Low-osmolarity Medium Research Articles

The ndvB locus of Rhizobium meliloti encodes a 319-kDa protein involved in the production of beta-(1----2)-glucan.

The ndvB locus of Rhizobium meliloti was sequenced and found to encode a 319-kDa protein involved in the production of beta-(1----2)-glucan. Transposon Tn5 mutagenesis revealed that a large portion of the downstream half of this gene is not essential for symbiosis but is required for optimal production of beta-(1----2)-glucan. A high molecular weight inner membrane protein, believed to be the ndvB gene product, was absent from two different upstream ndvB::Tn5 mutants. This protein could be labeled in vitro with UDP-[U-14C]glucose in the wild type but not in the symbiotically defective mutants. Inner membrane preparations from the symbiotically competent downstream mutants labeled less well than did those from wild type with UDP-[U-14C] glucose and did not show distinct bands after polyacrylamide gel electrophoresis and fluorography, suggesting that C-terminal truncations of NdvB might affect the stability of this molecule. These downstream mutants had reduced amounts of periplasmic beta-(1----2)-glucan and exhibited several vegetative defects seen also in the upstream mutants. These included alterations in phage and antibiotic sensitivity, in motility, and in growth in low osmolarity media. Bacteroids produced by two of the downstream mutants were morphologically abnormal, indicating that ndvB is involved not only in invasion but also in bacteroid development.

Osmoregulation in Rhizobium meliloti : Production of Glutamic Acid in Response to Osmotic Stress

Rhizobium meliloti, like many other bacteria, accumulates high levels of glutamic acid when osmotically stressed. The effect was found to be proportional to the osmolarity of the growth medium. NaCl, KCI, sucrose, and polyethylene glycol elicited this response. The intracellular levels of glutamate and K began to increase immediately when cells were shifted to high-osmolarity medium. Antibiotics that inhibit protein synthesis did not affect this increase in glutamate production. Cells growing in conventional media at any stage in the growth cycle could be suspended in medium causing osmotic stress and excess glutamate accumulated. The excess glutamate did not appear to be excreted, and the intracellular level eventually returned to normal when osmotically stressed cells were suspended in low-osmolarity medium. A glt mutant lacking glutamate synthase and auxotrophic for glutamate accumulated excess glutamate in response to osmotic stress. Addition of isoleucine, glutamine, proline, or arginine stimulated glutamate accumulation to wild-type levels when the mutant cells were suspended in minimal medium with NaCl to cause osmotic stress. In both wild-type and mutant cells, inhibitors of transaminase activity, including azaserine and aminooxyacetate, reduced glutamate levels. The results suggest that the excess glutamate made in response to osmotic stress is derived from degradation of amino acids and transamination of 2-ketoglutarate.

DNA supercoiling in Escherichia coli: topA mutations can be suppressed by DNA amplifications involving the tolC locus

The level of DNA supercoiling is crucial for many cellular processes, including gene expression, and is determined, primarily, by the opposing actions of two enzymes: topoisomerase I and DNA gyrase. Escherichia coli strains lacking topoisomerase I (topA mutants) normally fail to grow in the absence of compensatory mutations which are presumed to relax DNA. We have found that, in media of low osmolarity, topA mutants are viable in the absence of any compensatory mutation, consistent with the view that decreased extracellular osmolarity causes a relaxation of cellular DNA. At higher osmolarity most compensatory mutations, as expected, are in the gyrA and gyrB genes. The only other locus at which compensatory mutations arise, designated toc, is shown to involve the amplification of a region of chromosomal DNA which includes the tolC gene. However, amplification of tolC alone is insufficient to explain the phenotypes of toc mutants. tolC insertion mutations alter the distribution of plasmid topoisomers in vivo. This effect is probably indirect, possibly a result of altered membrane structure and an alteration in the cell's osmotic barrier. As tolC is a highly pleiotropic locus, affecting the expression of many genes, it is possible that some of the TolC phenotypes are a direct result of this topological change. The possible relationship between toc and tolC mutations, and the means by which tolC mutations might affect DNA supercoiling, are discussed.

