sequences further upstream are required for the proper osmoregulation of proU transcription. Experiments with himA and fis mutant strains indicated that the IHF and FIS proteins are not required for the normal osmoregulation of proU expression.

Bacteriophage lambda↗
PubMed1994

micF antisense RNA has a major role in osmoregulation of OmpF in Escherichia coli.

micF RNA, produced from a multicopy plasmid, was originally shown to be a major factor in negative osmoregulation of the OmpF outer membrane protein in Escherichia coli. However, subsequent experiments with a micF deletion strain suggested that chromosomal micF RNA was not a key component in this process. We report here that micF RNA is essential for the reduction in OmpF levels in cells grown in media of low-to-intermediate levels of osmolarity. Under these conditions, the amount of OmpF was reduced up to 60% in the parent strain while OmpF levels were not altered in the micF deletion mutant. In medium of higher osmolarity, OmpF synthesis was strongly inhibited in both strains. RNA measurements showed that micF RNA levels rose rapidly in cells grown in low-to-intermediate levels of osmolarity concomitant with the reduction in OmpF protein, while ompF mRNA decreased strongly only during high-osmolarity conditions. Taken together, these results strongly suggest that the negative osmoregulation of OmpF at low-to-intermediate osmolarity levels requires micF RNA and that this is masked at higher osmolarity by the known strong inhibition of OmpF transcription by OmpR. Results consistent with this model were also obtained by using procaine, a compound reported to inhibit ompF expression by a mechanism very similar to that involved in osmoregulation.

Bacterial Outer Membrane Proteins↗
PubMed2003

Osmoregulation of vasopressin release and gene transcription under acute and chronic hypovolemia in rats.

Although acute decreases in plasma volume are known to enhance the osmotically induced arginine vasopressin (AVP) release, it is unclear whether there is also such interaction at the level of gene transcription. It also remains to be established how sustained changes in plasma volume affect the osmoregulation. In this study, we examined how acute and chronic decreases in blood volume affected the osmoregulation of AVP release and gene transcription in rats. Acute hypovolemia was induced by intraperitoneal injection of polyethylene glycol (PEG), and chronic hypovolemia was induced by 3 days of water deprivation (WD) or 12 days of salt loading (SL). Rats were injected with isotonic or hypertonic saline, and plasma AVP levels and AVP heteronuclear (hn)RNA expression in the supraoptic and paraventricular nuclei, an indicator of gene transcription, were examined in relation to plasma osmolality in each group. Plasma AVP levels were correlated with plasma Na levels in all groups. Whereas the regression lines relating plasma AVP to Na were almost identical among control, WD, and SL groups, the thresholds of plasma Na for AVP release were significantly decreased only in the PEG group. AVP hnRNA levels were also correlated with plasma Na levels in control and PEG groups, and the thresholds were significantly decreased in the PEG group. In contrast, there was no significant correlation of AVP hnRNA and plasma Na levels in WD and SL groups. Thus it was demonstrated that acute and chronic reduction in plasma volume affected the osmoregulation of AVP release and gene transcription in different ways.

Acute Disease↗
PubMed1976

DDE feeding and plasma osmoregulation in ducks, guillemots, and puffins.

To assess the possibility that organochlorine pesticide disruption of osmoregulation is responsible for recent large kills of young seabirds, we have studied the effects of DDE feeding (10-250 ppm) on plasma osmoregulation and nasal gland function in the following species: mallared and white Pekin ducks (both Anas platyrhynchos), black guillemot (Cepphus grylle), and common (Fratercula arctica). Other investigators have recently reported that dietary DDE (10-1,000 ppm) inhibits nasal gland secretion in freshwater-maintained mallards; our initial experiments with white Pekins showed no such inhibition during either freshwater or seawater maintainance. Moreover, DDE had minimal effects on plasma electrolyte levels and total nasal gland Na-K-ATPase activities in all species studied. Liver DDE levels in experimental ducks and guillemots were comparable to those reported for seabirds found dead after kills; levels in starved experimental puffins were much higher. Thus DDE at environmental levels does not affect osmoregulation or nasal gland Na-K-ATPase either in ducks or in two species of oceanic birds.

Adenosine Triphosphatases↗
PubMed1987

Osmoregulation and control of vasopressin secretion in healthy humans.