Role of protein F in maintaining structural integrity of the Pseudomonas aeruginosa outer membrane

To investigate the functional role of protein F of the outer membrane of Pseudomonas aeruginosa, we isolated mutants devoid of protein F, and the defective gene was transferred to a wild-type strain by plasmid FP5-mediated conjugation. Chemical analyses of the protein F-deficient outer membrane revealed that the amount of outer membrane protein was reduced to 72 to 74% of that of the protein F-sufficient strain and that lipopolysaccharides and phospholipids increased to 117 to 123% and 135 to 136%, respectively. The mutants and the transconjugant showed the following characteristics: (i) growth rates of protein F-deficient strains in low-osmolarity medium (e.g., L broth containing 0.1% NaCl) were less than 1/10 the rate of the protein F-sufficient strain; (ii) protein F-deficient cells were rounded, and the outer membrane formed large protruded blebs; and (iii) the outer membrane became physically fragile, since a significant amount of periplasmic proteins leaked out and the cells became highly sensitive to osmotic shock. The results suggested that protein F plays an important role in morphogenesis and in maintaining the integrity of the outer membrane. Determination of the diffusion rates of saccharides and beta-lactam antibiotics showed that the protein F-deficient outer membrane had no detectable transport defect compared with the protein F-sufficient outer membrane. The MICs of antibiotics for the protein F-deficient strains were nearly identical to those for the protein F-sufficient strain.

Metabolism of periplasmic membrane-derived oligosaccharides by the predatory bacterium Bdellovibrio bacteriovorus 109J.

Membrane-derived oligosaccharides (MDO), a class of osmotically active carbohydrates, are the major organic solutes present in the periplasm of Escherichia coli and many other gram-negative bacteria when cells are grown in a medium of low osmolarity. Analyses of growing cells of Bdellovibrio bacteriovorus, a gram-negative predator of other bacteria, have confirmed that they also synthesize a characteristic MDO-like class of oligosaccharides. The natural growth environment of bdellovibrios is the periplasm of other gram-negative bacteria. Because of this location, prey cell MDO constitute a potential source of organic nutrients for growing bdellovibrios. Using cells of E. coli whose MDO were 3H labeled, we examined the extent to which B. bacteriovorus 109J metabolizes these prey cell components. Interestingly, there was neither significant degradation nor incorporation of prey cell MDO by bdellovibrios during the course of their intracellular growth. In fact, bdellovibrios had little capability either to degrade extracellular MDO that was made available to them or to transport glucose, the major monomeric constituent of prey cell MDO. Instead, periplasmic MDO were irreversibly lost to the extracellular environment during the period of bdellovibrio attack and penetration. Thus, although prey cell periplasmic proteins are retained, other important periplasmic components are released early in the bdellovibrio growth cycle. The loss of these MDO may aid in the destabilization of the prey cell plasma membrane, increasing the availability of cytoplasmic constituents to the periplasmic bdellovibrio.

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.

The Role of Divalent Cations and Ionic Strength in the Osmotic Sensitivity of Glutamate Oxidation in Oyster Gill Mitochondria

ABSTRACT The biochemical mechanism responsible for the osmotic sensitivity of glutamate oxidation in oyster (Crassostrea virginica Gmelin) gill mitochondria was examined. The relative roles of osmotically induced changes in matrix divalent cation concentration and ionic strength were determined. The calcium-magnesium ionophore A23187 inhibited glutamate oxidation in both low-(40% of control rate) and high-(25 % of control rate) osmolarity media. Addition of MgCl2 reversed A23187 inhibition, but at each MgCl2 concentration (1–5 mmoll−1) the rate of glutamate oxidation in the high-osmolarity medium was about 45 % that in the low-osmolarity medium. EDTA (4 mmol l−1) stimulated glutamate oxidation at both osmolarities (more pronounced at high osmolarity), removing the dependency on assay medium osmolarity. The stimulation in response to EDTA was correlated with mitochondrial swelling, which required the presence of monovalent cations for maximal effect. These data suggest that osmotically induced changes in matrix [Mg2+] or [Ca2+] are not responsible for the osmotic sensitivity of glutamate oxidation by oyster gill mitochondria in vitro. Changes in extramitochondrial [Mg2+] may be involved in regulating the rate of glutamate oxidation in vivo, through effects on mitochondrial volume. The maximal rate of glutamate dehydrogenase was not very sensitive to the ionic strength of the assay medium. The rate of electron transport was highly dependent on ionic strength, with the maximal rate occurring in 50 mmol l−1 salt. The osmotic sensitivity of glutamate oxidation in oyster gill mitochondria is apparently due to the effects of changes in matrix ionic strength on the rate of electron transport.

Transfer of phosphoethanolamine residues from phosphatidylethanolamine to the membrane-derived oligosaccharides of Escherichia coli.

The membrane-derived oligosaccharides (MDO) of Escherichia coli are periplasmic constituents composed of glucose residues linked by beta-1,2 and beta-1,6 glycosidic bonds. MDO are substituted with phosphoglycerol, phosphoethanolamine, and succinic acid moieties. The phosphoglycerol residues present on MDO are derived from phosphatidylglycerol (B. J. Jackson and E. P. Kennedy, J. Biol. Chem. 258:2394-2398, 1983), but evidence as to the source of the phosphoethanolamine residues has been lacking. We now report that phosphatidylethanolamine, exogenously added to intact cells of E. coli, provides a source of phosphoethanolamine residues that are transferred to MDO. The biosynthesis of phosphoethanolamine-labeled MDO is osmotically regulated, with maximum synthesis occurring during growth in medium of low osmolarity.

Regulation of diacylglycerol kinase biosynthesis in Escherichia coli. A trans-acting dgkR mutation increases transcription of the structural gene.

The mechanism of a trans-acting mutation, dgkR1, which causes a 7-fold elevation of diacylglycerol kinase activity in membranes (Raetz, C. R. H., Kantor, G. D., Nishijima, M., and Jones, M. L. (1981) J. Biol. Chem. 256, 2109-2112) was investigated by direct measurement of diacylglycerol kinase polypeptide by high performance liquid chromatography and by construction of fusions of the dgkA promoter to beta-galactosidase and galactokinase. The dgkR1 mutation was demonstrated to act by increasing the transcription of the structural gene for diacylglycerol kinase, dgkA. Additionally, sn-glycerol-3-phosphate acyltransferase activities were shown to be decreased 30-50% in membranes from dgkR1 mutant strains. Increased diacylglycerol levels occurred when cells were grown on low osmolarity media. This did not affect dgkA expression. In a dgkR+ background, enhanced expression of sn-1,2-diacylglycerol kinase activity in cells containing a high copy number plasmid bearing dgkA decreased sn-1,2-diacylglycerol levels. However, overproduction of diacylglycerol kinase in a dgkR1 genetic background did not affect diacylglycerol levels, suggesting that the dgkR1 mutation affects diacylglycerol metabolism by mechanisms additional to enhancement of dgkA transcription.

Lysis of Vibrio cholerae cells: direct isolation of the outer membrane from whole cells by treatment with urea.

Cells of Vibrio cholerae underwent rapid autolysis when suspended in media of low osmolarity under non-growing conditions. Chaotropes like urea and guanidine. HCl which are potent protein denaturants caused complete and immediate lysis of whole cells. This unique sensitivity of V. cholerae to protein denaturants led to the development of a rapid method for the selective isolation of the outer membrane upon treatment of whole cells with urea. The composition of the outer membrane isolated from both whole cells and crude envelopes by treatment with urea was comparable with that of the outer membrane isolated by other conventional methods.

Osmoregulation in Rhizobium meliloti: inhibition of growth by salts

A defined medium of low osmolarity was developed permitting growth of Rhizobium meliloti with generation times of approximately 2.8 h doubling-1. The effects of sodium, potassium, magnesium, ammonium, chloride, sulfate, phosphate, bicarbonate and acetate ions on the growth rate of R. meliloti were determined. Sodium, potassium and ammonium ions had little effect on growth at concentrations of 100 mEq or less; magnesium ion inhibited growth severely at concentrations of 50 mEq (25 mM). Of the anions, chloride and sulfate appeared to have little effect while phosphate, bicarbonate, and acetate inhibited growth at concentrations of as little as 25 mEq. The addition of proline, glutamate, or betaine to cells growing in inhibitory concentrations of NaCl did not relieve the inhibition. When grown in the presence of inhibitory levels of NaCl, the intracellular concentration of glutamate but not of proline or gamma amino butyric acid increased 5-fold.

Osmotic regulation and the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

The membrane-derived oligosaccharides (MDO) of Escherichia coli are periplasmic constituents containing 8-10 glucose units in a highly branched structure, linked by beta 1-2 and beta 1-6 bonds [Schneider, J. E. Reinhold, V., Rumley, M. K. & Kennedy, E. P. (1979) J. Biol. Chem. 254, 10135-10138]. The MDO are multiply substituted with sn-1-phosphoglycerol residues (derived from membrane phosphatidylglycerol) and with O-succinyl ester residues and, thus, are high anionic. Experiments in this paper offer evidence that the biosynthesis of MDO is an important aspects of osmoregulation in E. coli. Cells grown in medium of low osmolarity (ca. 50 mosM) synthesize 16 times more MDO than those grown in the same medium with 0.4 M NaCl. In cells grown in medium of low osmolarity, it appears that MDO is the principal source of fixed anion in the periplasmic space and, thus, acts to maintain the high osmotic pressure and Donnan membrane potential of the periplasmic compartment Regulation of MDO synthesis in response to changes in osmolarity of the medium appears to occur at the genetic level because the synthesis of new protein is needed to permit the production of MDO at high rates after shift of cells to medium of low osmolarity.