The functional characteristics of osmoregulated vasopressin secretion can be defined in terms of an osmotic threshold for its release and a sensitivity of the osmoreceptor and vasopressin-secreting unit. Osmotically stimulated thirst has features similar to osmoregulated vasopressin. There are wide individual variations in the functional characteristics of both thirst and vasopressin release in healthy humans, probably genetic in origin. The influence of aging appears to enhance the sensitivity of vasopressin secretion but blunt thirst appreciation. Yet in many physiological situations changes in osmoregulated vasopressin release and thirst occur in parallel. The fall in plasma osmolality associated with human pregnancy is accounted for entirely by a lowering of the osmotic thresholds for thirst and vasopressin release. Similar but less marked alterations accompany the ovulatory luteal phase of the menstrual cycle. A major nonosmotic stimulus to vasopressin secretion is hypotension and/or hypovolemia, mediated by high- (carotid sinus) and low- (left atrial) pressure receptors. Circulating catecholamines influence the release of vasopressin by alpha- and beta-adrenergic pathways. Drinking by hypertonic humans provides immediate reduction in thirst and vasopressin secretion probably mediated by pathways from the oropharynx. The modest but variable rise in plasma vasopressin in response to hypoglycemia appears to be due to cellular neuroglycopenia and is independent of parasympathetic pathways. Although osmotic and hemodynamic stimuli to vasopressin release do not act independently of each other, the precise subtle interactions between them and other nonosmotic stimuli remain to be clarified.

Drinking↗
PubMed1989

Effect of blood glucose concentration on osmoregulation in diabetes mellitus.

Poorly controlled insulin-dependent diabetes mellitus is associated with considerable elevations of plasma vasopressin concentrations, although well-controlled diabetics have normal osmoregulated thirst and vasopressin release. We studied the effect of blood glucose concentration on osmoregulated thirst and vasopressin secretion in insulin-dependent diabetes mellitus. Blood glucose was maintained overnight, and for the duration of the study, in either the euglycemic (4-5 mmol/l) or hyperglycemic (10-12 mmol/l) range, and patients underwent infusion of hypertonic (855 mmol/l) sodium chloride solution. Plasma sodium was lower during the hyperglycemic study, but elevation in plasma sodium concentration by infusion of saline caused progressive linear increases in both thirst and plasma vasopressin concentrations in both studies. Linear regression analysis defined lowered plasma sodium thresholds for both thirst appreciation and vasopressin release during the hyperglycemic study, although the sensitivity of the osmoreceptors remained unchanged. Analysis of the data in terms of plasma osmolality, corrected for the increase in blood glucose in the hyperglycemic study, revealed no differences in the osmotic thresholds for thirst or vasopressin release; sensitivity of the osmoreceptors also remained the same. Drinking abolished thirst and lowered plasma vasopressin concentrations before major changes in plasma sodium were observed. These results show that insulin-dependent diabetic patients osmoregulate appropriately when moderately hyperglycemic but that the threshold plasma sodium for vasopressin secretion and thirst appreciation is lowered by an unknown mechanism.

Adult↗
PubMed1990

Nonosmotic stimuli alter osmoregulation in patients with spinal cord injury.

Studies on two quadriplegic patients who developed severe hyponatremia during episodes of acute respiratory distress were performed to determine whether differences in osmoregulation of vasopressin release could be identified in these patients compared to other quadriplegic subjects previously studied in a similar manner. Both patients were clinically stable and normonatremic, with no signs or symptoms of respiratory distress, when the studies were performed. However, both exhibited evidence of hemodynamic instability in the sitting posture. Linear regression analysis of the plasma vasopressin/plasma osmolality (Pavp:Posm) relationship during infusions of 0.85 M sodium chloride showed no significant differences in either the slope (sensitivity) or abscissal intercept (osmotic threshold) of this relationship compared to that of other quadriplegic subjects when the patients were supine. In contrast, when the patients were studied in the sitting posture there was a marked shift in the relationship of Pavp:Posm indicative of increased sensitivity and reduced osmotic threshold for vasopressin release. The slopes of the Pavp:Posm relationships were 0.249 and 0.178 for the two patients, respectively, compared to 0.092 +/- 0.03 ( +/- SD) for previously studied quadriplegic subjects. Oral water-loading studies performed on one patient revealed marked impairment of urine-diluting ability and free water clearance in the sitting posture compared with observations in similar studies performed when the patient was supine. Impairment of renal water excretion could not be attributed to an effect of vasopressin, which was reduced to unquantifiable levels by water loading. These studies have shown that hemodynamic stress related to autonomic dysfunction in quadriplegic patients may result in marked alteration of osmoregulation of vasopressin release in more severely affected individuals. Such altered osmoregulation, which may also be associated with vasopressin-independent impairment of renal water excretion in the sitting posture, may be a predisposing factor in the development of hyponatremia, especially in the presence of other potent nonosmotic stimuli.