Separation of Newborn Rat Epidermal Cells on Discontinuous Isokinetic Gradients of PERCOLL

A one-step procedure has been developed for the separation of epidermal cells using PERCOLL (a new colloidal silica medium of low viscosity, osmolarity, and toxicity) for density gradient centrifugation. Newborn rat epidermal cells were dispersed with trypsin-EDTA and separated into 4 fractions in discontinuous isokinetic gradients. The cell fractions were characterized by their appearance in photomicrographs and their distribution by number and size. Preferential incorporation of 3H-thymidine and 14C-glycine, by basal and granular cells respectively, confirmed the identification of cell types. The basal cells, which were collected in the densest fraction (1.090), were the most homogeneous population with a mean diameter between 7-8 mum and showed 98% viability. The granular cells predominated in the least dense fraction (1.023). The intermediate fractions contained spinous cells admixed with the other cell types.

Synthesis of phosphatidylglycerol by chloroplasts from leaves of Spinacia oleracea L. (spinach)

Purified chloroplasts from leaves of Spinacia oleracea L. (spinach) incorporated glycerol 3-phosphate into diacylglycerol, monoacylglycerol, phosphatidylglycerol, phosphatidic acid, and lysophosphatidic acid. The omission of ATP or CTP, CoA or illumination decreased the incorporation markedly. The fraction of incorporated glycerol 3-phosphate found in phosphatidylglycerol was greatly reduced by the omission of bicarbonate, acetate, and ATP, or in darkness, low-osmolarity medium, or high magnesium ion concentration (10 m M). Incorporation of glycerol 3-phosphate into lipid and specifically into phosphatidylglycerol was optimal at a Mg 2+ CTP ratio of 1, whereas the optimal ratio for Mg 2+ ATP was closer to 2. The Mg 2+ CTP gave lower total incorporation but a higher fraction of incorporation in phosphatidylglycerol. Triton X-100 inhibited incorporation of glycerol 3-phosphate into lipid, especially into phosphatidylglycerol.

Clarification of factors influencing the nature and magnitude of the protonmotive force in bovine heart submitochondrial particles.

The magnitude of the protonmotive force, and its division between pH gradient and membrane potential components has been further characterised in submitochondrial particles. In a reaction medium containing sucrose for osmotic support and 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (Hepes) as buffer, with succinate as substrate, the total protonmotive force reached a maximum value of 245 mV. The presence of Cl- enhanced the pH gradient with a partial but not fully compensating decrease in the membrane potential. When submitochondrial particles were suspended in a medium of low osmolarity consisting of phosphoric acid neutralised with Tris, again with succinate as substrate, the protonmotive force was lower and did not exceed 185 mV, and the pH gradient component was equivalent to 25 mV or less. The final phosphorylation potential, delta Gp, (formula: see text); maintained by the particles was higher in the phosphate/Tris medium (46--47.7 kJ mol-1) than in the sucrose/Hepes/KCl medium (43.7 kJ mol-1). Thus, comparison of the phosphorylation potential with the protonmotive force would suggest that the mechanistic stoichiometry H+/ATP (H+ translocated per molecule of ATP synthesied) for the ATPase enzyme is 3 in the former medium and 2 in the latter, which might be taken to indicate two different types of mechanism required for ATP synthesis. However it is questioned whether a comparison of the protonmotive force with delta Gp in terms of equilibrium thermodynamics ought not to be complemented by analysis in terms of linear non-equilibrium thermodynamics. The latter treatment shows that it is possible to estimate only a value for the product of a phenomenological stoichiometry and the degree of coupling, which can be variable, but not the mechanistic stoichiometry. This treatment can also rationalise the observation of the higher delta Gp in reaction conditions where the lower values for delta p are estimated. Irrespective of possible explanations, the data show how an unprejudiced choice of reaction conditions can lead to different conclusions about the relationship between the phosphorylation potential and the protonmotive force.

Subcellular localization of a rat liver enzyme converting thyroxine into tri-iodothyronine and possible involvement of essential thiol groups.