Aged↗
PubMed2006

Ontogeny of osmoregulation in embryos of intertidal crabs (Hemigrapsus sexdentatus and H. crenulatus, Grapsidae, Brachyura): putative involvement of the embryonic dorsal organ.

This study examined whether the existence of hyperosmotic internal fluids in embryos of euryhaline crabs (Hemigrapsus sexdentatus and H. crenulatus) in dilute seawater reflects osmotic isolation due to impermeability of the egg envelope, as proposed for other decapods, or active osmoregulation. When ovigerous crabs with eggs at gastrula stage were transferred from 100% seawater (osmolality 1000 mmol kg(-1)) to 50% seawater, embryogenesis and hatching of zoea were completed normally, but were delayed. Hatching failed if the transfer to 50% seawater occurred before gastrulation, and embryogenesis was abnormal in 25% seawater. In 100% seawater, embryos at all stages were internally hyperosmotic by 150-250 mmol kg(-1). On transfer to 50% seawater, osmolality initially decreased but remained 200-350 mmol kg(-1) hyperosmotic to the medium for several weeks until hatching. High efflux rates of tritium-labelled water (t((1/2)) 16-75 min) and (22)Na (t(1/2) 109-374 min) from H. crenulatus embryos were inconsistent with the osmotic isolation hypothesis. It is concluded that post-gastrula embryos were actively hyper-osmoregulating. The diffusional water permeability of the embryos decreased during development while the sodium efflux rate increased 10-fold. Very rapidly exchanging pools of water and sodium (t(1/2) a few seconds to minutes) probably corresponded to peri-embryonic fluid and implied that the egg envelope was a negligible barrier to diffusion of water and salts. Higher Na(+)/K(+)-ATPase activities in late embryos of H. crenulatus incubated in 50% seawater than in embryos incubated in full strength seawater were consistent with an acclimation response. An area of the embryonic surface located over the yolk in the region of the embryonic dorsal organ stained with AgNO(3). Staining appeared at gastrulation, persisted throughout development and was lost at hatching. Deposits of AgCl between the outer and inner membranes, identified by X-ray microanalysis, suggest that the dorsal organ was a site of chloride extrusion. A model for osmoregulation in post-gastrula embryos is proposed: osmotic uptake of water is balanced by excretion of water and salts via the dorsal organ and salt loss is balanced by active uptake over the general embryonic ectoderm.

Animals↗
PubMed1993

Osmoregulation in the parasitic nematode Pseudoterranova decipiens.

When subjected to hyper- or hypo-osmotic stress at 5 degrees C for 24 h, third-stage larvae of the parasitic nematode Pseudoterranova decipiens do not exhibit changes in mass or in the osmotic pressure of the pseudocoelomic fluid. Immersion in solutions containing 3H2O demonstrates that exchange with the water in the pseudocoelomic fluid is substantially complete within 24 h. Sacs composed of cylinders of body wall without the intestine and pseudocoelomic fluid do not gain weight when immersed for 24 h in hypotonic medium. Metabolic poisons abolish the ability of whole worms and sacs to maintain their weight when immersed in hypotonic media. These observations support the conclusion that the nematode is capable of at least short-term osmoregulation and that the site of osmoregulation is the body wall. The observations that more fluid is passed from the anus in some hypo-osmotically stressed worms and that worms ligatured at the tail exhibit a small increase in mass when exposed to hypo-osmotic conditions may indicate that the intestine plays a minor and subsidiary role in osmoregulation.

Anal Canal↗
PubMed1999

Osmoregulation in Drosophila melanogaster selected for urea tolerance.

Animals may adapt to hyperosmolar environments by either osmoregulating or osmoconforming. Osmoconforming animals generally accumulate organic osmolytes including sugars, amino acids or, in a few cases, urea. In the latter case, they also accumulate 'urea-counteracting' solutes to mitigate the toxic effects of urea. We examined the osmoregulatory adaptation of Drosophila melanogaster larvae selected to live in 300 mmol l(-)(1) urea. Larvae are strong osmoregulators in environments with high NaCl or sucrose levels, but have increased hemolymph osmolarity on urea food. The increase in osmolarity on urea food is smaller in the selected larvae relative to unselected control larvae, and their respective hemolymph urea concentrations can account for the observed increases in total osmolarity. No other hemolymph components appear to act as urea-counteractants. Urea is calculated to be in equilibrium across body compartments in both selected and control larvae, indicating that the selected larvae are not sequestering it to lower their hemolymph osmolarity. The major physiological adaptation to urea does not appear to involve increased tolerance or improved osmoregulation per se, but rather mechanisms (e.g. metabolism, decreased uptake or increased excretion) that reduce overall urea levels and the consequent toxicity.

Adaptation, Physiological↗