Experiments with rat liver homogenates showed that on subcellular fractionation the ability to catalyse the conversion of thyroxine into tri-iodothyronine was lost. The activity could in part be restored by addition of the cytosol to the microsomal fraction. Both components were found to be heat labile. The necessity of the presence of cytosol could be circumvented by incorporation of thiol-group-containing compounds in the medium. Optimal enzymic activity was observed in the presence of dithiothreitol and EDTA in medium of low osmolarity. By comparing the distribution of the converting enzyme over the subcellular fractions with a microsomal marker enzyme, glucose 6-phosphatase, it was demonstrated that the former is indeed of microsomal origin. Finally, it was shown that thiol groups play an essential role in the conversion of thyroxine into tri-iodothyronine.

Multiple transport components for putrescine in Escherichia coli.

Putrescine uptake was studied in cultures of Escherichia coli K-12 grown in media of high or low osmolarity. When grown in high osmolarity medium, a transport system of low K(m) and low V(max) was found. For cultures grown in a medium of low osmolarity, the kinetics of putrescine uptake was more complex and consistent with the existence of an additional transport system of higher K(m) and V(max). This conclusion is supported by the isolation of mutants in which one or the other system appears to be defective and by the ability of chloramphenicol to block the expression of the second transport system. Both systems appear to prefer putrescine over other compounds, since several basic amino acids and other polyamines competed only weakly for transport. The action of both uptake systems was shown to cause significant displacement of intracellular putrescine. Both systems also are at least partially energy dependent.

The effect of osmolarity on mouse parthenogenesis

ABSTRACT Eggs from (C57BI × A2G)F1 mice were activated by treatment with hyaluronidase, which removed the follicle cells, and cultured in vitro. Observations were made 6–8 h after hyaluronidase treatment to determine the frequency of activation and the types of parthenogenones induced. Cumulus-free eggs resulting from hyaluronidase treatment were incubated for in culture media of various osmolarities. The frequency of activation was found to be dependent on the postovulatory age of oocytes, while the types of parthenogenones induced were dependent on the osmolarity of the in vitro culture medium and their postovulatory age. Culture in low osmolar medium suppressed the extrusion of the second polar body (2PB). This decreased the incidence of haploid eggs with a single pronucleus and 2PB and immediately cleaved eggs from 97·5% to 42·3 % of the activated population. Where 2PB extrusion had been suppressed, 97·4 % of parthenogenones contained two haploid pronuclei. Very few were observed with a single and presumably diploid pronucleus. Serial observations from 11 to 18 h after hyaluronidase treatment were made on populations of activated eggs as they entered the first cleavage mitosis after incubation in medium either of normal (0·287 osmol) or low (0·168 osmol) osmolarity. A delay in the time of entry into the first cleavage mitosis similar to the duration of incubation in low osmolar medium was observed. Further, eggs were incubated in control and low osmolar culture media containing uniformly labelled [U−14C]amino acid mixture to examine the extent of protein synthesis in recently activated eggs subjected to these culture conditions. An hypothesis is presented to explain the effect of incubation in low osmolar culture medium in delaying the first cleavage mitosis.

Phospholipid turnover in a conditional polyamine auxotroph of Escherichia coli.

Polyamines are required for rapid phospholipid turnover in Escherichia coli growing in media of low osmolarity.

Polyamine requirements of a conditional polyamine auxotroph of Escherichia coli.

Escherichia coli MA-159 is deficient in agmatine ureohydrolase. After addition of exogenous arginine, the cellular putrescine content declines immediately and exponentially; however, the spermidine content remains normal for 3 h. The growth rate of such cultures, measured turbidometrically, slows gradually over many hours. Putrescine-depleted cultures grow especially slowly in media of low osmolarity, whereas nondepleted cultures grow at similar and rapid rates in media of either normal or low osmolarity. External osmolarity also affects the ability of various exogenous polyamines to stimulate growth of putrescine-depleted cultures. In medium of normal osmolarity, putrescine and spermidine both allow sustained rapid growth for many hours. In low osmolarity medium, putrescine allows sustained rapid growth, whereas cultures containing spermidine grow more slowly; this result cannot be explained by conversion of putrescine to spermidine, for cultures grown with exogenous putrescine contain smaller spermidine pools than do cultures grown with exogenous spermidine. Spermine greatly stimulates growth in medium of normal osmolarity; however, in medium of low osmolarity, spermine is much less effective and can block the action of putrescine. Several other polyamines have been studied in this system. These results confirm and expand previous reports that polyamines are necessary for growth of E. coli and suggest that putrescine may have a specific function during growth in media of low osmolarity.